Biology

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A laboratory Manual of Small-Scale Experiments for the independent Study of

Microbiology

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The experiments in this manual have been and may be conducted in a regular formal laboratory or classroom setting with the users providing their own equipment and supplies. However, this manual was especially written for the benefit of the independent study of students who do not have convenient access to such facilities. It allows them to perform college and advanced high school level experiments at home or elsewhere by using a LabPaq, a collection of experimental equipment and supplies specifically packaged to accompany this manual.

Use of this manual and authorization to perform any of its experiments is expressly conditioned upon the user reading, understanding and agreeing to fully abide by all the safety precautions contained herein.

Although the author and publisher have exhaustively researched many sources to ensure the accuracy and completeness of the information contained in this manual, we assume no responsibility for errors, inaccuracies, omissions or any other inconsistency herein. Any slight of people, organizations, materials, or products is unintentional.

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Table of contents 5 Important Information to Help Students with the Study of Microbiology

Experiments 49 observing Bacteria and Blood

73 Bacterial Morphology

86 Aseptic Technique & Culturing Microbes

105 Isolation of Individual Colonies

129 Differential Staining

141 Methyl red Voges-Proskauer Test

153 Antibiotic Sensitivity

167 Microbes in the Environment

Appendix 178 Preparation of Cultures

181 Preparation of Disinfecting Solution

183 Final Cleanup Instructions

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Important Information to Help Students with the Study of Microbiology

Welcome to the study of microbiology. Do not be afraid of taking this course. By the end of the semester you will be really proud of yourself and will wonder why you were ever afraid of the m-word, microbiology! After their first microbiology class, most students say they thoroughly enjoyed it, learned a lot of useful information for their lives, and only regret not having studied it sooner.

Microbiology is not some “mystery” science only comprehendible by eggheads. Microbiology is simply the study of microscopic living organisms. It will be easier for you to understand the world we live in and to make the multitude of personal and global decisions that affect our lives and our planet after you have learned about the characteristics of life around you and how organisms change and interact with each other, with the environment, and with you. Plus, having microbiology credits on your transcript will certainly be impressive, and your microbiology knowledge may create some unique job opportunities for you.

This lab manual of microbiology experiments was designed to accompany any entry level college or advanced high school level microbiology course. It can be used by all students, regardless of the laboratory facilities available to them. Its experiments have been and continue to be successfully performed in regular microbiology laboratories. With the special LabPaq experiments can be performed at home by independent-study students or at small learning centers that do not have formal laboratories. Throughout the manual there are references about campus-based and independent study, but all of the information and references herein are equally relevant to both types of students.

Introduction

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Micro- and Small-Scale Experiments You may be among the growing number of students to take a full-credit microbiology course through independent study. If so, you can thank the development and perfection of micro- and small-scale techniques in microbiology experimentation. Experimentation is essential and fundamental to fully understanding the concepts of microbiology. In the past, microbiology courses required that all classes be conducted on a campus because experiments had to be performed in the campus laboratory. This was due in part to the potential hazards inherent in some traditional experimentation.

These elements of danger, plus increasing chemical and material costs and environmental concerns about chemical and biological material disposal, made high schools, colleges, and universities reexamine the traditional laboratory methods used to teach subjects such as chemistry and microbiology. Scientists began to scale down the quantities of chemicals used in their experiments and found that reaction results remained the same, even when very tiny amounts of chemicals were used. Institutions also discovered that student learning was not impaired by studying small- sized reactions.

Over time, more and more traditional chemistry and microbiology experiments were redesigned for micro- and small-scale techniques. One of the primary pioneers and most prominent contributors to micro- and small-scale experimentation is Dr. Hubert Alyea of Princeton University. He not only reformatted numerous experiments, he also designed many of the techniques and equipment used in micro- and small-scale chemistry and microbiology today.

With decreased hazards, costs, and disposal problems, micro- and small-scale experimentation techniques were quickly adapted for use in scholastic laboratories. As these techniques continued to be further refined it became possible to perform basic experiments in the classroom and eventually outside the classroom. This slow but steady progression of micro- and small-scale techniques makes it possible for independent study students to take a full-credit microbiology course since they can now perform experiments at home.

Introduction

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How to Study Microbiology Microbiology is not the easiest subject to learn, but neither is it the hardest. As in any other class, if you responsibly apply yourself, conscientiously read your text, and thoughtfully complete your assignments, you will learn the material. Here are some basic hints for effectively studying microbiology – or any other subject – either on or off campus.

Plan to Study: You must schedule a specific time and establish a specific place in which to seriously, without interruptions or distractions, devote yourself to your studies. Think of studying like you would think of a job, except that now your job is to learn. Jobs have specific times and places in which to get your work done, and studying should be no different. Just as television, friends, and other distractions are not permitted on a job; you should not permit them to interfere with your studies. You cannot learn when you are distracted. If you want to do something well, you must be serious about it. Only after you’ve finished your studies should you allow time for distractions.

get in the right Frame of Mind: Think positively about yourself and what you are doing. Give yourself a pat on the back for being a serious student and put yourself in a positive frame of mind to enjoy what you are about to learn. Then get to work! Organize any materials and equipment you will need in advance so you don’t have to interrupt your thoughts to find them later. Look over your syllabus and any other instructions to know exactly what your assignment is and what you need to do. Review in your mind what you have already learned. Is there anything that you aren’t sure about? Write it down as a formal question, then go back over previous materials to try to answer it yourself. If you haven’t figured out the answer after a reasonable amount of time and effort, move on. The question will develop inside your mind and the answer will probably present itself as you continue your studies. If not, at least the question is already written down so you can discuss it later with your instructor.

Be Active with the Material: Learning is reinforced by relevant activity. When studying feel free to talk to yourself, scribble notes, draw pictures, pace out a problem, tap out a formula, etc. The more active things you do with study materials, the better you will learn. Have highlighters, pencils, and note pads handy. Highlight important data, read it out loud, and make notes. If there is a concept you are having problems with, stand up and pace while you think it through. See the action taking place in your mind. Throughout your day try to recall things you have learned, incorporate them into your conversations, and teach them to friends. These activities will help to imprint the related information in your brain and move you from simple knowledge to true understanding of the subject matter.

Do the Work and Think about What you are Doing: Sure, there are times when you might get away with taking a shortcut in your studies, but in doing so you will probably shortchange yourself. The things we really learn are the things we discover ourselves. That is why we don’t learn as much from simple lectures or when someone gives us the answers. And when you have an assignment, don’t just go through the motions. Enjoy your work, think about what you are doing, be curious, examine your results, and consider the implications of your findings. These “critical thinking” techniques will improve and enrich your learning process. When you complete your assignments independently and thoroughly you will have gained knowledge and you will be proud of yourself.

Introduction

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How to Study Microbiology Independently There is no denying that learning through any method of independent study is a lot different than learning through classes held in traditional classrooms. A great deal of personal motivation and discipline is needed to succeed in a course of independent study where there are no instructors or fellow students to give you structure and feedback. But these problems are not insurmountable and meeting the challenges of independent study can provide a great deal of personal satisfaction. The key to successful independent study is in having a personal study plan and the personal discipline to stick to that plan.

Properly Use your learning Tools: The basic tools for telecourses, web courses and other distance-learning methods are often similar and normally consist of computer software or videos, textbooks, and study guides. Double check with your course administrator or syllabus to make sure you acquire all the materials you will need. These items are usually obtained from your campus bookstore, library, or via the Internet. Your area’s public and educational television channels may even broadcast course lectures and videos. If you choose to do your laboratory experimentation independently, you will need the special equipment and supplies described in this lab manual and contained in its companion LabPaq. The LabPaq can be purchased on the Internet at www. LabPaq.com.

For each study session, first work through the appropriate sections of your course materials. These basically serve as a substitute for classroom lectures and demonstrations. Take notes as you would in a regular classroom. Actively work with any computer and/or text materials, carefully review your study guide, and complete all related assignments. If you do not feel confident about the material covered, repeat these steps until you do. It’s a good idea to review your previous work before proceeding to a new section. This reinforces what you previously learned and prepares you to absorb new information. Experimentation is the very last thing done in each study session and it will only be really meaningful if you have first absorbed the text materials that it demonstrates.

Plan to Study: A regular microbiology course with a laboratory component will require you to spend around 15 hours a week studying and completing your assignments. Remember, microbiology is normally a 5-credit hour course! To really learn new material there is a generally accepted 3-to- 1 rule that states that at least 3 hours of class and study time are required each week for each hour of course credit taken. This rule applies equally to independent study and regular classroom courses. On campus, microbiology students are in class for 4 hours and in the laboratory for 2 to 3 hours each week. Then they still need at least 8 hours to read their text and complete assignments. Knowing approximately how much time you need will help you to formulate a study plan at the beginning of the course and then stick with it.

Schedule your Time Wisely: The more often you interact with study materials and call them to mind, the more likely you are to reinforce and retain the information. Thus, it is much better to study in several short blocks of time rather than in one long, mind-numbing session. Accordingly, you should schedule several study periods throughout the week, or better yet, study a little each day. Please do not try to do all of your study work on the weekends! You will just burn yourself out, you won’t really learn much, and you will probably end up feeling miserable about yourself and microbiology. Wise scheduling can prevent such unpleasantness and frustration.

Introduction

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Choose the Right Place for Your Home Laboratory If you are experimenting at home, the best place to perform your micro- and small-scale microbiology experiments is in an uncluttered room that has these important features:

● a door that can be closed to keep out pets and children

● a window or door that can be opened for fresh air ventilation and fume exhaust

● a source of running water for fire suppression and cleanup

● a counter or table-top work surface

● a heat source such as a stove top, hot dish, or Bunsen burner

The kitchen usually meets all these requirements, but you must make sure you clean your work area well both before and after experimentation. This will keep foodstuff from contaminating your experiment and your experiment materials from contaminating your food. Sometimes a bathroom makes a good laboratory, but it can be rather cramped and subject to a lot of interruptions. Review the “Basic Safety” section of this manual to help you select the best location for your home-lab and to make sure it is adequately equipped.

Introduction

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Organization of the Lab Manual Before proceeding with the experiments you need to know what is expected of you. To find out, please thoroughly read and understand all the various sections of this manual.

laboratory Notes: Like all serious scientists you will record formal notes detailing your activities, observations, and findings for each experiment. These notes will reinforce your learning experiences and knowledge of microbiology. Plus, they will give your instructional supervisor a basis for evaluating your work. The “Laboratory Notes” section of this manual explains exactly how your lab notes should be organized and prepared.

Required Equipment and Supplies: This manual also contains a list of the basic equipment and supplies needed to perform all the experiments. Students performing these experiments in a non-lab setting must obtain the “LabPaq” specifically designed to accompany this manual. It includes all the equipment, materials, and chemicals needed to perform these experiments, except for some items usually found in the average home or obtainable in local stores. At the beginning of each experiment there is a “Materials” section that states exactly which items the student provides and which items are found in the LabPaq. Review this list carefully to make sure you have all these items on hand before you begin the experiment. It is assumed that campus- based students will have all the needed equipment and supplies in their laboratories and that the instructors will supply required materials and chemicals in the concentrations indicated.

Laboratory Techniques: While these techniques primarily apply to full-scale experiments in formal laboratories, knowledge of them and their related equipment is helpful to the basic understanding of microbiology and may also be applicable to your work with micro- and small- scale experimentation.

Basic Safety and Micro-scale Safety reinforcement: The use of this lab manual and the LabPaq, plus authorization to perform their experiments, are expressly conditioned upon the user reading, understanding and agreeing to abide by all the safety rules and precautions noted. Additional terms authorizing use of the LabPaq are contained in its purchase agreement. These safety sections are relevant to both laboratory and non-laboratory experimentation. They describe potential hazards plus the basic safety equipment and safety procedures designed to avoid such hazards. The Basic Safety and Micro-scale Safety Reinforcement sections are the most important sections of this lab manual and should always be reviewed before starting each new experiment.

Experiments: All experimental materials and procedures are fully detailed in the laboratory manual for each experiment. Chemicals and supplies unique for a specific experiment are contained in a bag labeled with the experiment number.

Introduction

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How to Perform an Experiment Although each experiment is different, the process for preparing, performing, and recording all the experiments is essentially the same.

Review Basic Safety: Before beginning reread the safety sections, try to foresee potential hazards, and take appropriate steps to prevent problems.

Read through the Entire Experiment before You Start: Knowing what you are going to do before you do it will help you to be more effective and efficient.

Organize Your Work Space, Equipment, and Materials: It is hard to organize your thoughts in a disorganized environment. Assemble all required equipment and supplies before you begin working. These steps will also facilitate safety.

outline your lab Notes: Outline the information needed for your lab notes and set up required data tables. This makes it much easier to concentrate on your experiment. Then simply enter your observations and results as they occur.

Perform the Experiment According to Instructions: Follow exactly all directions in a step-by-step format. This is not the time to be creative. DO NOT attempt to improvise your own procedures!

Think About What you Are Doing: Stop and give yourself time to reflect on what has happened in your experiment. What changes occurred? Why? What do they mean? How do they relate to the real world? This step can be the most fun and often creates “light bulb” experiences of understanding.

Complete Your Lab Notes and Answer Required Questions: If you have properly followed all the above steps, this concluding step will be easy.

clean-up: Blot any minute quantities of unused chemicals with a paper towel or flush them down the sink with generous amounts of water. Discard waste in your normal trash. Always clean your equipment immediately after use or residue may harden and be difficult to remove later. Return equipment and supplies to their proper place, and if working at home with a LabPaq, store it out of the reach of children and pets.

Introduction

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Antibiotic Sensitivity 24 – 48 hrs. ahead 1 hour 24 – 72 hours 1 hour

EXPEriMENT 11:

Fomite Transmission None 1 – 2 hours 24 – 72 hours Less than 1 hour

EXPEriMENT 12:

Microbes in the Environment None 1 – 3 hours 24 – 72 hours Less than 1 hour

EXPEriMENT 13: 24 hour intervals

Fungi None Less than 1 hour Up to 1 week 2 – 3 hours

Estimated Time Requirements for Each Experiment Note: These estimates are provided to help you plan and schedule your time. They are given per individual lab performed separately and do not consider time and step savings possible when several labs are grouped together. Of course, these are only estimates and your actual time requirements may differ.

Experiment No. / Title Preparation Experimenting Incubation After Incubation

EXPEriMENT 1:

Observing Bacteria & Blood None 3 – 4 hours None None

EXPEriMENT 2:

Bacterial Morphology None 3 – 4 hours None None

EXPEriMENT 3:

Aseptic Techniques & Culturing Microbes None 1 – 2 hours 24 – 48 hours Less than 1 hour

EXPEriMENT 4:

Isolation of Individual Colonies None-use Exp. 3 cultures 3 – 4 hours 24 – 48 hours Less than 1 hour

EXPEriMENT 5: 30 minutes

Differential Staining 24 – 48 hours ahead 3 – 4 hours 24 – 48 hours None

EXPEriMENT 6:

Methyl Red 30 minutes

Voges-Proskauer Test 24 – 48 hours ahead Less than 1 hour 48 – 72 hours 1 hour

EXPEriMENT 7: 30 minutes

Motility Testing 24 – 48 hours ahead Less than 1 hour 24 – 48 hours Less than 1 hour

EXPEriMENT 8:

Carbohydrate 30 minutes

Fermentation Testing 24 – 48 hrs. ahead Less than 1 hour 12 – 24 hours Less than 1 hour

EXPEriMENT 9: 30 minutes

Osmosis 24 – 48 hrs. ahead Less than 1 hour 24 – 72 hours Less than 1 hour

EXPEriMENT 10: 30 minutes

Introduction

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laboratory Notes and lab reports Normally two basic records are compiled during and from scientific experimentation activities. The first record is Lab Notes which you will record as you perform your actual experiments. Entries into your lab notebook will be the basis for your second record, the Lab Report. The Lab Report formally summarizes the activities and findings of your experiment and is what is normally submitted for instructor grading.

Scientists keep track of their experimental procedures and results through lab notes that are recorded in a journal-type notebook as they work. In laboratories these notebooks are often read by colleagues such as directors and other scientists working on a project. In some cases scientific notebooks have become evidence in court cases. Thus, lab notes must be intelligible to others and include sufficient information so that the work performed can be replicated and so there can be no doubt about the honesty and reliability of the data and of the researcher.

Notebooks appropriate for data recording are bound and have numbered pages that cannot be removed. Entries normally include all of the scientist’s observations, actions, calculations, and conclusions related to each experiment. Data is never entered onto pieces of scratch paper to later be transferred, but rather is always entered directly into the notebook. When erroneous data is recorded, a light diagonal line is drawn neatly through the error, followed by a brief explanation as to why the data was voided. Information learned from an error is also recorded. Mistakes can often be more useful than successes, and knowledge gained from them is valuable to future experimentation.

As in campus-based science laboratories, independent-study students are normally expected to keep a complete scientific notebook of their work that may or may not be periodically reviewed by their instructor. Paperbound 5×7 notebooks of graph paper usually work well as science lab notebooks. Since it is not practical to send complete notebooks back and forth between instructors and students for each experiment, independent-study students usually prepare formal Lab Reports that are submitted to their instructors along with regular assignments via e-mail or fax.

Lab notes of experimental observations can be kept in many ways. Regardless of the procedure followed, the key question for deciding what kind of notes to keep is this: “Do I have a clear enough record so that I could pick up my lab notebook or read my Lab Report in a few months and still explain to myself or others exactly what I did?” Laboratory notes normally include these components:

Title: This should be the same title stated in the laboratory manual.

Purpose: Write a brief statement about what the experiment is designed to determine or demonstrate.

Procedure: Briefly summarize what you did in performing this exercise and what equipment was used. Do not simply copy the procedure statement from the lab manual.

Data Tables: Tables are an excellent way to organize your observational data. Where

Introduction

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applicable, the “Procedures” section of the experiment often advises a table format for data recording. Always prepare tables before experimenting so they will be ready to receive data as it is accumulated.

Observations: What did you observe, smell, hear, or otherwise measure? Usually, observations are most easily recorded in table form.

Questions: Questions are asked frequently throughout and at the end of exercises. They are designed to help you think critically about the exercise you just performed. Answer thoughtfully.

conclusions: What did you learn from the experiment? Your conclusions should be based on your observations during the exercise. Conclusions should be written in your best formal English, using complete sentences, paragraphs, and correct spelling.

Here are some general rules for keeping a lab notebook on your science experiments:

Leave the first two to four pages blank so you can later add a “Table of Contents” at the front of the notebook. Entries into the table of contents should include the experiment number and name plus the page number where it can be found.

● Your records should be neatly written.

● The notebook should not contain a complete lab report of your experiment. Rather, it should simply be a record of what you did, how you did it, and what your results were. Your records need to be complete enough so that any reasonably knowledgeable person familiar with the subject of your experiment, such as another student or your instructor, can read the entries, understand exactly what you did, and if necessary, repeat your experiment.

● Organize all numerical readings and measurements in appropriate data tables as in the sample Lab Report presented later.

● Always identify the units for each set of data you record (centimeters, kilograms, seconds, etc.).

● Always iden tify the equipment you are using so you can find or create it later if needed to recheck your work.

● It is an excellent idea to document important steps and observations of your experiments via digital photos and also to include yourself in these photos. Such photos within your Lab Report will document that you actually performed the experiment as well as what you observed.

● In general, it is better to record more rather than less data. Even details that may seem to have little bearing on the experiment you are doing (such as the time and the temperature when the data were taken and whether it varied during the observations) may turn out to be information that has great bearing on your future analysis of the results.

● If you have some reason to suspect that a particular data set may not be reliable (perhaps you had to make the read ing very hurriedly) make a note of that fact.

Introduction

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● Never erase a reading or data. If you think an entry in your notes is in error, draw a single line through it and note the correction, but do not scratch it out completely or erase it. You may later find that it was significant after all.

Although experimental results may be in considerable error, there is never a “wrong” result in an experi ment for even errors are important results to be considered. If your observations and measurements were carefully made, your result will be correct. Whatever happens in nature, includ ing the laboratory, cannot be wrong. Errors may have nothing to do with your investigation, or they may be mixed up with so many other events you did not expect that your report is not use- ful. Yet even errors and mistakes have merit and often lead to our greatest learning experiences. Thus, you must think carefully about the interpretation of all your results, including your errors.

Finally, the cardinal rule in a laboratory is to fully carry out all phases of your experiments instead of “dry-labbing” or taking shortcuts. The Greek scientist, Archytas, summed this up very well in 380 BCE:

In subjects of which one has no knowl edge one must obtain knowledge either by learning from someone else or by discover ing it for oneself. That which is learned, there- fore, comes from another and by outside help; that which is discovered comes by one’s own efforts and independently. To discover without seeking is difficult and rare, but if one seeks it is frequent and easy. If, however, one does not know how to seek, discovery is im possible.

Introduction

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Science lab report Format This guide covers the overall format that formal Lab Reports normally follow. Remember that the Lab Report should be self-contained so that anyone, including someone without a science background and without a lab manual, can read it and understand what was done and what was learned. Data and calculation tables have been provided for many of the labs in this manual and students are encouraged to use them. Computer spreadsheet programs such as Excel® can greatly facilitate the preparation of data tables and graphs. One website with additional information on preparing lab reports is: http://www.ncsu.edu/labwrite/. Remember, above average work is necessary to receive above average grades!

Lab Reports are expected to be word processed and to look organized and professional. They should be free of grammar, syntax, and spelling errors and be a respectable presentation of your work. Writing in the first person should be avoided as much as possible. Lab Reports should generally contain these sections:

● Title Page

● Section 1: Abstract, Experiment Description, Procedures, and Observations including photos, drawings, and data tables

● Section 2: Analysis including calculations, graphs, and error analysis

● Section 3: Discussion of Results

Each of the above three sections is discussed in greater detail below. They should be clearly distinguished from each other in the actual report. The presentation and organization skills developed by producing science Lab Reports will be beneficial to all potential career fields.

Title Page: This is the first page of the lab report and consists of:

a. Experiment number and/or title

b. Your name

c. The names of any lab partner(s)

d. The date and time the experiment was preformed

e. The location should be included if work was performed in the field

f. The course number

Introduction

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Section 1: Abstract, Experiment, and Observation Abstract: Even though the abstract appears at the beginning of the report, it is written last and inserted into the beginning. An abstract is a very concise description of the experiment’s objective, results, and conclusions. It should be no longer than a paragraph.

Experiment and Observation: Carefully, yet concisely, describe, in chronological order, what was done, what was observed, and what, if any, problems were encountered. Describe what field and laboratory techniques and equipment were employed to collect and analyze the data upon which the conclusions are based. Photos and graphic illustrations are usually inserted in this section. Graphics should be in .jpg or .gif format to minimize their electronic file size.

Show all work for any calculations performed. Every graph must have a title and its axes must be clearly labeled. Curves through data points this should be “best-fit curves,” which are smooth straight or curved lines that best represents the data, rather than a dot-to-dot connection of data points.

Include all data tables, photos, graphs, lists, sketches, etc. in an organized fashion. Include relevant symbols and units with data. Generally a sentence or two explaining how data was obtained is appropriate for each data table.

Note any anomalies observed or difficulties encountered in collecting data as these may affect the final results. Include information about any errors observed and what was learned from them. Be deliberate in recording the experimental procedures in detail. Your comments may also include any preliminary ideas you have on explaining the data or trends you see emerging.

Introduction

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Section 2: Analysis including Calculations, Graphs, and Error Analysis Generally, the questions at the end of each lab will act as a guide for preparing results and conclusions. This section is normally written in paragraph form and not more than one or two pages long. Additional considerations are:

● What is the connection between the experimental measurements taken and the final results and conclusions? How do your results relate to the real world?

● What were the results of observations and calculations?

● What trends were noticed?

● What is the theory or model behind the experiment preformed?

● Do the experimental results substantiate or refute the theory? Why? Be sure to refer specifically to the results you obtained!

● Were the results consistent with your original predictions of outcomes or were you forced to revise your thinking?

● Did “errors” such as environmental changes (wind, rain, etc.) or unplanned friction occur? If so, how did they affect the experiment?

● Did any “errors” occur due to the equipment used such as estimates being skewed due to a lack of sufficient measurement gradients on a beaker?

● What recommendations might improve the procedures and results?

Errors: In a single paragraph comment on the accuracy and precision of the apparatus and include a discussion of the experimental errors and an estimate of the error in your final result. Remember, “errors” are not “mistakes!” Errors arise because the apparatus and/or the environment inevitably fail to match the “ideal circumstances” assumed when deriving a theory or equations. The two principal sources or error are:

Physical phenomena: Elements in the environment may be similar to the phenomena being measured and thus may affect the measured quantity. Examples might include stray magnetic or electric fields or unaccounted for friction.

Limitations of the observer, the analysis, and/or the instruments: Examples are parallax error when reading a meter tape, the coarse scale of a graph, and the sensitivity of the instruments.

Examples of “mistakes” and “human errors” that are not acceptable scientific errors include:

a. Misuse of calculator (pushing the wrong button, misreading the display)

b. Misuse of equipment

c. Faulty equipment

d. Incorrectly assembled circuit or apparatus

Introduction

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Section 3: Discussion, Results, and Conclusions Discussion: The discussion section should be carefully organized and include consideration of the experiment’s results, interpretation of results, and uncertainty in results as further described below. This section is normally written in paragraph form and no more than one to two pages in length. Occasionally it will be more appropriate to organize various aspects of the discussion differently for different labs. Not all of the following questions will apply to every lab.

results

● What is the connection between your observations, measurements, and final results?

● What were the independent or dependent variables in the experiment?

● What were the results of your calculations?

● What trends were noticeable?

● How did the independent variables affect the dependent variables? For example, did an increase in a given independent variable result in an increase or decrease in the associated dependent variable?

Interpretation of Results

● What is the theory or model behind the experiment you performed?

● Do your experimental results substantiate or agree with the theory? Why or why not? Be sure to refer specifically to YOUR experimental results!

● Were these results consistent with your original beliefs or were you forced to re-evaluate your prior conceptions?

Uncertainty in results:

● How much did your results deviate from expected values?

● Are the deviations due to error or uncertainty in the experimental method or are they due to idealizations inherent in the theory, or are they due to both?

● If the deviations are due to experimental uncertainties can you think of ways to decrease the amount of uncertainty?

● If the deviations are due to idealizations in the theory what factors has the theory neglected to consider? In either case, consider whether your results display systematic or random deviations.

All of these comments on lab notes and lab reports undoubtedly sound complex and overwhelming upon first reading. But do not worry; they will make more sense to you when you actually begin to perform the experiments and write reports. After writing the first few lab reports they will become second nature to you. This manual contains a sample lab report example of “A” level work to provide a better understanding of how a formal lab report is written.

Introduction

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Laboratory Drawings Laboratory work often requires findings to be illustrated in representational drawings. Clear, well organized drawings are an excellent way to convey observations and are often more easily understood than long textual descriptions. The adage “a picture is worth a thousand words” really is true when referring to science laboratory notes.

Students often say they can’t draw, but with a little care and practice, anyone can illustrate science lab observations. A trick most artist’s use is to place a mental grid over the object or scene and then approach their drawing from the standpoint of the grid areas. For instance, look at the diagram below and quickly make a free hand drawing of it. Then mentally divide the diagram into quarters and try drawing it again. In all likelihood, the second grid-based drawing will yield a better result.

Give yourself ample drawing space, and leave a white margin around the actual illustration so it can be seen clearly. Also, leave a broad margin along one side of your drawing to insert labels for the objects in the drawing. Use a ruler to draw straight lines for the labels and as connecting lines between the objects and their related labels. The following is a good example of how your lab drawings should look when they are included in a formal lab report.

SoUrcE oF DrAWiNg

Such as MUNG BEAN

your Name

Date of Drawing

TiTlE oF DrAWiNg

Such as CELL STRUCTURE

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Visual Presentation of Data Learning to produce good graphs and tables is important because like pictures they can quickly and clearly communicate information visually. That is why graphs and tables are often used to represent or depict data that has been collected. Graphs and tables should be constructed in such a way that they are able to “stand alone.” That means, all the information required to understand a graph or table must be included in it. A graph is composed of two basic elements: the graph itself and the graph legend. The legend adds the descriptive information needed to fully understand the graph. In the graph at right the legend shows that the red line represents Red Delicious apples, the brown line is the Gala apples, and the green line is the Wine Sap apples. Without the legend it would be difficult to interpret this graph.

One of the most important uses of a graph is to “predict” data that is not measured by the data. In interpolation a graph is used to construct new data points within the range of a discrete set of known data points. As an example, if the data points on the pH graph are recorded at pHs of 1, 3, 5, 7, 9, and 11 but the investigator wants to know what happens at pH 6 the information can be found by interpolating the data between the points of pH 5 and 7. Follow the red line up to interpolate the value, there would be 12 tadpoles living at a pH of 6.

Along the same lines, a graph line can be extended to extrapolate data that is outside of the measured data. For example, if the researcher wanted to know what would happen at a pH greater than 11, this can be extrapolated by extending the line. In the example at right, the blue line represents an extrapolation that allows scientists to predict what might happen. Why is extrapolation less reliable than interpolation?

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Concentration of Plant Fertilizer vs. Plant Height

x-axis y-axis Fertilizer % solution Plant Height in cm 0 25 10 34 20 44 30 76 40 79 50 65 60 40

Constructing a Table: A table allows for the data to be presented in a clear and logical way. The independent data is put at the left hand side of the table and the dependent data falls to the right of that. Keep in mind that there will be only one independent variable but there can be more than one dependent variable. The decision to present data in a table rather than a figure is often arbitrary. However, a table may be more appropriate than a graph when the data set is too small to warrant a graph, or it is large and complex and is not easily illustrated. Frequently, a data table is provided to display the raw data, while a graph is then used to make the visualization of the data easier.

Setting up a Graph: Consider a simple plot of the “Plant Fertilizer” versus the “Plant Height.” This is a plot of points on a set of X and Y coordinates. The x-axis or abscissa, runs horizontally, while the y-axis or ordinate, runs vertically. By convention, the x-axis is used for the independent variable which is defined as a manipulated variable in an experiment whose presence determines the change in the dependent variable. The y-axis is used for the dependent variable which is the variable affected by another variable or by a certain event. In our example, the amount of fertilizer is the independent variable and should go on the x-axis. The plant height is the dependent

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variable and should go on the y-axis since it may change as a result of or dependent on how the amount of fertilizer changes.

One way to help figure out which data goes on the x-axis versus the y-axis is to think about what affects what, so does fertilizer affect plant height or would plant height affect the fertilizer. Only one of these should make sense, plant height will not change the fertilizer but the fertilizer will have an effect on the plant height. So which ever causes the change is the independent or x-axis and which responds as a result of that change is the dependent or y-axis.

The rules for constructing a table are similar. The important point is that the data is presented clearly and logically. As shown in the prior table, the independent data is put at the left-hand side of the table and the dependent data falls to the right of that. Keep in mind that there will be only one independent variable, but there can be more than one dependent variable. The decision to present data in a table rather than a figure is often arbitrary. However, a table may be more appropriate than a graph when the data set is too small to warrant a graph, or it is large and complex and is not easily illustrated. Frequently, a data table is provided to display the raw data, while a graph is then used to make the visualization of the data easier.

If the data deals with more than one dependent variable such as the apple varieties seen in the first example, it would be represented with three lines and a key or legend would be needed to identify which line represents which data set. In all graphs each axis is labeled and the units of measurement are specified. When a graph is presented in a lab report, the variables, the scale, and the range of the measurements should be clear. Graphs are often the clearest and easiest way to depict the patterns in your data — they give the reader a “feel” for the data.

Use the table below to help set up a line graph. Once you have a good feel for how to create a graph on your own, explore computer graphing using MS Excel.

Another easy program to use is http://nces.ed.gov/nceskids/Graphing/Classic/line.asp

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Introduction

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Introduction to Microscopy Ever since their invention in the late 1500s, light microscopes have enhanced our knowledge of basic microbiology, biomedical research, medical diagnostics, and materials science. Light microscopes can magnify objects up to 1000 times, revealing a world unknown to the naked eye details. Light-microscopy technology has evolved far beyond the first microscopes of Robert Hooke and Antoni van Leeuwenhoek. Special techniques and optics have been developed to reveal the structures and biochemistry of living cells. Microscopes have even entered the digital age, using fluorescent technology and digital cameras, yet the basic principles of these advanced microscopes are a lot like those of the microscope you will use in this class.

A light microscope works very much like a refracting telescope but with some minor differences. Let’s briefly review how a telescope works.

A telescope must gather large amounts of light from a dim, distant object. Therefore, it needs a large objective lens to gather as much light as possible and bring it to a bright focus. Because the objective lens is large, it brings the image of the object to a focus at some distance away which is why telescopes are much longer than microscopes. The eyepiece of the telescope then magnifies that image as it brings it to your eye.

In contrast to a telescope, a microscope must gather light from a tiny area of a thin, well- illuminated specimen that is nearby. So the microscope does not need a large objective lens. Instead, the objective lens of a microscope is small and spherical, which means that it has a much shorter focal length on either side. It brings the image of the object into focus at a short distance within the microscope’s tube. The image is then magnified by a second lens, called an ocular lens or eyepiece, as it is brought to your eye.

The other major difference between a telescope and a microscope is that a microscope has a light source and a condenser. The condenser is a lens system that focuses the light from the source onto a tiny, bright spot of the specimen which is the same area that the objective lens examines.

Also, unlike a telescope, which has a fixed objective lens and interchangeable eyepieces, microscopes typically have interchangeable objective lenses and fixed eyepieces. By changing the objective lenses (going from relatively flat, low-magnification objectives to rounder, high- magnification objectives), a microscope can bring increasingly smaller areas into view — light gathering is not the primary task of the objective lens of a microscope, as it is with that of a telescope.

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The Parts of a light Microscope A light microscope hAs the following bAsic systems

● Specimen control – to hold and manipulate the specimen

● Stage – where the specimen rests

● clips – to hold the specimen still on the stage. Because you are looking at a magnified image, even the smallest movements of the specimen can move parts of the image out of your field of view

● Illumination – to shed light on the specimen. The simplest illumination system is a mirror that reflects room light up through the specimen

● lamp – to produce the light. Typically, lamps are tungsten-filament light bulbs. For specialized applications, mercury or xenon lamps may be used to produce ultraviolet light. Some microscopes even use lasers to scan the specimen

● condenser – a lens system that aligns and focuses the light from the lamp onto the specimen

● diaphragm or disc apertures – placed in the light path to alter the amount of light that reaches the condenser. Varying the amount of light alters the contrast in the image

● lenses – to form the image

● objective lens – to gather light from the specimen

● eyepiece – to transmit and magnify the image from the objective lens to your eye

● nosepiece – a rotating mount that holds many objective lenses

● tube – to hold the eyepiece at the proper distance from the objective lens and blocks out stray light

● Focus – to position the objective lens at the proper distance from the specimen

● coarse-focus knob – to bring the object into the focal plane of the objective lens

● fine-focus knob – to make fine adjustments to focus the image

● Support and alignment

● arm – a curved portion that holds all of the optical parts at a fixed distance and aligns them

● base – supports the weight of all of the microscope parts

● tube – connected to the arm of the microscope by way of a rack and pinion gear which allows you to focus the image when changing lenses or observers and to move the lenses away from the stage when changing specimens

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Some of the parts mentioned above may vary between microscopes. Microscopes come in two basic configurations: upright and inverted. The microscope shown in the diagram is an upright microscope, which has the illumination system below the stage and the lens system above the stage. An inverted microscope has the illumination system above the stage and the lens system below the stage. Inverted microscopes are better for looking through thick specimens, such as dishes of cultured cells, because the lenses can get closer to the bottom of the dish where the cells grow.

Light microscopes can reveal the structures of living cells and tissues as well as of non-living samples such as rocks and semiconductors. Microscopes can be simple or complex in design, and some can do more than one type of microscopy, each of which reveals slightly different information. The light microscope has greatly advanced our biomedical knowledge and continues to be a powerful tool for scientists

Some Microscope Terms:

● Depth of field – the vertical distance, from above to below the focal plane, that yields an acceptable image

● Field of view – the area of the specimen that can be seen through the microscope with a given objective lens

● Focal length – the distance required for a lens to bring the light to a focus (usually measured in millimeters)

● Focal point/focus – the point at which the light from a lens comes together

● Magnification – the product of the magnifying powers of the objective and eyepiece lenses (a 15x eyepiece and a 40x objective lens will give you 15×40=600 power magnification)

● Numerical aperture – the measure of the light-collecting ability of the lens

● Resolution – the closest two objects can be before they are no longer detected as separate objects (usually measured in nanometers)

● Image Quality – When you look at a specimen using a microscope, the quality of the image you see is assessed by the following:

● Brightness – How light or dark is the image? Brightness is related to the illumination system and can be changed by changing the wattage of the lamp and by adjusting the condenser diaphragm aperture. Brightness is also related to the numerical aperture of the objective lens; the larger the numerical aperture, the brighter the image.

● Focus – Is the image blurry or well-defined? Focus is related to focal length and can be controlled with the focus knobs. The thickness of the cover glass on the specimen slide can also affect your ability to focus the image if it is too thick for the objective lens. The correct thickness is usually written on the side of the objective lens.

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Image of pollen grain under good brightness (left) and poor brightness (right)

● Resolution – How close can two points in the image be before they are no longer seen as two separate points? Resolution is related to the numerical aperture of the objective lens (the higher the numerical aperture, the better the resolution) and by the wavelength of light passing through the lens (the shorter the wavelength, the better the resolution).

Image of pollen grain in focus (left) and out of focus (right)

● contrast – What is the difference in lighting between adjacent areas of the specimen? Contrast is related to the illumination system and can be adjusted by changing the intensity of the light and the diaphragm/pinhole aperture. Also, chemical stains applied to the specimen can enhance contrast.

Image of pollen grain with good resolution (left) and poor resolution (right)

When observing a specimen by transmitted light, light must pass through the specimen in order to form an image. The thicker the specimen the less light passes through and thereby the darker the image. The specimens must therefore be thin (0.1 to 0.5 mm). Many organic specimens must be cut into thin sections before observation. Specimens of rock or semiconductors are too thick

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to be sectioned and observed by transmitted light, so they are observed by the light reflected from their surfaces.

Image of pollen grain with good contrast (left) and poor contrast (right)

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Types of Microscopy A major problem in observing specimens under a microscope is that their images do not have much contrast. This is especially true of living things (such as cells), although natural pigments, such as the green in leaves, can provide good contrast. One way to improve contrast is to treat the specimen with colored pigments or dyes that bind to specific structures within the specimen. Different types of microscopy have been developed to improve the contrast in specimens. The specializations are mainly in the illumination systems and the types of light passed through the specimen. Brightfield is the basic microscope configuration (the images seen thus far are all from brightfield microscopes). This technique has very little contrast and much of the contrast is provided by staining the specimens. A darkfield microscope uses a special condenser to block out most of the bright light and illuminate the specimen with oblique light, much like the moon blocks the light from the sun in a solar eclipse. This optical set-up provides a totally dark background and enhances the contrast of the image to bring out fine details of bright areas at boundaries within the specimen.

Following are various types of light microscopy techniques. They achieve different results by using different optical components. The basic idea involves splitting the light beam into two pathways that illuminate the specimen. Light waves that pass through dense structures within the specimen slow down compared to those that pass through less dense structures. As all of the light waves are collected and transmitted to the eyepiece, they are recombined, so they interfere with each other. The interference patterns provide contrast. They may show dark areas (more dense) on a light background (less dense), or create a type of false three-dimensional (3D) image.

● Phase-contrast – A phase-contrast microscope is best for looking at living specimens, such as cultured cells. The annular rings in the objective lens and the condenser separate the light paths. Light passing through the central part of the light path is then recombined with light traveling around the periphery of the specimen. Interference produced by these two paths produces images in which dense structures appear darker than the background.

A phase-contrast image of a glial cell cultured from a rat brain

● Differential Interference Contrast (DIC) – DIC uses polarizing filters and prisms to separate and recombine the light paths, giving a 3D appearance to the specimen (DIC is also called Nomarski after the man who invented it).

● Hoffman Modulation Contrast – Hoffman modulation contrast is similar to DIC except that it uses plates with small slits in both the axis and the off-axis of the light path to produce two sets of light waves passing through the specimen. Again, a 3D image is formed.

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● Polarization – The polarized-light microscope uses two polarizers, one on either side of the specimen, positioned perpendicular to each other so that only light that passes through the specimen reaches the eyepiece. Light is polarized in one plane as it passes through the first filter and reaches the specimen. Regularly-spaced, patterned or crystalline portions of the specimen rotate the light that passes through. Some of this rotated light passes through the second polarizing filter, so these regularly spaced areas show up bright against a black background.

● Fluorescence – This type of microscope uses high-energy, short-wavelength light (usually ultraviolet) to excite electrons within certain molecules inside a specimen, causing those electrons to shift to higher orbits. When they fall back to their original energy levels, they emit lower-energy, longer-wavelength light (usually in the visible spectrum), which forms the image.

Care and Handling of the Microscope

● When you move your microscope, you should always use two hands. Place one hand around the arm, lift the scope, and put your other hand under the base of the scope for support. If you learn to carry the scope in this way, it will force you to carry it carefully, ensuring that you do not knock it against anything while moving from one place to another.

● When you put the scope down, do so gently. If you bang your scope down on the table eventually you could jar lenses and other parts loose. Your microscope seems like a simple instrument but each eyepiece and objective is actually made up of a number of lenses put together in a precise way to create wonderful magnification. If you bang your scope around, you are shaking upward of 15 to 20 lenses.

● Always have clean hands when handling your scope. It would be a shame to damage your scope with too much peanut butter!

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Storing the Microscope

● If you have a sturdy, stable desk, table, or shelf on which to keep your scope and it is a place where the scope will not be disturbed or bumped, this is the best place to store your scope. Just make sure that you keep it covered with a plastic or vinyl cover when it is not in use. Dust is an enemy to your lenses; always keep your scope covered when not in use.

● If you are unable to find a safe place where you can leave your scope out, store it in the fitted foam case it comes in.

cleaning the Microscope

● The first step in keeping your microscope clean is to keep it from getting dirty. Always keep your microscope covered with the dust cover when it is not in use.

● Your eyepiece will need cleaning from time to time. Due to its position on the scope, it will have a tendency to collect dust and even oil from your eyelashes. The eyepiece lens should be cleaned with a high-quality lens paper, such as is available from a camera shop or an eyeglass center. Brush any visible dust from the lens and then wipe the lens. You may wish to use a bit of lens solution, applied to the lens paper to aid in cleaning. A cotton swab can be used in place of lens paper but do not use facial tissues to clean your lenses.

● You will also need to occasionally clean the objective lenses. Use a fresh area of lens paper each time so that you don’t transfer dust from one lens to another.

● Clean the lenses in the glass condenser under the stage.

● Finally, clean the glass lens over your light, or the mirror, so that an optimal amount of light can shine through. You can also follow up by wiping down the whole scope with a soft, clean cotton towel.

Using the Microscope ● Take the microscope body from the case. Put the eyepiece in the opening in the tube at the

top of the microscope. Remove the objective lenses from their individual containers and screw them into the revolving nosepiece, placing each in the color-coded position that corresponds to the color band on the lens.

● Adjust the tension on the focusing control knobs to suit your touch or to compensate for normal wear over time. To increase tension, hold the right-hand knob firmly and turn the opposite knob clockwise, whereas turning it counter clockwise loosens the tension.

● Unplug the rotating mirror bracket from the base of the microscope, insert the mirror (packaged separately with the microscope) into the bracket so that it swivels freely, and plug it back into the base of the microscope.

● Tilt the arm of the microscope back until it is at a position where you can comfortably look into the microscope eyepiece.

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● Place a slide under the clips on the stage, with the area you wish to view in line, between the lens selected and the hole in the stage.

● Turn the nosepiece of the microscope to select the longest lens (usually the highest power lens). Lower the barrel of the microscope with the coarse-focus knob until it almost touches the slide. If it will not go that far then unscrew the focus stop screw under the arm of the microscope until the lens can almost touch the slide, and while it is in that position lightly tighten the screw and lock it in place with the knurled nut.

● Place a light source in front of the microscope, use the small lever on the sub-stage condenser to open the diaphragm fully, and adjust the mirror so that the light is brightest as seen through the microscope.

● Rotate the nosepiece to select the lowest power lens. Lower the barrel with the coarse-focus knob until the tip of the lens is near the slide. Now raise the barrel slowly with the coarse- focus knob until you see an image from the slide. Finish the focus with the fine-focus knob.

● With thumb and forefinger on each end of the slide, move it slowly on the stage until the object you wish to study is centered in your field of view.

● Rotate the nosepiece of the microscope to select the objective lens that will give you the higher magnification you need.

● Once one lens is focused properly any other objective lens on the nosepiece rotated into position will be roughly in focus, requiring only fine focus to bring the image with the new lens into correct focus.

● Move the lever for the diaphragm through its full range to select the amount of light that gives you the best contrast. Many details will be visible with good contrast which would otherwise be lost with much or too little light.

Using the Electric illuminator

Grasp the illuminating mirror with your fingers behind its bracket and pull to unplug the bracket and mirror from the base of the microscope. Insert the metal plug tip of the electric illuminator into the hole from which you have unplugged the mirror bracket. Rotate the fixture so that the glass opening over the bulb points up toward the light condenser under the stage. Plug the electric cord into a 115-volt outlet and turn on the switch in the cord.

Using the Oil Immersion Lens (purchased separately)

Install the oil immersion 100x objective lens in place of any of the other objective lenses. The 4x lens is a good choice. First, focus the microscope and center the slide using a lower magnification objective. Apply a drop of oil on the specimen slide and turn the revolving nosepiece to bring the 100x objective into position. If the barrel is too low to allow the 100x lens to move into position raise it with the coarse focus very slightly, position the lens, and then lower the barrel until the tip of the 100x lens touches the oil and the slide. The tip of the lens is able to move a short distance into the lens against a spring in order to keep from putting too much pressure on the slide. With the lens tip touching the oil and slide focus with the fine-focus knob. The working distance of the lens is very short so do not use the coarse-focus knob other than to position the lens. After using the oil immersion lens wipe off the oil carefully with alcohol.

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Preparation of Solid Media Microbiological media are used to grow microbes for study and experimentation. Most bacteria collected in the environment will not be harmful. However, once an isolated microbe multiplies by millions in a broth tube or Petri dish it can become more of a hazard. Be sure to protect open cuts with rubber gloves and never ingest or breathe in growing bacteria. Keep growing Petri dishes taped closed until your experiment is done. Then you should safely destroy the bacteria colonies using bleach.

Microbiological media may be prepared as either liquid or as a solid media. When a solid medium is prepared, a corresponding liquid broth is solidified by the addition of agar to the broth. Agar is a polysaccharide found in the cell walls of some algae. It is inert and degraded by very few microorganisms. In addition, the fact it melts at around 100oC and solidifies at approximately 45oC-50oC makes it an ideal solidifying agent for microbiological media.

proceDUres Preparation of Solid Media

1. Disinfect your work area with a 10% bleach solution.

2. Place the test tube rack into a pan of water and place your tubes of agar into the rack. The agar will melt more easily if the water level is above or at the level of the agar. If your pan is not deep enough to bring the water above the level of the agar you will need to shake the tubes during the melting process to mix the melted and unmelted portion of the agar.

3. Place the pan on the stove top and bring to a boil. Once the water begins to boil, the agar should melt within 10 to 15 minutes. Remember, if your water level is below the level of the agar you will need to shake the tubes to mix the unmelted agar into the melting agar. Be careful as the heating tubes will be hot!

4. Once the agar media is melted, remove the pan from the heat but do not remove the tubes from the hot water.

5. Allow the water to cool until the tubes are cool enough to handle but the agar media is still liquid (50°C-60°C).

6. Label the bottom of two Petri dishes (per tube) with the type of medium you are using (in this LabPaq you will use nutrient or MRS agar).

7. Using aseptic handling techniques pour the liquid agar from the 18-mL tube into the bottom of the labeled Petri dishes. If you are preparing both types of medium, be careful to pour each medium into the correctly labeled dish. Pour enough to cover the bottom of each dish 1/8”- 1/4” thick (approximately 9mL so each 18-mL tube will make two dishes). Cover each dish with its lid immediately.

8. When all the dishes are poured, cover them with a paper towel to help prevent contamination and allow them to cool and solidify.

9. The agar dishes are done when solid. You may store the cooled dishes in a zip baggie in the refrigerator for later use or use them immediately.

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Preparation of Cultures Culture tubes should remain lidded while incubating. Do not open them once inoculated unless under aseptic conditions and to perform a necessary experimental step.

Saccharomyces cervicae: Add 1/2 teaspoon dry Saccharomyces cervicae (active dry yeast envelope) to 1/8 cup warm water (you can use a sample cup or any household cup) and gently swirl to mix. Set the culture aside to activate for at least 10 minutes. Stir to mix prior to using. Escherichia coli: 10. Remove the tube labeled: Broth, Nutrient – 5 mL in Glass tube, from culture media bag #2

from the refrigerator and allow it to come to room temperature.. 11. Moisten a paper towel with a small amount of alcohol and wipe the work area down. 12. Once the nutrient broth media is at room temperature:

● Remove the numbered E-coli culture tube from the cultures bag and remove its cap. Set the cap upside down to avoid contamination.

● Uncap the nutrient broth; set its cap upside down to avoid contaminating it while the broth is open.

● Use sterile techniques and draw 0.25mL of the nutrient broth into a sterile graduated pipet. NOTE: To sterilize the pipet draw a small amount of 70% alcohol into the bulb, and then expel it into a sink. Remove any excess alcohol by forcefully swinging the pipet in a downward arch several times to ensure that the pipet is dry before drawing up the nutrient broth. Add the broth to the vial containing the lyophilized E-coli pellet. Recap the E-coli vial and shake to mix until the pellet has dissolved in the broth. Note that the vial should be about one-half full to allow for shaking and mixing the pellet.

● Once the pellet has dissolved, use the same sterile pipet to draw up the E. coli solution and expel it into the original tube of nutrient broth. Recap the broth. NOTE: If the pipet has become contaminated, simply draw a small amount of 70% alcohol into the bulb and then expel it into a sink. Remove any excess alcohol by forcefully swinging the pipet in a downward arch several times to ensure that the pipet is dry before drawing up the E. coli solution.

Recap the nutrient broth and incubate the now E-coli inoculated tube of nutrient broth at 37°C. The culture should show active growth between 24 to 48 hours; it can be left as a liquid culture or plated out. Most freeze dried cultures will grow within a few days however some may exhibit a prolonged lag period and should be given twice the normal incubation period before discarding as non-viable. Refer to Experiment 3 for a description of indicators of growth.

● Lactobacillus acidophilus: Remove a tube of MRS broth from the refrigerator and allow it to come to room temperature. Aseptically transfer a portion of a tablet of L. acidophilus into the tube of media. Allow the tube to set, swirling periodically, as the tablet dissolves. There will be a significant amount of sediment in the bottom of the tube. Mark the level of the sediment with a marker, pencil, or pen. Incubate the inoculated tube at 37°C. The culture should show active growth between 24 to 48 hours. Refer to Experiment 3 for a description of indicators of growth. L. acidophilus often sediments as it grows. An increase (above the sediment line you marked on the tube) in the sediment is an indication of growth. Swirl the tube to mix the

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organisms back into the broth prior to use. ● Staphylococcus epidermidis: You can culture S. epidermidis as a liquid or solid culture.

Because you are inoculating from an environmental source (your skin), your sample may contain bacteria other than S. epidermidis. Thus, broth cultures derived directly from sampling may not be pure cultures of S. epidermidis. With the exception of Experiments 3 and 4 (#3 establishes a broth culture and #4 uses it to establish a pure culture), use the dish culture method to ensure you are using a pure sample for your experiment.

● Broth cultures of S. epidermidis: Without contaminating the cotton tip, cut the length of the swab such that it will fit entirely into a capped test tube. Dampen the cotton tip sterile swab with distilled water and rub it vigorously on your skin. Do not try to obtain a bacterial culture soon after washing your skin. Additionally choose an area that is not as likely to have been scrubbed as recently (the inside of the elbow or back of the knee is generally a good site). Do not obtain a sample from any bodily orifice (mouth, nose, etc.) as you are not likely to culture the desired microbe (Staphylococcus epidermidis). Using aseptic technique, place the swab into a tube of nutrient media, label the tube accordingly. Incubate the inoculated tube at 37°C. The culture should show active growth between 24 to 48 hours. Refer to Experiment 3 for a description of indicators of growth.

● Dish cultures of S. epidermidis: Use a sterile swab to obtain a sample of S. epidermidis from your skin described in the generation of a broth culture. Rub the swab lightly on the surface of one dish of nutrient agar to inoculate it with S. epidermidis. As the swab may not contain a high number of bacteria, be sure to rub all sides of the swab on the dish to transfer as many individual bacterium as possible. Incubate the dish at 37°C for 24 to 48 hours. The S. epidermidis culture was not a pure culture (derived from a single organism) and will most likely contain colonies from several different organisms. You will need to identify and select a colony. Staphylococci produce round, raised, opaque colonies, 1 – 2 mm in diameter. S. epidermidis colonies are white in color. Below is a picture of S. epidermidis grown on blood agar. As the sample is of human origin, it potentially contains bacteria that can act as opportunistic pathogens. Do not select or use any colony that does not appear to be S. epidermidis. If your dish contains colonies other than S. epidermidis, soak it in a 10% bleach solution and discard. Do not attempt to save the dish for use in future experiments!

You can either use the S. epidermidis colonies directly or amplify growth in a broth culture. If you choose to amplify into nutrient broth, 24 hours beginning the experiment, choose a S. epidermidis colony from the incubated dish and aseptically transfer the colony using an inoculation loop into a tube of nutrient media. Be sure to mix the broth gently to disburse the clumped bacteria into the broth. Incubate the tube at 37°C for an additional 24 hours.

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Microbiology Safety ● Any microbe can be hazardous. While the majority of microorganisms are not pathogenic

to humans and have never been shown to cause illness, under unusual circumstances a few microorganisms that are not normally pathogenic can act as pathogens. These are called opportunistic pathogens. Treat all microorganisms—especially unknown cultures such as from skin swabs or environmental samples—as if they were pathogenic. A student who has a compromised immune system or has had a recent extended illness is at higher risk for opportunistic infections. Do not attempt to swab your throat or nasal passages when sampling for S. epidermidis. You are not likely to culture the correct organism. Additionally, you are more likely to culture an opportunistic pathogen from these areas!

● Sterilize equipment and materials. All materials, media, tubes, dishes, loops, needles, pipets, and other items used for culturing microorganisms should be sterilized. Most of the materials and media you will be using are commercially sterilized products. You will be given instruction for sterilization with either flame or with a 10% bleach solution for items that are not sterilized or that will be reused.

● Disinfect work areas before and after use. Use a disinfectant, such as a 10% bleach solution to wipe down benches and work areas both before and after working with cultures. Also be aware of the possible dangers of the disinfectant. Bleach, if spilled, can ruin your clothing and can be dangerous if splashed into the eyes. Students should work where a sink is located to facilitate immediate rinsing if bleach is splashed or spilled.

● Wash your hands. Use an antibacterial soap to wash your hands before and after working with microorganisms. Non-antibacterial soap will remove surface bacteria and can be used if antibacterial soap is not available. Gloves should be worn as an extra protection.

● Never pipet by mouth. Use pipet bulbs or pipet devices for the aspiration and dispensing of liquid cultures.

● Do not eat or drink while working with microorganisms. Never eat or drink while working with microorganisms. Keep your fingers out of your mouth, and wash your hands before and after the laboratory activity. Cover any cuts on your hands with a bandage. Gloves should be worn as an extra protection.

● Label everything clearly. All cultures, chemicals, disinfectants, and media should be clearly and securely labeled with their names and dates.

● Disinfect all waste material. All items to be discarded after an experiment, such as culture tubes, culture dishes, swabs, and gloves, should be covered with a 10% bleach solution and allowed to soak for at least 1 to 2 hours. After soaking, the materials can be rinsed and disposed of by regular means.

● Clean up spills with care. Cover any spills or broken culture tubes with a 10% bleach solution; then cover with paper towels. After allowing the spill to sit with the disinfectant, carefully clean up and place the materials in a bag for disposal. If you are cleaning up broken glass, place the materials in a puncture-proof container (such as a milk carton), and label the container “broken glass” before placing in the trash. Wash the area again with disinfectant. Never pick

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up glass fragments with your fingers or stick your fingers into the culture itself. Instead, use a brush and dustpan.

● Be certain to dispose of cultures properly. Liquid cultures should have bleach added to them (to create a solution that is approximately 10% bleach) and allowed to set for a minimum of one hour before disposal. The deactivated samples can be discarded in the sink. Be sure to flush with plenty of water to remove any bleach residue. Petri dishes or any solid culture material should be soaked in a 10% bleach solution for a minimum of one hour. They can then be bagged and discarded in the trash.

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Basic Safety guidelines This section contains vital information that must be thoroughly read and completely understood before a student begins to perform experiments.

PrEVENT iNJUriES AND AcciDENTS! Science experimentation is fun, but does involve potential hazards which must be acknowledged to be avoided. To safely conduct science experiments, students must first learn and then always follow basic safety procedures. Although there are certainly not as many safety hazards in experimenting with physics and geology as there are in chemistry and biology, safety risks exist in all science experimentation and science students need to be aware of safety issues relevant to all the disciplines. Thus, the following safety procedures review is relevant to all students regardless of their field of study.

While this manual tries to include all relevant safety issues, not every potential danger can be foreseen as each experiment involves slightly different safety considerations. Thus, students must always act responsibly, learn to recognize potential dangers, and always take appropriate precautions. Regardless of whether a student will be working in a campus or home laboratory setting, it is extremely important that he or she knows how to anticipate and avoid possible hazards and to be safety conscious at all times.

BASic SAFETy ProcEDUrES: Science experimentation often involves using toxic chemicals, flammable substances, breakable items, and other potentially dangerous materials and equipment. All of these things can cause injury and even death if not properly handled. These basic safety procedures apply when working in a campus or home laboratory.

● Because eyesight is precious and eyes are vulnerable to chemical spills and splashes, to shattered rocks and glass, and to floating and flying objects:

» Students must always wear eye protecting safety goggles when experimenting

● Because toxic chemicals and foreign matter may enter the body through digestion:

» Drinking and eating are always forbidden in laboratory areas

» Students must always wash their hands before leaving their laboratory

» Students must always clean their lab area after experimentation

● Because toxic substances may enter the body through the skin and lungs:

» The laboratory area must always have adequate ventilation

» Students must never “directly” inhale chemicals

» Students should wear long-sleeved shirts, pants, and enclosed shoes when in their lab area

» Students must wear gloves and aprons when appropriate

● Because hair, clothing, and jewelry can create hazards, cause spills, and catch fire while experimenting:

» Students should always tie or pin back long hair

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» Students should always wear snug fitting clothing (preferably old)

» Students should never wear dangling jewelry or objects

● Because a laboratory area contains various fire hazards:

» Smoking is always forbidden in laboratory areas

● Because chemical experimentation involves numerous potential hazards:

» Students must know how to locate and use basic safety equipment

» Students must never leave a burning flame or reaction unattended

» Students must specifically follow all safety instructions

» Students must never perform any unauthorized experiments

» Students must always properly store equipment and supplies and ensure these are out of the reach of small children and pets

● Because science equipment and supplies often include breakable glass and sharp items that pose potential risks for cuts and scratches and small items as well as dangerous chemicals that could cause death or injury if consumed:

» Students must carefully handle all science equipment and supplies

» Students must keep science equipment and supplies stored out of the reach of pets and small children

» Students must ensure pets and small children will not enter their lab area while they are experimenting

● Because science experimentation may require students to climb, push, pull, spin, and whirl:

» Students should undertake these activities cautiously and with consideration for people, property, and objects that could be impacted

» Students must ensure any stool, chair, or ladder used to climb is sturdy and take ample precautions to prevent falls

● Because students’ best safety tools are their own minds and intellectual ability:

» Students must always preview each experiment, and carefully think about what safety precautions need to be taken to perform the experiment safely

BASIC SAFETY EQUIPMENT: The following pieces of basic safety equipment are found in all campus laboratories. Informal and home laboratories may not have or need all of these items, but simple substitutes can usually be made or found. Students should know their exact location and proper use.

SAFETy gogglES – There is no substitute for this important piece of safety equipment! Spills and splashes do occur, and eyes can very easily be damaged if they come in contact with laboratory chemicals, shattered glass, swinging objects, and flying rock chips. While normal eyeglasses do provide some protection, these items can still enter the eyes from the side. Safety goggles

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cup around all sides of the eyes to provide the most protection and can be worn over normal eyeglasses if required.

EYEWASH STATION – All laboratories should have safety equipment to wash chemicals from the eyes. A formal eyewash station looks like a water fountain with two faucets directed up at spaces to match the space between the eyes. In case of an accident, the victim’s head is placed between the faucets while the eyelids are held open so the faucets can flush water into the eye sockets and wash away the chemicals. In an informal laboratory, a hand-held shower wand can be substituted for an eyewash station. After the eyes are thoroughly washed, a physician should be consulted promptly.

FIRE EXTINGUISHER – There are several types of fire extinguishers, at least one of which should be found in all types of laboratories. Students should familiarize themselves with and know how to use the particular type of fire extinguisher in their laboratory. At a minimum, home laboratories should have a bucket of water and a large pot of sand or dirt available to smother fires.

FirE BlANKET – This is a tightly woven fabric used to smother and extinguish a fire. It can cover a fire area or be wrapped around a victim who has caught on fire.

SAFETY SHOWER – This shower is used in formal laboratories to put out fires or douse people who have caught on fire or suffered a large chemical spill. A hand-held shower wand is the best substituted for a safety shower in a home laboratory.

FirST-AiD KiT – This kit of basic first-aid supplies is used for the emergency treatment of injuries and should be found in both formal and informal laboratories. It should be always well stocked and easily accessible.

SPill coNTAiNMENT KiT – This kit consists of absorbent material that can be ringed around a spilled chemical to keep it contained until the spill can be neutralized. The kit may simply be a bucket full of sand or other absorbent material such as kitty litter.

FUME HOOD – This is a hooded area containing an exhaust fan that expels noxious fumes from the laboratory. Experiments that might produce dangerous or unpleasant vapors are conducted under this hood. In an informal laboratory such experiments should be conducted only with ample ventilation and near open windows or doors. If a kitchen is used for a home laboratory, the exhaust fan above the stove substitutes nicely for a fume hood.

POTENTIAL LABORATORY HAZARDS: Recognizing and respecting potential hazards is the first step toward preventing accidents. Please appreciate the grave dangers the following laboratory hazards represent. Work to avoid these dangers and consider how to respond properly in the event of an accident.

FirES: The open flame of a Bunsen burner or any heating source combined, even momentarily, with inattention may result in a loose sleeve, loose hair, or some unnoticed item catching fire. Except for water, most solvents including toluene, alcohols, acetones, ethers, and acetates which are highly flammable and should never be used near an open flame. As a general rule NEVER LEAVE AN OPEN FLAME OR REACTION UNATTENDED. In case of fire, use a fire extinguisher, fire blanket and/or safety shower.

CHEMICAL SPILLS: Flesh burns may result if acids, bases, or other caustic chemicals are spilled and

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come in contact with skin. Flush the exposed skin with a gentle flow of water for several minutes at a sink or safety shower. Acid spills should be neutralized with simple baking soda, sodium bicarbonate. If eye contact is involved use the eyewash station or its substitute. Use the spill containment kit until the spill is neutralized. To better protect the body from chemical spills, wear long-sleeved shirts, full-length pants, and enclosed shoes, not sandals, when in the laboratory.

AciD SPlATTEr: When water is added to concentrated acid the solution becomes very hot and may splatter acid on the user. Splattering is less likely to occur if acid is slowly added to the water: Remember this AAA rule: Always Add Acid to water, NEVER add water to acid.

GLASS TUBING HAZARDS: Never force a piece of glass tubing into a stopper hole. The glass may snap and the jagged edges can cause a serious cut. Before inserting glass tubing into a rubber or cork stopper hole be sure the hole is the proper size. Lubricate the end of the glass tubing with glycerol or soap, and then while grasping it with a heavy glove or towel, gently but firmly twist the tubing into the hole. Treat any cuts with appropriate first-aid.

HEATED TEST TUBE SPLATTER: Splattering and eruptions can occur when solutions are heated in a test tube. Thus, you should never point a heated test tube toward anyone. To minimize this danger direct the flame toward the top, rather than the bottom, of the solution in a test tube. Gently agitate the tube over the flame to heat the contents evenly.

SHATTERED GLASSWARE: Graduated cylinders, volumetric flasks and certain other pieces of glassware are NOT designed to be heated. If heated, they are likely to shatter and cause injuries. Always ensure you are using heatproof glass before applying it to a heat source. Special caution should always be taken when working with any type of laboratory glassware.

INHALATION OF FUMES: To avoid inhaling dangerous fumes, partially fill your lungs with air and, while standing slightly back from the fumes, use your hand to waft the odors gently toward your nose and then lightly sniff the fumes in a controlled fashion. NEVER INHALE FUMES DIRECTLY! Treat inhalation problems with fresh air and consult a physician if the problem appears serious.

INGESTION OF CHEMICALS: Virtually all the chemicals found in a laboratory are potentially toxic. To avoid ingesting dangerous chemicals, never taste, eat, or drink anything while in the laboratory. All laboratories, especially those in home kitchens, should always be thoroughly cleaned after experimentation to avoid this hazard. In the event of any chemical ingestion immediately consult a physician.

HORSEPLAY: A laboratory full of potentially dangerous chemicals and equipment is a place for serious work, not for horseplay! Fooling around in the laboratory is just an invitation for an accident.

VEry iMPorTANT cAUTioN For WoMEN: If you are pregnant or could be pregnant, you should seek advice from your personal physician before doing any type of science experimentation.

If you or anyone accidentally consumes or otherwise comes into contact with something that is not easily washed away (such as splashed in the eyes) with a chemical that might be toxic, you should immediately call the National Poison Control Center for advice at:

1-800-332-3073

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Safety Quiz Refer to the illustration on the following page when answering the questions.

1. List three unsafe activities in the illustration and explain why each is unsafe.

2. List three correct procedures depicted in the illustration.

3. What should Tarik do after the accident?

4. What should Lindsey have done to avoid an accident?

5. Compare Ming and David’s laboratory techniques. Who is following the rules?

6. What are three things shown in the laboratory that should not be there?

7. Compare Joe and Tyler’s laboratory techniques. Who is working the correct way?

8. What will happen to Ray and Chris when the instructor catches them?

9. List three items in the illustration that are there for the safety of the students.

10. What is Consuela doing wrong?

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Science lab Safety reinforcement Agreement Any type of science experimentation involves potential hazards and unforeseen risks may exist. The need to prevent injuries and accidents cannot be over-emphasized!

Use of this lab manual and any LabPaq are expressly conditioned upon the student agreeing to follow all safety precautions and accept full responsibility for his or her own actions. Study the safety section of the manual until you can honestly state the following:

_ Before beginning an experiment, I will first read all directions and then assemble and organize all required equipment and supplies.

_ I will select a work area that is inaccessible to children and pets while experiments are in progress. I will not leave experiments unattended and I will not leave my work area while chemical equipment is set up unless the room will be locked.

_ To avoid the potential for accidents, I will clear my home-lab workspace of all non-laboratory items before setting up the equipment and supplies for my lab experiments.

_ I will never attempt an experiment until I fully understand it. If in doubt about any part of an experiment, I will first speak with my instructor before proceeding.

_ I will wear safety goggles when working with chemicals or items that get into my eyes.

_ I know that except for water, most solvents such as toluene, alcohols, acetone, ethers, ethyl acetate, etc. are highly flammable and should never be used near an open flame.

_ I know that the heat created when water is added to concentrated acids is sufficient to cause spattering. When preparing dilute acid solutions, I will always add the acid to the water (rather than the water to the acid) while slowly stirring the mixture.

_ I know it is wise to wear rubber gloves and goggles when handling acids and other dangerous chemicals, that acid spills should be neutralized with sodium bicarbonate (baking soda), and that acid spilled on the skin or clothes should be washed off immediately with a lot of cold water.

_ I know that many chemicals produce toxic fumes and that cautious procedures should be used when smelling any chemical. When I wish to smell a chemical I will never hold it directly under my nose but instead will use my hand to waft vapors toward my nose.

_ I will always handle glassware with respect and promptly replace any defective glassware because even a small crack can cause glass to break, especially when heated. To avoid cuts and injuries, I will immediately dispose of any broken glassware.

_ I will avoid burns by testing glass and metal objects for heat before handling. I know that the preferred first aid for burns is to immediately hold the burned area under cold water for several minutes.

_ I know that serious accidents can occur if the wrong chemical is used in an experiment. I will always carefully read the label before removing any chemical from its container.

_ I will avoid the possibility of contamination and accidents by never returning an unused chemical to its original container. To avoid waste, I will try to pour out only the approximate amount of chemicals required.

_ I know to immediately flush any chemical that spills on the skin with cold water and then consult a doctor if required.

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_ To protect myself from potential hazards I will wear long pants, a long-sleeved shirt, and enclosed shoes and I will tie up any loose hair, clothing, or other materials when performing chemical experiments.

_ I will never eat, drink, or smoke while performing experiments.

_ After completing all experiments, I will clean up my work area, wash my hands, and store the lab equipment in a safe place that is inaccessible to children and pets.

_ I will always conscientiously work in a reasonable and prudent manner so as to optimize my safety and the safety of others whenever and wherever I am involved with any type of science equipment or experimentation.

It is impossible to control students’ use of this lab manual and related LabPaqs or students’ work environments, the author(s) of this lab manual, the instructors and institutions that adopt it, and Hands-On Labs, Inc. the publisher of the manual and producer of LabPaqs authorize the use of these educational products only on the express condition that the purchasers and users accept full and complete responsibility for all and any liability related to their use of same. Please review this document several times until you are certain you understand it and will fully abide by its terms; then sign and date the agreement were indicated below.

I am a responsible adult who has read, understands, and agrees to fully abide by all safety precautions prescribed in this manual for laboratory work and for the use of a LabPaq. Accordingly, I recognize the inherent hazards potentially associated with science experimentation; I will always experiment in a safe and prudent manner; and I unconditionally accept full and complete responsibility for any and all liability related to my purchase and/or use of a science LabPaq or any other science products or materials provided by Hands-On Labs, Inc. (HOL).

____________________________________________________ ____________

Student’s Name (print) and Signature Date

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MSDS: Material Safety Data Sheets A Material Safety Data Sheet (MSDS) is designed to provide chemical, physical, health, and safety information on chemical reagents and supplies. An important skill in the safe use of chemicals is being able to read an MSDS. It provides information about how to handle store, transport, use, and dispose of chemicals in a safe manner.

MSDS also provide workers and emergency personnel with the proper procedures for handling and working with chemical substances. While there is no standard format for an MSDS, they all provide basic information about physical data (melting point, boiling point, flash point, etc.), toxicity, health effects, first aid procedures, chemical reactivity, safe storage, safe disposal, protective equipment required, and spill cleanup procedures. An MSDS is required to be readily available at any business where any type of chemical is used. Even day-care centers and grocery stores need MSDS for their cleaning supplies.

It is important to know how to read and understand the MSDS. They are normally designed and written in the following sections:

Section 1: Product Identification (Chemical Name and Trade Names)

Section 2: Hazardous Ingredients (Components and Percentages)

Section 3: Physical Data (Boiling point, density, solubility in water, appearance, color, etc.)

Section 4: Fire and Explosion Data (Flash point, extinguisher media, special fire fighting procedures, and unusual fire and explosion hazards)

Section 5: Health Hazard Data (Exposure limits, effects of overexposure, emergency and first aid procedures)

Section 6: Reactivity Data (Stability, conditions to avoid, incompatible materials, etc.)

Section 7: Spill or Leak Procedures (Steps to take to control and clean up spills and leaks, and waste disposal methods)

Section 8: Control Measures (Respiratory protection, ventilation, protection for eyes or skin, or other needed protective equipment)

Section 9: Special Precautions (How to handle and store, steps to take in a spill, disposal methods, and other precautions)

Summary: The MSDS is a tool that is available to employers and workers for making decisions about chemicals. The least hazardous chemical should be selected for use whenever possible, and procedures for storing, using, and disposing of chemicals should be written and communicated to workers.

View MSDS information at www.hazard.com/msds/index.php. You can also find a link to MSDS information at www.LabPaq.com. If there is ever a problem or question about the proper handling of any chemical, seek information from one of these sources.

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LabPaq by Hands-On Labs

experiments

observing Bacteria and Blood Cynthia Alonzo, M.S. Version 42-0249-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will learn how to use a microscope to observe prepared slides of three major types of bacteria, the protists Paramecium and Amoeba, yeast, and the fungi Penicillium. Students will prepare slides to observe bacterial cultures obtained from yogurt. They will also prepare and study blood smears to identify platelets and red and white blood cells.

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objectives ● Gain functional knowledge of microscope operations through practical applications of a

microscope in the observation of bacteria and blood

● Identify and observe various bacterial shapes and arrangements in a yogurt culture

● Identify and observe red and white blood cells in a blood smear

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materials

MATEriAlS QTY iTEM DEScriPTioN

Student provides 1 Plain active culture yogurt 1 Collection container 1 Microscope with 100x oil immersion lens 1 Immersion oil 1 Toothpick 1 Bandage 1 Distilled water

LabPaq provides 1 Gloves, Disposable 1 Lens-paper-pack-50-sheets 1 Slide – Cover Glass – Cover Slip Cube 1 Lancet 1 Form, Lancet, Sterile – Directions for Use 1 Alcohol Prep Pad 4 Pipet, Long Thin Stem 1 Slide – Amoeba proteus 1 Slide – Anabaena, w.m. 1 Slide – Ascaris eggs, w.m. 1 Slide – Bacteria bacillus form 1 Slide – Bacteria coccus form 1 Slide – Bacteria spirillum 1 Slide – Letter e Focusing Slide 1 Slide – Paramecium conjugation 1 Slide – Penicillium w/conidia 1 Slide – Yeast, w.m. 1 Slide – Yogurt bacteria 1 Slide-Box-MBK with Blank-Slides 1 Mask, Face with Earloops

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Discussion and review Since their invention in the late 1500s, light microscopes have enhanced our knowledge of basic microbiology, biomedical research, medical diagnostics, and materials science. Light microscopes can magnify objects up to 1500 times, revealing a world of details unknown to the naked eye. Light-microscopy technology has evolved far beyond the first microscopes of Robert Hooke and Antoni van Leeuwenhoek. Special techniques and optics have been developed to reveal the structures and biochemistry of living cells. Microscopes have even entered the digital age, using fluorescent technology and digital cameras.

A light microscope works similar to a refracting telescope with some minor differences. A telescope must gather large amounts of light from a dim, distant object. Therefore, the telescope needs a large objective lens to gather as much light as possible and bring it to a bright focus. Because the objective lens is large, it brings the image of the object at a distance to a focus, which is why telescopes are much longer than microscopes. Then the telescope eyepiece magnifies the image as it brings it to your eye.

In contrast to a telescope, a microscope must gather light from a tiny area of a thin, well-illuminated specimen that is nearby. Hence, the microscope does not need a large objective lens. Instead, the microscope’s objective lens is small and spherical, which means it has a much shorter focal length on either side. The lens brings the image of the object into focus at a short distance within the microscope’s tube. Then a second lens, called an ocular lens or eyepiece, magnifies the image as it brings it to your eye.

The other major difference between a telescope and a microscope is a microscope has a light source and a condenser. The condenser is a lens system that focuses the light from a source onto a tiny, bright spot of the specimen, which is the same area the objective lens examines.

Also, unlike a telescope, which has a fixed objective lens and interchangeable eyepieces, microscopes typically have interchangeable objective lenses and fixed eyepieces. By changing the objective lenses – moving from relatively flat, low-magnification objectives to rounder, high- magnification objectives – a microscope can bring increasingly smaller areas into view. Light gathering is not the primary task of a microscope objective lens, as it is with that of a telescope.

The Parts of a light Microscope

A light microscope has the following basic systems:

Specimen control: used to hold and manipulate the specimen.

● Stage: where the specimen rests.

● clips: holds the specimen on the stage. When looking at a magnified image, even moving the specimen slightly can move parts of the image out of view.

llumination: used to shed light on the specimen. The simplest illumination system is a mirror that reflects room light up through the specimen.

● lamp: produces light. Typically, lamps are tungsten-filament light bulbs. For specialized applications, mercury or xenon lamps may be used to produce ultraviolet light. Some microscopes use lasers to scan the specimen.

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Experiment Observing Bacteria and Blood

● condenser: a lens system that aligns and focuses the light from the lamp onto the specimen.

● Diaphragm or disc apertures: placed in the light path to alter the amount of light reaching the condenser. Varying the amount of light alters the image contrast.

lenses: used to form the image.

● Objective lens: gathers light from the specimen.

● Eyepiece: transmits and magnifies the image from the objective lens to your eye.

● Nosepiece: a rotating mount that holds many objective lenses.

● Tube: holds the eyepiece at the proper distance from the objective lens and blocks out stray light.

Focus: used to position the objective lens at the proper distance from the specimen.

● coarse-focus knob: brings the object into the focal plane of the objective lens.

● Fine-focus knob: makes fine adjustments to focus the image.

Support and alignment

● Arm: a curved portion that holds all of the optical parts at a fixed distance and aligns them.

● Base: supports the weight of all of the microscope parts.

● Tube: connects to the arm of the microscope by way of a rack and pinion gear, which allows for focusing the image when changing lenses or observers and moving the lenses away from the stage when changing specimens.

Some of the parts mentioned previously vary among microscopes. Microscopes come in two basic configurations: upright and inverted. The microscope shown in the Figure 1 is an upright microscope, which has the illumination system below the stage and the lens system above the stage. An inverted microscope has the illumination system above the stage and the lens system below the stage. Inverted microscopes are better for looking through thick specimens, such as dishes of cultured cells, because the lenses can get closer to the bottom of the dish where the cells grow.

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Experiment Observing Bacteria and Blood

Figure 1: Upright microscope.

Light microscopes can reveal the structures of living cells and tissues as well as of non-living samples such as rocks and semiconductors. Microscopes can be simple or complex in design, and some can do more than one type of microscopy, each of which reveals slightly different information. The light microscope has greatly advanced our biomedical knowledge and continues to be a powerful tool for scientists.

Microscope Terms

● Depth of field: The vertical distance from above to below the focal plane that yields an acceptable image.

● Field of view: The area of the specimen that can be seen through the microscope with a given objective lens.

● Focal length: The distance required for a lens to bring the light to a focus, (usually measured in millimeters).

● Focal point/focus: The point at which the light from a lens comes together.

● Magnification: The product of the magnifying powers of the objective and eyepiece lenses. For example, a 15x eyepiece and a 40x objective lens will give you 600 power magnification (15x x 40x = 600x).

● Numerical aperture: The measure of the lens’ light-collecting ability.

● Resolution: The closest two objects can be before they are no longer detected as separate objects (usually measured in nanometers).

● Image Quality: The quality of the microscope image is assessed as follows:

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Experiment Observing Bacteria and Blood

● Brightness: How light or dark is the image? Brightness is related to the illumination system. The brightness can be changed by changing the wattage of the lamp and by adjusting the condenser diaphragm aperture. Brightness is also related to the numerical aperture of the objective lens; the larger the numerical aperture, the brighter the image.

Figure 2: Pollen grain under proper brightness (left) and poor brightness (right).

● Focus: Is the image blurry or well-defined? Focus is related to focal length and can be controlled with the focus knobs. The thickness of the cover glass on the specimen slide can also affect the ability to focus the image if it is too thick for the objective lens. The correct thickness is usually written on the side of the objective lens.

Figure 3: Pollen grain in focus (left) and out of focus (right).

● Resolution: How close can two points in the image be before they are no longer seen as two separate points? Resolution is related to the numerical aperture of the objective lens – the higher the numerical aperture, the better the resolution; and the wavelength of light passing through the lens – the shorter the wavelength, the better the resolution.

Figure 4: Pollen grain with proper resolution (left) and poor resolution (right).

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Experiment Observing Bacteria and Blood

● contrast: What is the difference in lighting between adjacent areas of the specimen? Contrast is related to the illumination system and can be adjusted by changing the intensity of the light and the diaphragm/pinhole aperture. Chemical stains applied to the specimen can also enhance contrast.

Figure 5: Pollen grain with proper contrast (left) and poor contrast (right).

When specimens are observed by transmitted light, light must pass through the specimen in order to form an image. The thicker the specimen, the less light that passes through, which creates a darker image. Therefore, the specimens must be thin (0.1 to 0.5 mm). Many organic specimens must be cut into thin sections before observation. Specimens of rock or semiconductors are too thick to be sectioned and observed by transmitted light, so they are observed by the light reflected from their surfaces.

Figure 6: Glial cell cultured from a rat brain.

Types of Microscopy

A major problem in observing specimens under a microscope is that their images do not have much contrast. This is especially true of living things, although natural pigments, such as the green in leaves, can provide good contrast. One way to improve contrast is to treat the specimen with colored pigments or dyes that bind to specific structures within the specimen.

Different types of microscopy have been developed to improve the contrast in specimens. The specializations are mainly in the illumination systems and the types of light passed through the specimen. Brightfield is the basic microscope configuration, and the images to this point are from brightfield microscopes. This technique provides very little contrast, and much of the contrast is

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Experiment Observing Bacteria and Blood

provided by staining the specimens. A darkfield microscope uses a special condenser to block out most of the bright light and illuminate the specimen with oblique light, much like the moon blocks the light from the sun in a solar eclipse. This optical setup provides a totally dark background and enhances the contrast of the image to bring out fine details of bright areas at boundaries within the specimen.

Following are various types of light microscopy techniques. These techniques achieve different results by using different optical components. The basic idea involves splitting the light beam into two pathways that illuminate the specimen. Light waves that pass through dense structures within the specimen slow down compared to those that pass through less dense structures. As all of the light waves are collected and transmitted to the eyepiece, they are recombined, so they interfere with each other. The interference patterns provide contrast. They may show dark areas (more dense) on a light background (less dense), or create a type of false three-dimensional (3-D) image.

● Phase-contrast: A phase-contrast microscope is best for looking at living specimens, such as cultured cells. The annular rings in the objective lens and the condenser separate the light paths. Light passing through the central part of the light path is then recombined with light traveling around the periphery of the specimen. Interference produced by these two paths produces images in which dense structures appear darker than the background.

Figure 7: Phase contrast.

● Differential Interference Contrast (DIC): DIC uses polarizing filters and prisms to separate and recombine the light paths, giving a 3-D appearance to the specimen. DIC is also called Nomarski after its inventor.

● Hoffman Modulation Contrast: Hoffman modulation contrast is similar to DIC; however, it uses plates with small slits in both the axis and the off-axis of the light path to produce two sets of light waves passing through the specimen. Again, a 3-D image is formed.

● Polarization: The polarized-light microscope uses two polarizers, one on either side of the specimen, positioned perpendicular to each other so that only light that passes through the specimen reaches the eyepiece. Light is polarized in one plane as it passes through the first filter and reaches the specimen. Regularly spaced, patterned, or crystalline portions of the

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specimen rotate the light that passes through. Some of this rotated light passes through the second polarizing filter, so these regularly spaced areas show up bright against a black background.

● Fluorescence: This type of microscope uses high-energy, short-wavelength light (usually ultraviolet) to excite electrons within certain molecules inside a specimen, causing those electrons to shift to higher orbits. When they fall back to their original energy levels, they emit lower-energy, longer-wavelength light (usually in the visible spectrum), which forms the image.

Care and Handling of the Microscope

● When moving a microscope, always use two hands. Place one hand around the arm, lift the scope, and put your other hand under the base of the scope for support. Learning to carry the scope in this way will force you to carry it carefully and ensure you do not knock it against anything while moving it.

● When putting the scope down, do so gently. If you bang your scope down on the table, eventually lenses and other parts will jar loose. The microscope seems like a simple instrument, but each eyepiece and objective is made up of a number of lenses put together in a specific way to create wonderful magnification. If you bang the scope around, you are shaking upward of 15 to 20 lenses.

● When handling the scope, always have clean hands. It would be a shame to damage the scope with too much peanut butter!

Storing the Microscope

● The best place to store the scope is on a sturdy desk, table, or shelf where the scope will not be disturbed. Make sure to keep the scope protected with a plastic or vinyl cover when it is not in use. Dust is an enemy to the lenses, so always cover the scope.

● If you are unable to find a safe place where you can leave the scope out, store it in its original fitted, foam case packaging.

cleaning the Microscope

● The first step in keeping the microscope clean is to keep it from getting dirty. Always keep the microscope covered with the dust cover when it is not in use.

● The eyepiece will need cleaning from time to time. Due to its position on the scope, it will have a tendency to collect dust and oil from your eyelashes. The eyepiece lens should be cleaned with a high quality lens paper, available from a camera shop or an eyeglass center. Brush any visible dust from the lens and then wipe the lens. Apply a bit of lens solution to the lens paper to aid in cleaning. Use a cotton swab in place of lens paper, but do not use facial tissue to clean the lenses.

● Occasionally, the objective lenses will need cleaning. Use a fresh area of lens paper for each lens to avoid transferring dust from one lens to another.

● Clean the lenses in the glass condenser under the stage.

● Clean the glass lens over the light or the mirror, so an optimal amount of light can shine through. Follow up by wiping down the whole scope with a soft, clean, cotton towel.

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Using the Microscope

● Take the microscope body from the case. Put the eyepiece in the opening in the tube at the top of the microscope. Remove the objective lenses from their individual containers and screw them into the revolving nosepiece, placing each lens in its respective color coded position.

● Adjust the tension on the focusing control knobs to suit your touch or to compensate for normal wear over time. To increase tension, hold the right-hand knob firmly and turn the opposite knob clockwise; turning the knob counterclockwise loosens the tension.

● Unplug the rotating mirror bracket from the base of the microscope, insert the mirror (packaged separately with the microscope) into the bracket so that it swivels freely, and plug the bracket back into the base of the microscope.

● Tilt the arm of the microscope back until it is at a position where you can comfortably look into the microscope eyepiece.

● Place a slide under the clips on the stage with the area you wish to view positioned between the lens selected and the hole in the stage.

● Turn the nosepiece to select the longest lens (usually the highest power lens). Lower the barrel of the microscope with the coarse-focus knob until it almost touches the slide. If the barrel will not go that far, unscrew the focus stop-screw under the arm of the microscope until the lens can almost touch the slide. When the lens is in position, lightly tighten the screw and lock it in place with the knurled nut.

● Place a light source in front of the microscope; use the small lever on the sub-stage condenser to fully open the diaphragm; and adjust the mirror so the light is brightest when seen through the microscope.

● Rotate the nosepiece to select the lowest power lens. Lower the barrel with the coarse-focus knob until the tip of the lens is near the slide. Now raise the barrel slowly with the coarse- focus knob until you see an image from the slide. Finish the focus with the fine-focus knob.

● With your thumb and forefinger on each end of the slide, move it slowly on the stage until the object you wish to study is centered in your field of view.

● Rotate the nosepiece of the microscope to select the objective lens that will give you the higher magnification you need.

● Once one lens is focused properly, any other objective lens on the nosepiece when rotated into position will be roughly in focus and require only fine focus to bring the image into correct focus.

● Move the lever for the diaphragm through its full range to select the amount of light that gives you the best contrast. Many details will be visible with good contrast which would otherwise be lost with too much or too little light.

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Using the Electric illuminator

With your fingers, grasp the illuminating mirror behind its bracket and pull to unplug the bracket and mirror from the base of the microscope. Insert the metal plug tip of the electric illuminator into the hole from which you unplugged the mirror bracket. Rotate the fixture so that the glass opening over the bulb points up toward the light condenser under the stage. Plug the electric cord into a 115-volt outlet and turn on the switch in the cord.

Using the Oil Immersion Lens (purchased separately)

Install the oil immersion 100x objective lens in place of any of the other objective lenses. The 4x lens is a good choice. First, focus the microscope and center the slide using a lower magnification objective. Apply a drop of oil on the specimen slide and turn the revolving nosepiece to bring the 100x objective into position. If the barrel is too low to allow the 100x lens to move into position, raise it very slightly with the coarse focus, position the lens, and then lower the barrel until the tip of the 100x lens touches the oil. The tip of the lens is able to move a short distance into the lens against a spring in order to keep from putting too much pressure on the slide. With the lens tip touching the oil, focus with the fine-focus knob. The working distance of the lens is very short, so do not use the coarse-focus knob other than to position the lens. After using the oil immersion lens, wipe off the oil carefully with alcohol.

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Exercise 1: Viewing Prepared Slides procedure Part I: Viewing Prepared Slides

1. Set up your microscope. Refer to the Discussion and Review section for more information.

2. Clean the ocular lenses and objectives with lens paper prior to use.

3. Place the prepared e focusing slide, cover slip up, on the stage within the spring loaded lever.

4. Turn the rotating nosepiece until the 10x objective is above the ring of light coming through the slide.

5. Move the slide using the X and Y stage travel knobs until the specimen is within the field of view.

6. Adjust the focus by looking into the eyepiece and focusing the specimen with the coarse then fine focus knobs.

7. Bring the condenser up to the bottom of the slide and then slightly back for maximum light.

8. Adjust the iris diaphragm until there is sufficient light passing through the specimen. This will take practice. Begin with the diaphragm closed and slowly open it while observing the specimen. Choose the level at which there is enough light to allow good resolution, but not so much light that there is a glare or whitening of the field of view.

9. Repeat the previous steps with six different prepared slides with 10x and 40x objectives. Refer to Figure 8 for image comparisons.

Figure 8: Comparisons of slides with 10x and 40x objective lenses.

Fungi – 10x lens Fungi – 40x lens

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Yeast – 10x lens Yeast – 40x lens

Paramecium – 10x lens Paramecium – 40x lens

Part ii: Using an immersion oil lens

The most important objective used in microbiology is the oil immersion lens, 100x. Many bacteria cannot be visualized clearly without the use of oil immersion. When using an oil immersion lens, oil is placed between the objective and the slide to prevent the loss of light due to the bending of light rays as they pass through air. This enhances the resolving power of the microscope.

1. After focusing with a high, dry objective, turn the 40x objective away from the specimen.

2. Place a drop of oil on the slide.

3. Rotate the oil immersion objective, 100x, into the oil, then past the oil and back. This ensures there are no air bubbles between the objective and the oil.

4. Use only the fine focus to bring the object into focus.

5. Practice viewing at least six prepared slides at 10x, 40x, and 100x with oil. Refer to Figure 9 for image comparisons.

a. When replacing slides on the stage, start with the 10x or the 40x before going to oil. Do not let oil get on the 10x and 40x objectives.

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Experiment Observing Bacteria and Blood

b. Always rotate the oil immersion objective away before removing a slide.

c. Never use the coarse focus with the oil objective in place. The slide could break and the objective could get damaged.

6. Clean the oil off the oil objective with lens paper. Then clean all the objectives with clean lens paper.

Figure 9: Comparisons of slides with the 10x, 40x, and 100x (oil immersion) lenses

Yeast – 10x lens Yeast – 40x lens Yeast – 100x oil immersion lens

Fungi – 10x lens Fungi – 40x lens Fungi – 100x oil immersion lens

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Questions A. Identify the following parts of the microscope and describe the function of each.

B. Define the following microscopy terms:

● Focus:

● Resolution:

● Contrast:

C. What is the purpose of immersion oil? Why does it work?

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Experiment Observing Bacteria and Blood

Exercise 2: observing Bacteria cultures in yogurt Bacteria occur in a variety of different shapes. By far, the most numerous are spheres, rods, commas, and spirals. Spherical bacteria, called cocci, and rod shaped bacteria, called bacillus, are the most common shapes.

Figure 10: Bacteria shapes.

In addition to shape, the way individual bacteria are arranged is an identifying feature. For example, bacteria can occur in pairs (diplo), strands (strepto), or clusters (staphylo). A common inhabitant of yogurt is a paired, round bacteria – diplococcus.

Figure 11: Bacteria arrangements.

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Experiment Observing Bacteria and Blood

procedure 1. Locate a small, sealable container made of glass or plastic. Clean the container thoroughly

with soap and then rinse the container several times to remove all the soap.

2. Place a teaspoon of yogurt in the container.

3. Cover the container and place it in a dark, relatively warm area. Leave the container undisturbed for 12–24 hours.

4. Use a toothpick to take a sample of yogurt from the container and place the sample on a clean slide. If the sample on the slide seems too thick, dilute it with a drop of water.

5. Place a cover slip on top of the sample.

6. Observe the bacteria under the microscope at 10x, 40x, and 100x oil immersion. The diaphragm setting should be very low, because the fresh bacteria will appear nearly transparent.

7. Next, view the prepared stained yogurt slide from the kit. Compare your observations of the fresh, live slide to the prepared, stained slide.

8. Clean the collection vials and slides thoroughly after use.

Questions

A. Describe your observations of the fresh yogurt slide.

B. Were there observable differences between your fresh yogurt slide and the prepared yogurt slide? If so, explain.

C. Describe the four main bacterial shapes.

D. What are the common arrangements of bacteria?

E. Were you able to identify specific bacterial morphologies on either yogurt slide? If so, which types?

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Experiment Observing Bacteria and Blood

Exercise 3: Preparing and observing a Blood Slide procedure Part i: Preparing a Blood Slide

WArNiNg: Blood can carry diseases that can be transferred from person to person. Avoid contact with another person’s blood. When necessary to contact blood, wear rubber gloves.

1. Thoroughly wash your hands with soap and warm water.

2. Clean a finger tip with the alcohol prep pad and allow to dry.

3. Quickly and lightly poke the inside of your sterilized finger with the lancet.

4. Squeeze your finger to place a drop of blood on a clean slide in accordance with the following directions.

a. Drop the blood toward one end of a slide as shown in Figure 12.

b. Tilt the cover slip toward the drop. Then slowly move the slip toward the drop until it contacts the blood and grabs the drop.

c. Without changing the tilt of the cover slip, move the slip back over the slide, drawing the blood across the slide.

d. Lay the cover slip flat across the blood smear.

Figure 12: Blood smear preparation.

5. Place a bandage on your finger to prevent infection.

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Experiment Observing Bacteria and Blood

Part ii: observing Blood

Human blood appears to be a red liquid to the naked eye, but under a microscope it contains four distinct elements:

● plasma

● red blood cells

● white blood cells

● platelets

The plasma is the liquid part of blood and is actually straw-yellow in color. The red blood cells give blood its red color. White blood cells are interspersed in the sea of red blood cells and help fight infection. The platelets are fragments of red blood cells and function in clotting. While red blood cells should be visible on the slide, white blood cells and platelets may be harder to find.

1. Place the blood slide on the microscope stage and bring it into focus on low power. Adjust the lighting and then switch to a 40x magnification. To view individual cells, use 100x oil immersion.

You should see hundreds of tiny red blood cells. There are billions circulating throughout your blood stream. Red blood cells contain no nucleus, which means they can’t divide. Red blood cells are constantly produced by the bone marrow and the spleen.

You should also be able to find a few white blood cells. They are slightly larger than red blood cells and have a nucleus. Some, macrophages, often resemble an amoeba and can contort their body in any way they like to engulf foreign objects. Others are spherical. White blood cells fight infection by consuming foreign bodies or injecting them with enzymes that induce cell death or apoptosis. Platelets are fragments of red blood cells and are very small.

Figure 13: Blood smear slides at 10x, 40x, and 100x (oil immersion) lenses.

10x lens 40x lens 100x lens

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Experiment Observing Bacteria and Blood

Questions A. Describe the cells you were able to see in the blood smear.

B. Are the cells you observed in your blood smear different than the bacterial cells you have observed? Why or why not?

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Experiment Observing Bacteria and Blood

observing Bacteria and Blood Cynthia Alonzo, M.S. Version 42-0249-00-01

lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

Exercise 1: Viewing Prepared Slides Questions A. Identify the following parts of the microscope and describe the function of each.

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Experiment Observing Bacteria and Blood

Define the following microscopy terms:

● Focus: Is the image blurry or well-defined?

● Resolution:

● Contrast:

B. What is the purpose of immersion oil? Why does it work?

Exercise 2: observing Bacteria cultures in yogurt Questions A. Describe your observations of the fresh yogurt slide.

B. Were there observable differences between your fresh yogurt slide and the prepared yogurt slide? If so, explain.

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Experiment Observing Bacteria and Blood

C. Describe the four main bacterial shapes.

Cocci –

Bacillus –

Spirillum –

Vibrio –

D. What are the common arrangements of bacteria?

Diplo –

Strepto –

Staphylo –

E. Were you able to identify specific bacterial morphologies on either yogurt slide? If so, which types?

Exercise 3: Preparing and observing a Blood Slide Questions A. Describe the cells you were able to see in the blood smear.

B. Are the cells you observed in your blood smear different than the bacterial cells you have observed? Why or why not?

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Experiment Observing Bacteria and Blood

Bacterial Morphology Cynthia Alonzo, M.S. Version 42-0240-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will observe various bacterial morphologies using prepared slides. They will prepare live culture smears of Saccharomyces cerevisiae and cheek cells, and view these specimens under a microscope using direct and indirect staining techniques. Students will also learn how to prepare disinfectants and use them to decontaminate working surfaces.

ExpErimEnt

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objectives ● Observe bacterial morphologies by preparing wet-mount slides

● Learn and employ direct and indirect staining techniques

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Experiment Bacterial Morphology

materials

MATEriAlS QTY iTEM DEScriPTioN Student provides 1 10%-bleach solution

1 Microscope 1 Immersion Oil 3 Toothpicks 1 Warm water 1 Paper towels 1 Clothespin, tweezers, or test tube holder

LabPaq provides 1 Gloves, disposable (1 pair) 1 Goggles, safety 1 Apron, plastic 1 Slide – Cover Glass – Cover Slip Cube (3) 1 Lens-paper-pack-50-sheets 1 Cup, Plastic, 9 oz Tall 1 Pencil, marking

1 Tray-Staining tray 2 Candle, tea size (flame source) 1 Congo Red Stain, 0.1% – 1 mL in Pipet 2 Baker’s Yeast Packet – Saccharomyces cerevisiae 2 Pipet, Long Thin Stem 1 Gram Stain Solution #-1, Crystal Violet – 15mL in Dropper Bottle 2 Sterile Swabs – 2 per Pack 1 Slide – Bacteria Bacillus form 1 Slide – Bacteria Coccus form 1 Slide – Bacteria spirillum 1 Slide-Box-MBK with Blank Slides 1 Mask, Face with Earloops

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment Bacterial Morphology

Discussion and review The size, shape, and arrangement of bacteria and other microbes are the result of their genes, a defining characteristic called morphology. Bacteria come in a variety of sizes and shapes and new ones are discovered all the time. Nature loves variety in its life forms. The most common bacterial shapes are rods, cocci, and spiral. However, within each of these groups are hundreds of unique variations. Rods may be long, short, thick, thin, have rounded or pointed ends, or be thicker at one end than the other. Cocci may be large, small, or oval-shaped to various degrees. Spiral-shaped bacteria may be fat, thin, loosely spiraled or tightly spiraled.

The group associations of microbes, both in liquid and on solid medium, are also defining. Bacteria may exist as single cells or in a common grouping such as chains, uneven clusters, pairs, tetrads, octads, or other packets. Bacteria may exist as masses embedded within a capsule. A description of the physical qualities – form and structure – of bacteria constitutes its individual morphology and is an identifying quality of the specific bacteria. There are square bacteria, star-shaped bacteria, stalked bacteria, budding bacteria that grow in net-like arrangements, and many other morphologies. When observing bacteria, describe as many of these characteristics as possible.

In this experiment, bacterial morphology will be examined by:

● Observing living, unstained organisms

● Observing killed, stained organisms

Because bacteria are almost colorless and show little contrast with the broth in which they are suspended, they are difficult to observe when unstained. Staining microorganisms allows you to:

● See greater contrast between the organism and the background

● Differentiate various morphological types by shape, arrangement, gram reaction, etc.

● Observe certain structures such as flagella, capsules, endospores, etc.

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Experiment Bacterial Morphology

Exercise 1: Viewing Prepared Slides of Common Bacterial Shapes procedure 1. Set up the microscope.

2. View the prepared slides of bacterial morphology. Record your observations.

3. Use each morphological type as a comparative tool for the remainder of the exercise.

Spiral bacteria – 100x magnification Bacillus – 100x magnification bacillus

Spiral bacteria – 400x magnification Bacillus – 400x magnification

Spiral bacteria – 1000x oil immersion Bacillus – 1000x oil immersion bacillus Figure 1: Morphological Examples

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Experiment Bacterial Morphology

Exercise 2: Wet-Mount Preparations procedure Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic. Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

Pre-Experiment Preparation: Prepare an S. cerevisiae culture in accordance with the Preparation of Cultures section in the Appendix.

1. Disinfect your work area with a 10%-bleach solution using the Procedures in the Preparation of Disinfecting Solution section in the Appendix.

2. Use the marker pencil to make a dime-sized circle on each of the three slides.

3. Use a clean pipet to add a drop of warm water to the circle on the first slide.

4. Open the sterile cotton swab. Vigorously scrape the inside of your mouth and gums.

5. Smear the swab inside the circle on the first slide, transferring as much material to the drop of water as possible. Cover the drop with a cover slip.

6. View the slide under the microscope. Record the observations.

7. Use the pipet to add a drop of water to the circle on the second slide.

8. Use the toothpick to scrape a sample of plaque from your teeth.

9. Transfer the plaque from the toothpick to the drop of water, mixing well to dissolve any clumps. Cover the drop with a cover slip.

10. View the slide under the microscope. Record the observations.

11. Use the pipet to add a drop of the S. cerevisiae mixture to the circle of the third slide. Cover the drop with a cover slip.

12. View the slide with your microscope. Record the observations.

13. Set the cup of yeast mixture and its pipet aside for later use. Wash the slides for use in the next exercise. Make sure to remove all markings and specimen residue. Discard the used cover slips.

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Yeast – 100x wet-mount Cheek smear – 40x wet-mount Cheek smear – 100x wet-mount Figure 2: Wet-Mount Samples

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Experiment Bacterial Morphology

Exercise 3: Direct Staining In order to understand how staining works, it will be helpful to know a little about the physical and chemical nature of stains. Stains are generally salts in which one of the ions is colored. A salt is a compound composed of a positively charged ion and a negatively charged ion. For example, the dye methylene blue, is actually the salt methylene blue chloride. Methylene blue chloride dissociates in water into a positively charged methylene blue ion which is blue in color and a negatively charged chloride ion which is colorless.

Dyes or stains may be divided into two groups: basic and acidic. If the chromophore or colored portion of the dye resides in the positive ion, it is called a basic dye – methylene blue, crystal violet, and safranin. If the chromophore is in the negatively charged ion, it is called an acidic dye – India ink, nigrosin, and Congo red.

Because of their chemical nature, the cytoplasm of all bacterial cells have a slight negative charge when growing in a medium of near neutral pH. Therefore, when using a basic dye, the positively charged chromophore of the stain combines with the negatively charged bacterial cytoplasm – opposite charges attract – and the organism becomes directly stained. An acidic dye, due to its chemical nature, reacts differently. Because the chromophore of the dye is on the negative ion, it will not readily combine with the negatively charged bacterial cytoplasm – like charges repel. Instead, it forms a deposit around the organism, leaving the organism itself colorless. Since the organism is seen indirectly, this type of staining is called indirect or negative and is used to get a more accurate view of bacterial sizes, shapes, and arrangements.

Before direct staining bacteria, the organism must be fixed to the glass slide. If the preparation is not fixed, the organisms will be washed off the slide during staining. Simple methods to fix a slide include air-drying and heat-fixing. The organisms are heat-fixed by passing an air-dried smear of the organism through flame. The heat coagulates the organism’s proteins causing the bacteria to stick to the slide.

Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic. Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

procedure Note: Because most stains are strong and can damage clothing and furniture, wear gloves and an apron to protect skin and clothes. Use a staining tray for this work.

1. Use the marker pencil to make a dime-sized circle in the middle of each of the three slides. Label the slides #1, #2, and #3.

2. Use the pipet to place a small drop of the prepared yeast culture onto slide #1. Spread the sample into a thin layer. Set the slide aside to dry.

3. Use a sterile cotton swab to vigorously scrape the inside of your mouth and gums.

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Experiment Bacterial Morphology

4. Smear the swab onto the center of slide #2 in a thin layer. Set the slide aside to dry.

5. Use a toothpick to scrape a sample of plaque from your teeth. Smear the sample from the toothpick in the center of slide #3 and set it aside to dry.

Note: If necessary, create a thin, smooth layer by adding a small drop of water to the slide.

6. When the slides are completely dry, heat-fix each slide with a flame source.

a. Grasp the slide, sample side up, with a clothespin or test tube holder.

b. Leisurely pass the slide over the flame 3–4 times.

Caution: Too much heat may distort the organism. Keep the slide out of the direct flame but close to the heat. The slide should feel very warm but not too hot to hold.

7. Place the first slide in the staining tray. Add a drop or two of Gram Stain Solution #1, crystal violet, to the slide to cover the sample.

8. Allow the crystal violet to sit on the slide for 30 seconds. Then gently rinse the slide with water.

9. Blot the slide dry with a paper towel. Do not wipe the slide.

10. Repeat steps 7-9 for the remaining slides.

11. Use the microscope to view the stained specimens. Record observations for each sample.

12. Set the cup of yeast mixture and its pipet aside for later use. Wash the slides for use in the next exercise. Make sure to remove all markings and specimen residue.

Cheek Smear – 100x direct stain Yeast – 100x direct stain

Plaque smear – 100x direct stain Cheek smear – 100x direct stain Figure 3: Direct Stain Examples

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Experiment Bacterial Morphology

Exercise 4: indirect Staining In negative staining, the negatively charged color portion of the acidic dye is repelled by the negatively charged bacterial cell. Therefore the background will be stained and the cell will remain colorless. Indirect, or negative staining, does not require heat-fixing; thus, it is less likely to create abnormal cellular images or staining artifacts. Congo red is a common negative stain used in this exercise.

1. Use the marker pencil to label three slides #1, #2, and #3.

2. Place a small drop of Congo red on the side of slide #1.

3. Use a pipet to add a drop of yeast culture to the drop of Congo red.

4. Place a clean cover slip over the preparation. Press the slip down and blot gently with a paper towel to get a thin, even film under the cover slip. Set the slide aside.

5. Place a small drop of Congo red on slide #2.

6. Use a toothpick to scrape a sample of plaque from your teeth.

7. Transfer the sample from the toothpick into the drop of Congo red. Mix the sample into the Congo red until it is completely disbursed.

8. Place a clean cover slip over the preparation, press down, and blot gently.

9. Place a drop of Congo red on the side of slide # 3.

10. Use the sterile cotton swab to vigorously scrape the inside of your mouth and gums.

11. Roll the swab through the Congo red, transferring as much of the sample as possible.

12. Place a clean cover slip over the preparation, press down, and blot gently.

13. Use the microscope to examine the stained specimens. Record the results.

14. Mix 1 tablespoon of bleach into the yeast culture and let it stand for at least 30 minutes to ensure all organisms have been destroyed. Then discard the contents.

15. Clean your slides and disinfect your area thoroughly with a 10%-bleach solution.

Cheek smear – 40x indirect stain Cheek smear – 100x indirect stain

Yeast – 100x indirect stain Plaque smear – 100x indirect stain Figure 4: Indirect Stain Examples

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Experiment Bacterial Morphology

Questions A. List three reasons why you might choose to stain a particular slide rather than view it as a

wet-mount.

B. Define the following terms:

● chromophore:

● acidic dye:

● basic dye:

C. What is the difference between direct and indirect staining?

D. What is heat fixing?

E. Why is it necessary to ensure that your specimens are completely air dried prior to heat fixing?

F. Describe what you observed in your plaque smear wet-mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences?

G. Describe what you observed in your cheek smear wet-mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences?

H. Describe what you observed in your yeast wet-mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences?

I. Were the cell types the same in all three specimen sets: yeast, plaque, and cheek? How were they similar? How were they different?

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Experiment Bacterial Morphology

Chromophore:

Acidic Dye:

Basic Dye:

Bacterial Morphology Cynthia Alonzo, M.S. Version 42-0240-00-01

lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

Questions A. List three reasons why you might choose to stain a particular slide rather than view it as a wet-mount.

B. Define the following terms:

C. What is the difference between direct and indirect staining?

D. What is heat fixing?

E. Why is it necessary to ensure that your specimens are completely air dried prior to heat fixing?

F. Describe what you observed in your plaque smear wet-mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences?

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Experiment Bacterial Morphology

G. Describe what you observed in your cheek smear wet-mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences?

H. Describe what you observed in your yeast wet-mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences?

I. Were the cell types the same in all three specimen sets: yeast, plaque, and cheek? How were they similar? How were they different?

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Experiment Bacterial Morphology

Aseptic Technique & culturing Microbes Cynthia Alonzo, M.S. Version 42-0239-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will use aseptic techniques to transfer cultures, including Lactobacillus acidophilus and Staphylococcus epidermidis. They will learn about culture media and how to distinguish various types of microbial growth. Students will also learn about variable conditions that are required for microbial growth, including oxygen levels and temperature.

ExpErimEnt

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objectives ● Learn and employ aseptic technique

● Become familiar with basic requirements of microbial growth

● Learn the basic forms of culture media

● Become familiar with methods used to control microbial growth

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Experiment Aseptic Technique and Culturing Microbes

materials

MATEriAlS QTY iTEM DEScriPTioN Student provides 1 Small cardboard box or Styrofoam cooler

1 Microscope 1 Immersion Oil 1 Desk lamp or heating pad 1 Aluminum foil 1 10%-bleach solution 1 Paper towels 1 S. epidermidis sample

LabPaq provides 2 Gloves, Disposable (1 pair) 1 Goggles, safety 1 Apron, plastic 1 Thermometer-in-cardboard-tube 2 Candles (flame source)

1 Test-tube-rack-6×21-mm 1 Slide-Box-MBK with Blank-Slides 1 Slide, Cover Slip (20 pcs)

1 Broth, MRS – 9 mL in Glass Tube 1 Broth, Nutrient – 5 mL in Glass Tube

1 Lactobacillus acidophilus – capsule in Bag 2″x 3″ 1 Gram Stain Solution #1, Crystal Violet – 15 mL in Dropper Bottle 1 Swab, Sterile (pkg of 2) 1 Loop, inoculation, plastic 1 Mask, Face with Earloops

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment Aseptic Technique and Culturing Microbes

Discussion and review Controlling microbial growth is necessary in many practical situations. Significant advances in agriculture, medicine, and food science have been made through the study of this area of microbiology.

Control of growth refers to the prevention of growth of microorganisms. This control is affected in two basic ways: by killing microorganisms or by inhibiting the growth of microorganisms. Control of growth usually involves the use of physical or chemical agents which either kill or prevent the growth of microorganisms. Agents that kill cells are called cidal agents; agents that inhibit the growth of cells without killing them are called static agents. Thus the term bactericidal refers to killing bacteria, and bacteriostatic refers to inhibiting the growth of bacterial cells. A bactericide kills bacteria; a fungicide kills fungi, and so on.

Sterilization is the complete destruction or elimination of all viable organisms in or on an object. There are no degrees of sterilization; an object is either sterile or it is not. Sterilization Procedures involve the physical removal of cells or the use of heat, radiation, or chemicals.

Methods of Killing Microbes

Heat is the most important and widely used method of killing microbes. For sterilization always consider the type of heat, the time of application, and the temperature to ensure the destruction of all microorganisms. Endospores of bacteria are considered the most thermoduric of all cells, so their destruction guarantees sterility.

● Incineration burns organisms and physically destroys them. This method is used for needles, inoculating wires, glassware, etc.

● Boiling at 100oC for 30 minutes kills almost all endospores. Very long or intermittent boiling is required to kill endospores and sterilize a solution.

Note: For the purpose of purifying drinking water, boiling at 100oC for five minutes is probably adequate. However, there have been some reports that Giardia cysts can survive this process.

● Autoclaving (steam under pressure or pressure cooker) at 121oC for 15 minutes (15lbs/in2 pressure) is good for sterilizing almost anything; however, autoclaving will denature or destroy heat-labile substances.

● Dry heat (hot air oven) at 160oC for 2 hours or 170oC for 1 hour is used for glassware, metal, and objects that will not melt.

You can refer to Table 1: Recommended Use of Heat to Control Bacterial Growth in the Lab Report Assitant to find further information on using heat with bacteria.

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Table 1: Recommended Use of Heat to Control Bacterial Growth Treatment Temperature Effectiveness

Incineration >500oC Incineration vaporizes any organic material on nonflammable surfaces but may destroy many substances in the process.

Boiling 100oC 30 minutes of boiling kills microbial pathogens and vegetative forms of bacteria but may not kill bacterial endospores.

Intermittent boiling 100oC Three 30-minute intervals of boiling followed by periods of cooling kills bacterial endospores.

Autoclave and pressure cooker (steam under pressure)

121oC/15 minutes at 15# pressure

Autoclaving kills all forms of life including bacterial endospores. The item being sterilized must be maintained at the effective temperature for the full time.

Dry heat (hot air oven) 160oC/2 hours

Dry heat is used for materials that must remain dry and which are not destroyed at temperatures between 121oC and 170oC. The method is good for glassware and metal, but not plastic or rubber items.

Dry heat (hot air oven) 170oC/1 hour The effects are the same as above. Note that increasing the temperature by 10o shortens the sterilizing time by 50%.

Pasteurization (batch method) 63

oC/30 minutes Pasteurization kills most vegetative bacterial cells including pathogens such as streptococci, staphylococci, and Mycobacterium tuberculosis.

Pasteurization (flash method) 72

oC/15 seconds

The effect on bacterial cells is similar to the batch method. For milk, this method is more conducive to the industry and has fewer undesirable effects on quality or taste.

Irradiation usually destroys or distorts nucleic acids. Ultraviolet light is generally used to sterilize the surfaces of objects, although x-rays and microwaves can be useful.

Filtration involves the physical exclusion and removal of all cells in a liquid or gas, and is especially important to sterilize solutions which would be denatured by heat (antibiotics, injectable drugs, vitamins, etc.).

Toxic chemicals and gas such as formaldehyde, glutaraldehyde, and ethylene oxide can kill all forms of life in a specialized gas chamber.

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Experiment Aseptic Technique and Culturing Microbes

In natural environments, microorganisms usually exist as mixed populations. However, if we are to study, characterize, and identify microorganisms, we must have the organisms in the form of a pure culture. A pure culture is one in which all the organisms are descendants of the same organism. Techniques for obtaining pure cultures from a mixed population will be described in the Isolation of Individual Colonies experiment.

To culture microorganisms we must have a sterile, nutrient-containing medium in which to grow the organisms. Anything in or on which we grow microorganisms is termed a medium. A medium is usually sterilized by heating it to a temperature at which all contaminating microorganisms are destroyed.

Finally, in working with microorganisms, we must have a method of transferring growing organisms, called the inoculum, to a sterile medium without introducing any unwanted, outside contaminants. This method of preventing unwanted microorganisms from gaining access is termed aseptic technique.

The first step of aseptic technique is awareness – awareness that microbes are found on virtually every surface and in the air itself. Take care to minimize the culture’s exposure to environmental microbes. This is not as difficult as it may seem. Using gloves prevents contamination of the culture with the bacteria on our skin. Using a mask when handling cultures prevents contamination of the culture from microbes contained in our breath and minimizes the air currents our breath causes towards the culture tube. Think of how your breath affects a nearby candle. Take care not to touch caps or tube tops to counter tops or other surfaces. Use both disinfectant and a flame source to remove potential contaminants and to prevent further possible contamination.

Aseptically Inoculating a Broth Medium

1. Put on gloves and a mask and disinfect the work area.

2. Place the sample source and the target source (tube of sterile medium) in front of you. A primary goal of aseptic transfer is to avoid the possibility of contamination, so it is important to minimize the time the samples are exposed.

3. Pick up the instrument you are using to inoculate your new culture, such as a sterile swab, in one hand, taking care not to touch the microbe containing area.

4. Pick up the target medium tube in the other hand, and remove the cap with the hand holding the inoculation instrument. Do not to touch the inner surface of the cap. Keep the cap in your hand; do not set the cap down on the counter.

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Experiment Aseptic Technique and Culturing Microbes

Figure 1: Uncapping Medium Tube with Inoculation Instrument

5. Light a candle. Then run the top of the tube through the tip of the flame (flame the lip). The flame will sterilize the lip of the tube, and the heat will create an updraft which takes air contaminants away from the tube entrance.

Figure 2: Top of the Tube in the Flame

6. Quickly transfer the bacterial sample to the tube.

Figure 3: Transferring Bacterial Sample

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Experiment Aseptic Technique and Culturing Microbes

7. Use the candle flame to sterilize the top of the tube again to eliminate potential contamination and replace the cap.

Figure 4: Sterilizing the Tube

8. Disinfect your work area.

Forms of culture Media

Nutrient Broth is a liquid medium. A typical nutrient broth medium, such as Trypticase soy broth, contains substrates for microbial growth such as pancreatic digest of casein, pancreatic digest of soybean meal, sodium chloride, and water. After incubation, growth, the development of many cells from a few cells, may be observed as one or a combination of three forms:

● Pellicle: A mass of organisms floats in or on top of the broth. Smaller masses or clumps of organisms that are dispersed throughout the broth form an even pattern called flocculent.

Figure 5: Pellicle Form

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Experiment Aseptic Technique and Culturing Microbes

● Turbidity: The organisms appear as a general cloudiness throughout the broth.

Figure 6: Turbidity Form

● Sediment: A mass of organisms appears as a deposit at the bottom of the tube.

Figure 7: Sediment Form

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Experiment Aseptic Technique and Culturing Microbes

Agar slant tubes are tubes containing a nutrient medium plus a solidifying agent, called agar. The medium has been allowed to solidify at an angle in order to generate a flat inoculating surface.

Figure 8: Slant Tube

Stab tubes, called deeps, are tubes of hardened agar medium which are inoculated by stabbing the inoculum into the agar.

Figure 9: Stab Tube

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Experiment Aseptic Technique and Culturing Microbes

Agar dishes are sterile Petri dishes aseptically filled with a melted sterile agar medium and allowed to solidify. Dishes are much less confining than slant and stab tubes and are commonly used when culturing, separating, and counting microorganisms.

Figure 10: Agar Dishes

Requirements for Microbial Growth

oxygen: Microorganisms show a great deal of variation in their requirements for gaseous oxygen. Most microorganisms can be placed in one of the following groups.

● obligate aerobes are organisms that grow only in the presence of oxygen. They obtain energy from aerobic respiration.

● Microaerophiles are organisms that require a low concentration of oxygen for growth. They obtain energy from aerobic respiration.

● obligate anaerobes are organisms that grow only without oxygen; oxygen inhibits or kills them. They obtain energy from anaerobic respiration or fermentation.

● Aerotolerant anaerobes, like obligate anaerobes, cannot use oxygen for growth, but they tolerate oxygen fairly well. They obtain energy from fermentation.

● Facultative anaerobes are organisms that grow with or without oxygen, but generally better with oxygen. They obtain energy from aerobic respiration, anaerobic respiration, and fermentation. Most bacteria are facultative anaerobes.

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Experiment Aseptic Technique and Culturing Microbes

Temperature: Microorganisms are divided into groups based on their preferred ranges of temperature.

● Psychrophiles are cold-loving bacteria. Their optimum growth temperature is between -5°C and 15°C. They are usually found in the Arctic and Antarctic regions and in streams fed by glaciers.

● Mesophiles are bacteria that grow best at moderate temperatures. Their optimum growth temperature is between 25°C and 45°C. Most bacteria are mesophilic and include common soil bacteria and bacteria that live in and on the body.

● Thermophiles are heat-loving bacteria. Their optimum growth temperature is between 45°C and 70°C. They are commonly found in hot springs and compost heaps.

● Hyperthermophiles are bacteria that grow at very high temperatures. Their optimum growth temperature is between 70°C and 110°C. They are usually members of the Archaea and are found growing near hydrothermal vents at great depths in the ocean.

Before culturing microbes, ensure the necessary nutritional and environmental conditions are present.

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Experiment Aseptic Technique and Culturing Microbes

Exercise: culturing Microbes Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic. Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

procedure Pre-Experiment Preparation: Find an incubation site or construct an incubator at least 24 hours in advance of the experiment to allow for time to monitor temperatures.

Part I: Set Up Incubation Site

Each bacterium has an optimal temperature at which it grows best. You can estimate the optimal growth temperature by considering the bacteria’s natural environment. Through the course of this experiment series, you will be culturing microbes from various sources, but most will fall into two main categories:

● Microbes from the environment that grow best at room temperature

● Microbes from our bodies that grow best at physiological or body temperature

You will need to establish sites that you can use to incubate both types of organisms. The sites should be free from draft and maintained at a consistent temperature. You will need to incubate your samples for 24 – 72 hour periods, so the site should be out of the way and free from interference. Use the thermometer to test the temperature of various areas in your home.

Some household areas that often closely approximate body temperature are the tops of water heaters or refrigerators. If you do not have access to these types of areas, use a desktop lamp or heating pad as a heat source to construct an incubator. Each lamp or pad is a bit different, so use the thermometer and test how far from the bulb or pad to keep the samples to keep them at 35°C–37°C (physiological temperature).

To construct an incubator:

1. Use a small box that is tall enough to hold the test tube rack with the broth tubes upright (6 inches minimum) and wide enough to set agar filled Petri dishes.

It is best if the box has a lid to reduce air drafts and help maintain a consistent temperature. However, you can line a piece of cardboard with foil to lie across the top of the box in place of a lid.

Alternately, use a small Styrofoam cooler in place of a box. Cut the lid in half to allow space to direct the lamp into the box.

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Experiment Aseptic Technique and Culturing Microbes

2. Line the interior of the box with foil. If available, line pieces of Styrofoam with foil to fit in the box and provide greater insulation.

Figure 11: Foil Lined Box

3. Cut a small hole in the side of the box to fit a thermometer for monitoring temperature. Place the hole so the bulb of the thermometer will be at the same level as the cultures.

Figure 12: Thermometer Hole

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Experiment Aseptic Technique and Culturing Microbes

4. When using a desk lamp as the heating source, place the lamp so the light is aimed into the opening of the box. If the box has a lid, cut a hole in the lid and aim the lamp bulb through the hole.

Figure 13: Lamp Heat Source

5. Insert the thermometer into the box and monitor the temperature to determine the optimum distance to keep the lamp bulb. Monitor the temperature at different times of the day to ensure the temperature remains stable as environmental temperatures change.

6. When using a heating pad as the heating source, place the heating pad in the bottom of the box. Then, place a towel or folded paper towels on top of the heating pad. Insert the thermometer and monitor the temperature to determine both the optimal setting for the heating pad and the amount of padding to put between the pad and the samples to achieve the appropriate temperature. Monitor the temperature at different times of the day to ensure the temperature remains stable as environmental temperatures change.

Part ii: Determine Medium Type

Two types of media will be used to grow the microbial specimens: nutrient medium and MRS medium. Though both media are available in liquid broth and solidified agar form, this experiment will use the liquid broth. Nutrient medium is the standard growth medium used for culturing most microbes. It consists of heat-stable digestive products of proteins (called peptones) and beef extract. These ingredients provide amino acids, minerals, and other nutrients used by a wide variety of bacteria for growth.

The MRS culture medium contains polysorbate, acetate, magnesium, and manganese which are known as a rich nutrient base and act as special growth factors for lactobacilli. The MRS medium will be used to culture Lactobacillus acidophilus. L. acidophilus will not grow sufficiently in nutrient media. Label the tubes carefully so the MRS medium will be easily identified for experiments using L. acidophilus. Remember, L. acidophilus needs to be cultured in MRS medium to grow!

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Experiment Aseptic Technique and Culturing Microbes

Part iii: generate Microbial cultures

1. Disinfect the work area.

2. Use the ASEPTIC TRANSFER TECHNIQUE described above to generate liquid broth cultures of L. acidophilus, and S. epidermidis.

3. S. epidermidis – Refer to the Preparation of Cultures section in the Appendix.

a. Label a tube of Nutrient Broth “S. epidermidis.”

b. Use a sterile swab to obtain a sample of bacteria from your skin.

c. Aseptically transfer the swab into the tube of sterile media.

d. Incubate the culture tube at 37°C for 24 – 72 hours.

4. L. acidophilus. – Refer to the Preparation of Cultures section in the Appendix.

a. Label a tube of MRS broth “L. acidophilus.”

b. Taking care not to touch the contents, open a capsule of L. acidophilus.

c. Aseptically transfer the contents of the capsule into the sterile MRS media.

d. Incubate the culture tube at 37°C for 24 – 72 hours.

Part iV: observe your Microbial cultures

1. Observe the organisms after 24 hours and again after 48 hours to assess the growth pattern of each tube. Record your macroscopic observations.

Note: If there is no observable growth after 48 hours, allow the tubes to incubate an additional 24 hours.

Figure 14: Broth Growth Patterns

2. Prepare wet-mount slides of both the S. epidermidis and L. acidophilus cultures.

3. Prepare direct stained slides of both the S. epidermidis and L. acidophilus cultures.

4. Observe the slides microscopically at both 40X and 100X oil immersion magnification. Record the results.

5. Store both cultures in the refrigerator for use in future experiments.

6. Disinfect the work area.

Questions A. What is the difference between a bactericidal and bacteriostatic agent? What is the difference

between sterilization and disinfection?

B. List five microbial killing methods, how they work, and what they are used for.

C. What is a pure culture? Why is it important to work with a pure culture?

D. What is aseptic technique? Why is it so critical?

E. Describe three common forms of growth that you are likely to see in a broth culture.

F. What is the difference between an aerobe and an anaerobe?

G. Describe the difference between facultative and obligate.

H. Which two types of media did you use in this experiment? Why did you need two types of media instead of only one?

I. Describe your microscopic observations of the cultures.

J. Define the following terms:

● psychrophile:

● mesophile:

● thermophile:

● hyperthermophile:

K. Which type of organisms did you use in this experiment?

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Experiment Aseptic Technique and Culturing Microbes

Aseptic Technique & Culturing Microbes Cynthia Alonzo, M.S. Version 42-0239-00-01

lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

Questions A. What is the difference between a bactericidal and bacteriostatic agent? What is the difference

between sterilization and disinfection?

B. List five microbial killing methods, how they work, and what they are used for.

C. What is a pure culture? Why is it important to work with a pure culture?

D. What is aseptic technique? Why is it so critical?

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Experiment Aseptic Technique and Culturing Microbes

E. Describe three common forms of growth that you are likely to see in a broth culture.

F. What is the difference between an aerobe and an anaerobe?

G. Describe the difference between facultative and obligate.

H. Which two types of media did you use in this experiment? Why did you need two types of media instead of only one?

I. Describe your microscopic observations of the cultures.

J. Define the following terms:

K. Which type of organisms did you use in this experiment?

Psychrophile:

Mesophile:

Thermophile:

Hyperthermophile:

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Experiment Aseptic Technique and Culturing Microbes

Isolation of Individual colonies Cynthia Alonzo, M.S. Version 42-0245-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will learn about two types of culture growth media and colony morphology. Students will use several isolation techniques, including the pour plate method, the dilution method, and the streak plate method to prepare pure cultures. They will also learn how to maintain stock cultures.

ExpErimEnt

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objectives ● Become familiar with subtypes of culture media and the uses for each

● Learn and employ the streak and pour dish techniques

● Generate a pure culture of a specific organism

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Experiment Isolation of Individual Colonies

materials

MATEriAlS QTY iTEM DEScriPTioN Student provides 1 Distilled water

1 Paper towels 1 10%-bleach or 70% alcohol solution 1 Match or lighter 1 Marker, pemanent, black 1 Incubator 1 Zip bag 1 Pan to heat agar 1 Isopropyl alcohol (rubbing alcohol) 1 Cultures: S. epidermidis and L. acidophilus

LabPaq provides 2 Gloves, Disposable (1 pair) 1 Pencil, marking

11 Petri dish, 60 mm 2 Candles (flame source) 1 Thermometer-in-cardboard-tube 6 Test Tube(6), 16 x 125 mm in Bubble Bag

1 Test tube holder 1 Test-tube-rack-6×21-mm 1 Pipet Graduated Small (5 mL) 1 Baker’s Yeast Packet – Saccharomyces cerevisiae 1 Agar, MRS – 18 mL in Glass Tube 4 Agar, Nutrient – 18 mL in Glass Tube 1 Broth, Nutrient – 5 mL in Glass Tube 2 Inoculation Loop, Plastic 1 Mask, Face with earloops

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment Isolation of Individual Colonies

Discussion and review Bacteria are everywhere! They are on bench tops, in water, soil, and food, on your skin, and in your ears, nose, throat, and intestinal tract (normal flora). The diversity of bacteria present in our environment and on and in our bodies is incredible. When trying to study bacteria from the environment, we quickly discover that bacteria usually exist in mixed populations. It is only in very rare situations that bacteria occur as a single species.

However, to study the cultural, morphological, and physiological characteristics of an individual species, we must separate the organism from other species normally found in its habitat by creating a pure culture of the microorganism. A pure culture is defined as a population containing only a single species or strain of bacteria. Contamination means more than one species is present in a culture that is supposed to be pure. Contamination does not imply that the contaminating organism is harmful. It simply means the contaminating organism is unwanted in the culture being isolated and studied.

Petri dishes or plates are covered dishes used to culture microorganisms. The sterile Petri dish is filled with a solidified nutrient medium.

Media Composition and Function

In addition to its physical state (liquid or solid), microbiological media are categorized by composition and/or function.

● Chemically Defined or Synthetic Media: In a synthetic medium, the exact amount of pure chemicals used to formulate the medium is known.

● complex Media: A complex medium is composed of a mixture of proteins and extracts in which the exact amount of a particular amino acid, sugar, or other nutrient is not known.

● Enrichment Media: An enrichment medium contains some important growth factor (vitamin, amino acid, blood component, or carbon source) necessary for the growth of fastidious organisms. The MRS medium used in the Aseptic Technique & Culturing Microbes experiment is an enriched medium due to the presence of growth factors that encourage Lactobacillus acidophilus growth.

● Selective Media: Selective media allow for the selection of particular microorganisms that may be present in a mixed culture. Selective media usually contain a component that enhances the growth of the desired organism or inhibits the growth of competing organisms.

● Differential Media: Differential media allow for the separation of organisms based on some observable change in the appearance of the medium or by an observable effect on the microbe.

Any single medium may be a combination of the previous categories. For example, Mannitol Salt Agar (MSA), used for the isolation and identification of Staphylococcus, is a complex, selective, and differential medium. MSA contains NaCl, mannitol (a simple sugar), pancreatic digest of soy bean meal, potassium phosphate, and phenol red (a pH indicator). The presence of the pancreatic digest in the medium makes it a complex medium, because the exact composition of the pancreatic digest is not known. The relatively high concentration of salt in the medium

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is designed to inhibit many organisms and to select salt tolerant organisms. This makes MSA a selective medium. Staphylococcus is commonly found on the human skin – a salty environment due to sweat. Finally, the inclusion of mannitol and phenol red makes MSA a differential medium. Staphylococci metabolize mannitol and produce acid as a waste product. This acid lowers the pH of the agar in the immediate vicinity of the organism. Phenol red changes color from red to yellow when the pH falls. Thus mannitol fermenting organisms can be differentiated from other organisms because the area around colonies of mannitol fermenters changes color from red to yellow as the organisms grow.

Microbiological media may be prepared as either liquid broth or solid medium. When a solid medium is prepared, the corresponding broth is solidified by the addition of agar. Agar is a gelatin type substance that is extracted from red-purple marine algae. Though it is possible to use standard gelatin, agar is preferred because it is stronger than gelatin and will not be degraded (eaten) by the bacteria. Agar is a solid gel at room temperature and melts at approximately 85°C. A particularly useful feature of agar is that while it melts at 85°C, it does not solidify until it cools to 32oC-40°C. This allows the agar to remain in liquid form long enough and at a cool enough temperature to be managed. Agar is added to liquid nutrient medium, generally in a final concentration of 1%-2%, to obtain a solid culture medium.

colony Morphology

To obtain a pure culture, it is necessary to separate individual cells of a particular microbe. This requires the use of a solid medium that provides a surface for the individual cells to be separated and isolated from the other microbial cells that may be present in the original sample.

A colony is a visible mass of microorganisms growing on a solid medium. A colony is considered to form from reproduction of a single cell. Thus, all the members of a colony are descendents from that original cell.

The colonies of different types of bacteria will have a distinct appearance. The visual characteristics of a colony (shape, size, pigmentation, etc.) are referred to as the colony morphology and can be used to identify bacteria. Bergey’s Manual of Determinative Bacteriology, a standard resource used by many microbiologists, describes the majority of bacterial species identified by scientists so far. Bergey’s Manual provides descriptions for the colony morphologies of each bacterial species.

Though there are many identifiable characteristics that a colony may posses, there are six main criteria that comprise a standard morphology:

● Shape: What is the basic formation of the colony? Is it circular, irregular, or filamentous?

Figure 1: Shape Characteristics

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● Elevation: What is the cross-sectional form of the colony when viewed from the side? Is it flat, raised, or convex?

Figure 2: Elevation Characteristics

● Margin: How does the edge of the colony appear when magnified? Is it smooth, lobed, or curled?

Figure 3: Margin Characteristics

● Surface: What is the appearance of the surface of the colony? Is it glistening, rough, or dull?

Figure 4: Surface Characteristics

● Pigmentation: Is the colony colored? Is it white, cream colored, pink, etc.?

Figure 5: Pigmentation Characteristics

● opacity: Is the colony transparent, opaque, translucent, or iridescent?

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There are three additional characteristics that are sometimes used for identification but should be examined only in a controlled setting such as in a laboratory containment hood. These characteristics are consistency, emulsifiability, and odor.

Pure Cultures and Microbial Enumeration

Several different methods for getting a pure culture from a mixed culture are available including streak plate, pour plate, and dilution to extinction. All of these techniques depend on the physical isolation of a single bacterial cell on or in a solid medium. These cells give rise to isolated pure colonies of the bacteria.

In addition to the isolation of pure colonies, diluting to extinction also allows for the enumeration or determination of the number of organisms present in the original culture or sample. Microbial enumeration is routinely used in public health. Public safety officials test food, milk, or water and calculate the number of microbial pathogens present to determine if these products are safe for human consumption. Microbial counting techniques are also used to determine the number of microbes present in a given culture in commercial or scientific settings. For example, if the number of bacteria present in a fermentation culture is known, it is then possible to calculate the amount of fermentation product (such as insulin) that can be harvested from that population.

Several methods can be used to determine the number of microbes in a given sample. Viable counts include cells that can be cultured or are metabolically active. Total counts include all cells present, including dead or inactive cells. Direct methods count actual cells or colonies; indirect methods estimate the number of cells present based on the measurement of an indicator such as light absorption. Some of the more commonly used techniques are to measure the optical density of the population using a spectrophotometer, directly count the microorganisms using a hemocytometer, or serial dilute the bacteria and plate the diluted bacteria on a medium that supports the growth of the micro-organisms.

Optical Density: Spectroscopy Enumeration Method

Optical density is an indirect method of determining the cell concentration in a bacterial culture. Bacterial cells absorb light well at the wavelength of 686 nm when grown in standard media. A spectrophotometer is used to measure the amount of light at a wavelength of 686 nm that is transmitted through a bacterial culture. Because the bacteria absorb the light of that wavelength, the amount of light transmitted through the culture, rather than absorbed by it, is inversely proportional to the number of bacteria present in the sample. The more bacteria present, the less light that will transmit through the sample.

Figure 6: Optical Density

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Measurement of light transmitted through the culture can be used to determine the number of bacteria present by graphing absorbance against known bacterial counts to obtain a standard curve.

Figure 7: Light Absorbance

The measurements can also be converted to Optical Density (OD) which is a quantitative method of describing the cellular mass of a culture. The measurements obtained through spectrophotometer readings are considered total count measurements because they include all cells present, both viable and nonviable.

Aseptically Inoculating from a Liquid Culture

When working with bacterial cultures, it is essential to use proper aseptic culture techniques. Remember, aseptic techniques are the precautionary measures used to avoid contamination of cultures and manipulate microorganisms to prevent contamination by undesirable organisms. Aseptic techniques not only protect a laboratory culture from becoming contaminated, but also protect the experimenter and the environment from becoming contaminated by the microorganisms.

1. Disinfect the inoculating loop. Never lay the loop down once it is disinfected or it may become contaminated. To disinfect the plastic inoculation loop, swish the loop in a 10%-bleach solution or 70%-alcohol solution for about 10 seconds. Then rinse the loop with distilled water and allow it to completely air dry before using. Do not use a flame for disinfecting the plastic loop.

2. Hold the culture tube in one hand and the inoculating loop in the other hand as if it were a pencil.

3. Remove the cap of the culture tube with the little finger of your loop hand. Never lay the cap down or it may become contaminated.

4. Light a candle and briefly flame the lip of the culture tube.

5. Keeping the culture tube at an angle, insert the inoculating loop into the tube and remove a loop full of inoculum.

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6. Flame the lip of the culture tube again. Then replace the cap.

7. Pick up the sterile tube of medium. For the purposes of this experiment, you will be inoculating sterile agar medium.

8. Briefly flame the lip of the tube.

9. Place the loop full of inoculum into the medium. Withdraw the loop, but do not lay the loop down!

10. Flame the lip of the tube again.

11. Disinfect the inoculation loop.

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Exercise 1: Isolation Using the Pour Plate Method Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic. Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

procedure Part I: Preparation of Solid Media

1. Disinfect the work area.

2. Melt the agar tubes. Refer to the Preparation of Solid Media section in the Introduction for further instruction.

Note: When you remove melted agar tubes from the water bath, they cool rapidly. Agar will solidify at about 45oC. If the agar solidifies, boil the tubes again to re-melt. It is time consuming and inconvenient if the agar deeps solidify in the tube before they are inoculated.

If the agar solidifies after inoculation but before it is poured into Petri dishes, re-melt the agar to kill the inoculated organisms. Under these circumstances, there is nothing to do but start over. The key to successful pour plates is to be well organized and work quickly.

3. Leave the 18 mL tube of MRS agar in hot water (50°C) for use in Part II.

4. Use the marking pencil to label the bottom of one Petri dish S. epidermidis. Pour one half (9 mL) of the contents of a tube of nutrient agar into the S. epidermidis Petri dish and the other half into the bottom of an unmarked Petri dish. Cover the dishes and allow them to solidify for use in Part IV.

5. Pour the remaining melted nutrient agar into the unmarked Petri dishes (half a tube per dish). Cover the dishes and allow them to solidify for use in Part III.

Note: There will be one extra nutrient agar dish. Store the dish in the refrigerator for use in the Antibiotic Sensitivity experiment. Invert the dish in a zip bag to protect it from contamination and dry-out.

Part II: Isolation Using the Pour Plate Method

The pour plate technique, sometimes called the loop dilution method, involves the successive transfer (serial dilution) of bacteria from the original culture to a series of tubes of liquefied agar. A loop of the original culture is transferred to a tube of liquid agar and mixed. As a result of this transfer, the concentration of bacteria in the tube is lower than the concentration in the original culture – in effect, a dilution of the original culture. A loop of material from the first tube of liquefied agar is then transferred to a second tube, effecting an additional dilution of the bacterial

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culture. The process is repeated for a third tube of agar. Following inoculation of the tubes of liquid agar, the contents of each tube are poured into separate Petri dishes. After incubation, one of the dishes should have a low enough concentration of microbes to allow separation and isolation of individual colonies.

1. Disinfect the work area.

2. Label the bottom surface of three sterile Petri dishes L. acidophilus #1, #2, and #3, respectively.

Figure 8: Part II Pour Plates

3. Disinfect three test tubes by submerging them in boiling water for 5 minutes. The tubes will be hot, so use tongs or tweezers to lift them out of the water. Be careful not to contaminate the tubes by touching their lips or interiors. When the tubes are cool, label them to match the L. acidophilus Petri dishes.

4. Divide the liquid MRS agar into the three test tubes marked L. acidophilus. If the agar has begun to solidify, reheat it until it is fully melted. Set the test tubes of agar in the hot water to prevent them from solidifying.

5. After ensuring the tubes of agar are cool enough not to kill the bacterial culture but are still fully liquid, use aseptic techniques to inoculate the tube labeled L. acidophilus #1 with one loop full of the saved L. acidophilus culture. Gently mix and return the tube to the hot water.

6. Inoculate L. acidophilus #2 with one loop full of the bacteria media mix from tube #1. Gently mix and return the tubes to the hot water.

7. Inoculate L. acidophilus #3 with one loop full of the bacteria media mix from tube #2. Gently mix and return the tubes to the hot water.

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Figure 9: Inoculated Test Tubes

8. Pour the contents of L. acidophilus #1 into the corresponding Petri dish and cover the dish immediately. Repeat for L. acidophilus #2 and #3.

9. Allow the agar to solidify at room temperature.

Figure 10: Inoculated Petri Dishes

10. Incubate the dishes in an inverted position for 24–72 hours at 35oC–37oC.

11. Examine the dishes for isolated colonies. Record the appearance of each dish.

12. Store the culture in the refrigerator for use in future experiments.

13. Soak the Petri dishes in a 10%-bleach solution for 1 hour and then discard them.

14. Soak the test tubes in a 10%-bleach solution for 1 hour and then discard the contents. Clean and rinse the test tubes for future use.

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Exercise 2: Isolation and Enumeration by Dilution to Extinction procedure To use serial dilution to enumerate a population, make dilutions of a solution containing an unknown number of microbes and then plate a sample of each dilution. The total number of organisms in the original solution is calculated by counting the number of colony forming units (organisms capable of forming a colony) and comparing them to the dilution factor. Each colony forming unit represents a single microbe that was present in the diluted sample. The numbers of Colony Forming Units (CFUs) are divided by the product of the dilution factor and the volume of the plated diluted suspension to determine the number of organisms per mL that were present in the original solution.

CFU = CFU/mL original Volume Plated (mL) x dilution factor

1. Disinfect the work area.

2. Prepare an S. cerevisiae culture. Refer to the Preparation of Cultures section in the Appendix for further instruction.

3. Label six test tubes 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6.

4. Label six unmarked agar dishes from Part I 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6.

Figure 11: Labeled Agar Dishes

5. Use a plastic graduated pipet to add 2.25 mL of distilled water to each test tube. NOTE: To sterilize the pipet draw a small amount of 70% alcohol into the bulb, and then expel it into a sink. Remove any excess alcohol by forcefully swinging the pipet in a downward arch several times to ensure that the pipet is dry before drawing up water.

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Figure 12: Plastic Pipet Measurements

6. Mix the yeast-water solution well to disperse the organisms evenly.

7. Transfer 0.25 mL of the yeast solution into the test tube labeled 10-1. Pipet the solution up and down several times to mix it thoroughly and ensure all organisms are rinsed from the pipet into the solution.

8. Transfer 0.25 mL of the 10-1 yeast solution into the test tube labeled 10-2. Pipet the solution up and down several times to mix it thoroughly and ensure all organisms are rinsed from the pipet into the solution.

9. Repeat the transfer process to transfer the yeast solution from the 10-2 tube to the 10-3 tube; from the 10-3 tube to the 10-4 tube; from the 10-4 tube to the 10-5 tube; and from the 10-5 tube to the 10-6 tube as shown in Figure 13.

Thoroughly rinse the pipet in water to remove all organisms, inside and out.

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Figure 13: Test Tube Transfer Process

10. Beginning with the 10-1 tube, mix the sample by pipetting the solution up and down several times. Then pipet 0.125 mL (four drops) onto the corresponding 10-1 agar dish.

11. Place the cover on the dish and swirl the dish gently to spread the solution evenly over the surface.

12. Repeat the steps for the remaining dilutions as shown in Figure 14.

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Figure 14: Petri Dish Transfer Process

13. Incubate the dishes at 37oC for 48–72 hours. Leave the agar dishes right-side up for the first 12 hours to let the liquid culture set into the dish. After the first 12 hours, invert the dishes to protect the growing colonies from condensation.

14. After incubation, you should see a gradient of growth on the dishes, representing where the highest concentration is producing the heaviest growth. The growth pattern will likely look similar to the dilution plate series pictured in Figure 15.

Figure 15: Dilution Plate Series

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15. For each dilution, count the number of colony-forming units on the dishes. Mark the position of each colony on the bottom of the dish with a marking pen as you count it. Dishes containing more than a few hundred colonies are considered Too Numerous To Count (TNTC) and are recorded as TNTC in data records. Dishes with only a few colonies are considered Too Few To Count (TFTC) and recorded as TFTC.

Using the formula provided earlier, use the number of CFUs per dish to calculate the number of organisms per mL in the original sample.

For example: For the 1×10-6 dilution dish, you plated 0.125 mL of the diluted cell suspension. If you counted 100 colonies, the calculation would be:

100 CFU ÷ (0.125mLx10-6) → 100 CFU ÷ 1.25-6 mL → 8 x 107 CFU/mL 16. Mix 1 tablespoon of bleach into the yeast culture and let it stand for at least 30 minutes to

ensure all organisms have been destroyed. Then discard the contents.

17. Soak the Petri dishes in a 10%-bleach solution for 1 hour and then discard them.

18. Soak the test tubes in a 10%-bleach solution for 1 hour and then discard the contents. Clean and rinse the test tubes for future use.

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Exercise 3: Isolation by the Streak Plate Method procedure In the streaking Procedure, a disinfected loop or sterile swab is used to obtain a microbial culture. The inoculating instrument is then streaked lightly over an agar surface. On the initial section of the streak, many microorganisms are deposited, resulting in confluent (solid) growth, which is growth over the entire surface of the streaked area. However, because the loop is sterilized or disinfected between streaking different sections, or zones, fewer and fewer microorganisms are deposited as the streaking progresses. Finally, only an occasional microorganism is deposited, because the streaking process dilutes the sample placed in the initial section.

Figure 16: Streak Pattern

During incubation, the isolated microbes multiply, giving rise to individually isolated colonies in the lightest inoculated areas. Colonies appear as piles of material on the agar surface, and they come in a variety of shapes, sizes and textures which are characteristic of individual microorganisms. For example, if a single Escherichia coli cell is deposited on a nutrient agar dish and incubated at 37°C, the cell and its progeny will divide every 30–40 minutes. In 10–12 hours, the colony will have reached a population of one million, and a pinpoint colony will be visible. To obtain good results with this technique, the agar surface should be smooth, moist, and free of contamination. However, excessive moisture from the condensation of water, derived from the initial cooling of the hot sterile medium, can collect on the inside of the lid and sides of the dish. If the water drops onto the agar surface, spreading and merging of colonies can occur. Always invert the dishes after streaking them and when incubating them.

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1. Disinfect the work area.

2. Use the nutrient agar dish labeled S. epidermidis from Exercise 1.

3. Use aseptic techniques to obtain a loop full of the saved liquid S. epidermidis culture.

4. Streak the inoculum into the first quadrant as shown in Figure 16.

5. Disinfect the inoculation loop. Do not obtain a new inoculum. Instead, use the disinfected inoculation loop to streak several times through Quadrant 1 to pick up some organisms on the loop. Then streak from Quadrant 1 to Quadrant 2 as shown in Figure 16.

6. Repeat the Procedure for Quadrants 3 and 4, respectively. Be sure to disinfect the inoculation loop between each quadrant.

7. Disinfect the inoculation loop.

8. Cover the dish, invert it, and incubate it for 48–72 hours at 35oC–37oC.

9. Identify an S. epidermidis colony. The S. epidermidis culture was not a pure culture (derived from a single organism) and will most likely contain colonies from several different organisms. Staphylococci produce round, raised, opaque colonies 1–2 mm in diameter. S. epidermidis colonies are white in color.

Figure 17: S. Epidermidis

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Exercise 4: Stock cultures procedure When a particular organism is going to be used more than once, create a stock culture of that organism. A stock culture is maintained for as long as the organism is needed. The use of stock cultures is beneficial for several reasons including:

● They maintain consistency by ensuring the same strain of organism is used.

● They save time and money by eliminating the need to recreate the same culture.

In the following steps you will make a stock culture of S. epidermidis to use in the remaining experiments.

1. Label a tube of nutrient broth S. epidermidis Stock Culture.

2. Aseptically transfer an S. epidermidis colony from your Petri dish into the nutrient broth. The culture that grows in the broth will be a pure culture because it originated from only a single colony, which originated from a single organism.

3. Incubate the stock culture for 24–48 hours to establish the culture. Then store the culture in a zip bag in the refrigerator for use in future experiments. You may also store dish cultures in a similar manner.

4. Mix 1 tablespoon of bleach into the original S. epidermidis culture and let it stand for at least 30 minutes to ensure all organisms have been destroyed. Then discard the contents.

5. Soak the Petri dishes in a 10%-bleach solution for 1 hour and then discard them.

6. Disinfect the work area.

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Questions A. Define the following:

● enriched media:

● selective media:

● differential media:

● complex media:

● synthetic media:

B. Why is it necessary to use a solid agar medium to obtain a pure culture of S. epidermidis?

C. Compare your L. acidophilus pour plates and spread plate. Which method do you think worked better to isolate individual colonies? Why?

D. What are the six qualities included in a description of colony morphology?

E. Describe the colony morphology seen on your S. epidermidis dish.

F. What is the difference between a viable and total count? What is the difference between direct and indirect counts?

G. What is a spectrophotometer? How is it used to enumerate microbes?

H. What is a hemocytometer? How is it used to enumerate microbes?

I. Define the following acronyms:

● CFU:

● TNTC:

● TFTC:

● OD:

J. When serial dilution is used to enumerate microbes in a real life application, such as in a water quality study, each dilution is plated on a series of dishes. The data from each dish (the number of CFUs) is pooled together and an average CFU per dish is generated for the dilution. It is this average, rather than the actual plate counts, that is used to calculate the final CFU/ mL result.

Why do you think an average is used rather than the actual plate counts? Why might there be differences in the number of CFUs on each dish when they are grown from the same dilution?

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Isolation of Individual Colonies Cynthia Alonzo, M.S. Version 42-0245-00-01

lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

observations

Questions A. Define the following:

Enriched Media:

Selective Media:

Differential Media:

Complex Media:

Synthetic Media:

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B. Why is it necessary to use a solid agar medium to obtain a pure culture of S. epidermidis?

C. Compare your L. acidophilus pour plates and spread plate. Which method do you think worked better to isolate individual colonies? Why?

D. What are the six qualities included in a description of colony morphology?

E. Describe the colony morphology seen on your S. epidermidis dish.

F. What is the difference between a viable and total count? What is the difference between direct and indirect counts?

G. What is a spectrophotometer? How is it used to enumerate microbes?

H. What is a hemocytometer? How is it used to enumerate microbes?

I. Define the following acronyms: CFU

TNTC TFTC OD

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J. When serial dilution is used to enumerate microbes in a real life application, such as in a water quality study, each dilution is plated on a series of dishes. The data from each dish (the number of CFUs) is pooled together and an average CFU per dish is generated for the dilution. It is this average, rather than the actual plate counts, that is used to calculate the final CFU/mL result.

Why do you think an average is used rather than the actual plate counts? Why might there be differences in the number of CFUs on each dish when they are grown from the same dilution?

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Experiment Isolation of Individual Colonies

Differential Staining Cynthia Alonzo, M.S. Version 42-0242-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will use Gram’s stain techniques to differentiate between types of bacteria and explore the difference between Gram-positive and Gram- negative bacteria. Students will explore what properties differentiate microorganisms including Escherichia coli, Staphylococcus epidermidis, Lactobacillus acidophilus, and Saccharomyces cerevisiae.

ExpErimEnt

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objectives ● Understand and employ differential staining techniques

● Describe the differences between Gram-negative and Gram-positive bacteria

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materials

MATEriAlS QTY iTEM DEScriPTioN Student provides 1 10%-bleach solution

1 Isopropyl rubbing alcohol 1 Microscope 1 Immersion Oil 1 Tap water 1 Paper towels 1 Clothespin, tweezers, or test tube holder 1 Stock culture: S. epidermidis 1 Saved culture: L. acidophilus

LabPaq provides 1 Goggles, safety 1 Gloves, Disposable (1 pair)

1 Apron, plastic 1 Tray – Staining tray 2 Candles (flame source)

1 Broth, Nutrient – 5 mL in Glass Tube 2 Baker’s Yeast Packet – Saccharomyces cerevisiae 1 E. coli culture 1 Pipet, Graduated Small (5 mL) 1 Gram Stain Solution #1, Crystal Violet – 15mL in Dropper Bottle 1 Gram Stain Solution #2, PVP-Iodine – 15 mL in Dropper Bottle 1 Gram Stain Solution #3, Decolorizer – 30 mL in Dropper Bottle 1 Gram Stain Solution #4, Safranin – 15 mL in Dropper Bottle 1 Sterile Swabs – 2 per Pack 1 Mask, Face with Earloops 1 Slide-Box-MBK with Blank-Slides (4) 1 Slide, cover slip (20 pcs)

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment Differential Staining

Discussion and review There are several staining methods used routinely with bacteria. These methods are generally classified as either simple, nonspecific, or differential (specific). Simple stains will react with all microbes in an identical fashion. They are used solely for increasing contrast, so an organism’s morphology, size, and arrangement can be determined. Differential stains provide varying results depending on the organism being treated. These results are often helpful in identifying the microbe. This exercise will focus on one of the most commonly used differential stains – the Gram’s stain.

The Gram’s stain is the most widely used staining Procedure in bacteriology. It is called a differential stain because it differentiates between Gram-positive and Gram-negative bacteria. Bacteria that stain purple are termed Gram-positive. Those that stain pink are termed Gram-negative.

Figure 1: Gram-Positive (left); Gram-Negative (right)

Gram-positive and Gram-negative bacteria stain differently because of fundamental differences in the structure of their cell walls. The bacterial cell wall serves to give the organism its size and shape as well as to prevent osmotic lysis. The material in the bacterial cell wall that confers rigidity is peptidoglycan. The Gram-positive cell wall appears thick and consists of numerous interconnecting layers of peptidoglycan. Also interwoven in the cell wall of Gram-positive bacteria are teichoic acids. Generally, 60%-90% of the Gram-positive cell wall is peptidoglycan.

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Figure 2: Gram-Positive Peptidoglycan

The Gram-negative cell wall contains a much thinner section of peptidoglycan – only two or three layers thick. This section is surrounded by an outer membrane composed of phospholipids, lipopolysaccharide, and lipoprotein. Only 10% – 20% of the Gram-negative cell wall is peptidoglycan.

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Figure 3: Gram-Negative Peptidoglycan

gram Staining Procedure:

1. The bacteria are first stained with the basic dye, crystal violet. Both Gram-positive and Gram- negative bacteria become directly stained and appear purple.

2. Then the bacteria are treated with Gram’s iodine solution. The solution helps retain the stain by forming an insoluble crystal violet-iodine complex. Both Gram-positive and Gram-negative bacteria remain purple.

3. Then the bacteria are treated with Gram’s decolorizer, a mixture of ethyl alcohol and acetone. This is the differential step. Gram-positive bacteria retain the crystal violet-iodine complex in their thick peptidoglycan layer. The complex washes out of the thinner peptidoglycan layer of Gram-negative bacteria which become decolorized.

4. Finally, the bacteria are treated with the counterstain, safranin. Because the Gram-positive bacteria are already stained purple, they are not affected by the counterstain. The Gram- negative bacteria are colorless and become directly stained pink by the safranin. Consequently, the Gram-positive bacteria appear purple and the Gram-negative bacteria appear pink.

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Experiment Differential Staining

Exercise 1: Differential Staining Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic. Therefore, be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

procedure Pre-Experiment Preparation: Place the stock culture of S. epidermidis and the saved culture of L. acidophilus in an incubator 12–24 hours prior to the start of the experiment. Prepare an E. coli culture in accordance with the Preparation of Cultures section in the Appendix 24–48 hours prior to the start of the experiment. Prepare an S. cerevisiae culture in accordance with the Preparation of Cultures section in the Appendix.

Part I: Differential Staining

Note: Because most stains are strong and can damage clothing and furniture, wear gloves and an apron to protect skin and clothes. Use a staining tray for this work.

1. Disinfect the work area.

2. Label four slides E. coli, S. epidermidis, L. acidophilus, and S. cerevisiae.

3. Make a slide (smear and heat-fix) for each organism.

4. Place the first slide in the staining tray.

5. Flood the slide with crystal violet and let the slide sit for approximately 1 minute.

6. Drain the excess dye into the sink and gently rinse the slide with tap water.

Figure 4: Crystal Violet Slide Before (left) and After (right) Rinsing

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7. Next, flood the slide with iodine solution and allow it to sit for 1 minute.

8. Rinse the slide gently but thoroughly with water.

Figure 5: Iodine Slide Before (left) and After (right) Rinsing

9. Lean the slide against the side of the staining tray.

10. Decolorize the slide by applying drops of acetone-alcohol to the slide until no more color washes off.

Note: Be careful not to over decolorize.

11. Rinse the slide gently with water.

Figure 6: Decolorizing the Slide with Acetone-Alcohol

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Experiment Differential Staining

12. Place the slide back in the staining tray.

13. Flood the slide with safranin and allow it to sit for 30–60 seconds.

14. Rinse the slide gently with water and gently blot the slide dry with paper towels.

Figure 7: Safranin Slide Before (left) and After (right) Rinsing

15. Observe each slide under the microscope. Record the observations.

16. Save the E. coli culture and the S. epidermidis stock culture in the refrigerator for use in later experiments.

17. Mix 1 tablespoon of bleach into the yeast culture and let it stand for at least 30 minutes to ensure all organisms have been destroyed. Then discard the contents.

18. You will not need the L. acidophilus culture for future experiments. Mix 1 tablespoon of bleach into the culture and let it stand for at least 30 minutes to ensure all organisms have been destroyed. Then discard the contents.

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Experiment Differential Staining

Questions A. What is a differential stain? How is it different from a simple stain?

B. What is the difference between Gram-positive and Gram-negative cell walls?

C. What is the purpose of crystal violet in the Gram’s stain procedure?

D. What is the purpose of iodine in the Gram’s stain procedure? What is a mordant?

E. What is the purpose of acetone-alcohol in the Gram’s stain procedure?

F. What is the purpose of safranin in the Gram’s stain procedure?

G. Why do Gram-positive cells stain purple?

H. Why do Gram-negative cells stain pink?

I. Which of the organisms stained Gram-negative?

J. Which of the organisms stained Gram-positive?

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Experiment Differential Staining

Differential Staining Cynthia Alonzo, M.S. Version 42-0242-00-01

lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

observations

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Experiment Differential Staining

Questions A. What is a differential stain? How is it different from a simple stain?

B. What is the difference between Gram-positive and Gram-negative cell walls?

C. What is the purpose of crystal violet in the Gram’s stain Procedure?

D. What is the purpose of iodine in the Gram’s stain Procedure? What is a mordant?

E. What is the purpose of acetone-alcohol in the Gram’s stain Procedure?

F. What is the purpose of safranin in the Gram’s stain Procedure?

G. Why do Gram-positive cells stain purple?

H. Why do Gram-negative cells stain pink?

I. Which of the organisms stained Gram-negative?

J. Which of the organisms stained Gram-positive?

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Experiment Differential Staining

Methyl red Voges- Proskauer Test Cynthia Alonzo, M.S. Version 42-0246-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will learn how different biochemical tests, including methyl red, Voges-Proskauer, and catalase, are used to differentiate microorganisms. Students will test Escherichia coli and Staphylococcus epidermidis with these biochemicals to analyze the bacteria’s use of various sugars and different biochemical pathways.

ExpErimEnt

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objectives ● Become familiar with and perform the MR-VP biochemical test

● Learn some variations in how different organisms metabolize glucose

● Become familiar with and perform the catalase biochemical test

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Experiment Methyl Red Voges-Proskauer Test

materials

MATEriAlS QTY iTEM DEScriPTioN Student provides 1 Hydrogen peroxide

1 10%-bleach solution 1 Paper towels 1 Match or lighter 1 Stock culture: S. epidermidis 1 Saved culture: E. coli

LabPaq provides 1 Gloves, Disposable (1 pair) 1 Goggles, safety

2 Candles (flame source) 4 Test Tube, 16 x 125 mm in Bubble Bag

1 Test-tube-rack-6×21-mm 1 Pipet, Long Thin Stem 1 Slide-Box-MBK with Blank-Slides

2 Broth, MR-VP – 5 mL in Glass Tube 1 Barritt’s A Reagent – 3 mL in Pipet 1 Barritt’s B Reagent – 3 mL in Pipet 1 Methyl Red Reagent, 0.1% – 1 mL in Pipet 1 Inoculation Loop, Plastic 1 Mask with Earloops

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment Methyl Red Voges-Proskauer Test

Discussion and review Different bacteria may have similar morphologies or produce colonies that are indistinguishable from those of other types of bacteria. Staining techniques provide additional opportunities to gather information such as bacterial morphology, cell wall composition, and the presence of capsules, flagella, or endospores. However, visual examination, both macroscopic and microscopic is often not enough to identify a specific bacterial species. In such cases, we must rely on biochemical characteristics to differentiate between organisms.

All organisms utilize a vast array of biochemical pathways to perform metabolic functions. Each pathway consists of a series of chemical reactions. Specialized proteins called enzymes are used to catalyze these reactions. Many enzymes require dietary minerals, vitamins, and other cofactors in order to function properly. As you can imagine, each pathway can be quite elaborate and require many different proteins, minerals, vitamins, or other molecules. The metabolic processes used by a cell are similar from organism to organism. However, the specific pathway or molecules used in the pathway can and do vary. The specific pathways or molecules used by a specific organism comprise its biochemical profile or “fingerprint” and can be used to identify a particular species. Microbiologists have developed series of biochemical tests that use the biochemical profile of a particular microbe to differentiate between even closely related species.

Figure 1: Biochemical Test Series

There are many types of commonly used biochemical tests that test for either the presence of a particular enzyme or for a byproduct or end product of a particular pathway. The tests can be done individually or as a series. There are a number of commercially produced test strips that combine tests designed to identify specific groups of organisms. Each strip is a collection of mini chambers, each of which contains the reagents necessary to test for a specific biochemical characteristic. The strip is designed so that a microbe of interest can be inoculated into a groove or tube that carries the microbe into each chamber without intermixing. Once inoculated, the strip is incubated and the results of the tests can be observed. The interpretation of positive and

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Experiment Methyl Red Voges-Proskauer Test

negative test results allows for identification of the bacteria to the species level. Additionally, newer methods of testing identify organisms using other characteristics, such as DNA sequence or reaction to monoclonal antibodies.

In this experiment, you will perform three common biochemical tests: the Methyl Red test, the Voges-Proskauer test, and the catalase test.

Methyl red Test

The Methyl Red (MR) test is used to identify bacteria based on their pattern of glucose metabolism. Most bacteria that ferment glucose produce pyruvic acid as an early step in metabolism; however, not all bacteria metabolize pyruvic acid like other acids, such as lactic acid and formic acids. Methyl Red broth contains glucose, peptone, and a phosphate buffer. Bacteria that produce mixed-acids as an end product of glucose fermentation overwhelm the buffer in the broth and cause a decrease in pH. Bacteria that utilize other fermentation pathways and produce other, non-acidic end products do not cause a drop in the pH of the broth.

Figure 2: MR Results After incubation, Methyl Red, a pH indicator, is added to the broth. Methyl Red turns red when the pH is below 4.4; yellow when the pH is above 6.0; and orange when the pH is between 4.4 and 6. A positive Methyl Red test result, indicating the production of stable acidic end products, is evidenced when the incubated broth turns red. A yellow color is a negative result indicating acidic end products were not produced. If the incubated tube turns orange, the result is inconclusive. It is likely that the bacteria are producing acidic products but not in large enough quantities to overwhelm the phosphate buffer in the broth. In these cases, the tube should be incubated for an additional 24 hours to see if more acid is produced.

Voges-Proskauer Test

The Voges-Proskauer (VP) test is an assay for the presence of acetyl methyl carbinol (acetoin). Acetoin can be produced as an intermediate product in the fermentation of pyruvate to 2,3-Butanediol. It can also be produced by some organisms that ferment glucose to form unstable acid products which can be converted to acetoin. After incubation of the organism in the MR- VP broth, Barritt’s Reagent A (a-napthol) and B (40% KOH) are added. The reagents react with

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Experiment Methyl Red Voges-Proskauer Test

acetoin, creating a maroon band at the top of the broth. The appearance of the band is a positive VP result, indicating the production of acetoin.

Figure 3: VP Results

catalase Test

Hydrogen peroxide is a harmful byproduct of many normal metabolic processes. To prevent damage, hydrogen peroxide must be quickly converted into other, less dangerous substances. Like many other organisms, microorganisms may produce enzymes which neutralize toxic forms of oxygen such as hydrogen peroxide. One such enzyme is catalase, which facilitates the breakdown of hydrogen peroxide into water and molecular oxygen.

2 H2O2 → 2 H2O + O2

Microbes which produce catalase will bubble when placed into hydrogen peroxide, as the enzyme speeds the decomposition of the hydrogen peroxide to water and gaseous oxygen.

Figure 4: Catalase Test

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Experiment Methyl Red Voges-Proskauer Test

Exercise 1: Methyl red-Voges Proskauer Tests procedure Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic. Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

The MR and VP tests use the same base broth as the medium. The MR-VP broth contains peptone, buffers, and glucose. Because they use the same broth, the tests are usually done together.

Pre-Experiment Preparation: Place the saved E. coli culture and the S. epidermidis stock culture in an incubator 12–24 hours prior to the start of the experiment.

1. Disinfect the work area.

2. Remove the tubes of MR-VP broth from the refrigerator and allow them to come to room temperature.

3. Label the MR-VP broth tubes E. coli and S. epidermidis.

4. Use aseptic techniques to inoculate each MR-VP broth tube with the corresponding organism.

Figure 5: MP-VP Test Tubes

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Experiment Methyl Red Voges-Proskauer Test

5. Incubate the tubes for 48 hours at 35°C–37°C.

6. Allow the reagents to warm to room temperature.

7. Sterilize and label two test tubes E. coli and two test tubes S. epidermidis.

8. Transfer half (2.5 mL) of the incubated MR-VP broth labeled E. coli into each of the corresponding test tubes. Repeat for the broth labeled S. epidermidis.

9. Choose one tube for each organism for the Methyl Red test and label it accordingly. Use a pipet to add six to eight drops of Methyl Red reagent to each of the tubes. If the test is positive, the red-pink color of acid presence from glucose use will appear within seconds.

10. Use the remaining tubes for the Voges-Proskauer test. Add 12 drops of Barritt’s A Reagent to each tube and mix gently.

11. Add four drops of Barritt’s B Reagent to each tube. Shake the tube gently for 30 seconds. The broth must be exposed to oxygen for a color reaction to occur.

12. Allow the tubes to stand for 30 minutes before interpreting.

Figure 6: MR Test

Note: The reagents must be added in the correct order and in the correct amounts. The tubes must sit undisturbed and open to the air (no cap) for at least 30–45 minutes as the light pink color intensifies at the top of the tube. Do not shake the tube after sitting it down for the waiting period. Do not read test results more than one hour after adding the reagents.

13. Record the results.

14. Soak the test tubes in a 10%-bleach solution for 1 hour and then discard the contents. Clean and rinse the test tubes for future use.

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Experiment Methyl Red Voges-Proskauer Test

Exercise 2: catalase Test procedure 1. Disinfect the work area.

2. Label two microscope slides E. coli and S. epidermidis.

3. Use a plastic inoculation loop to transfer a sample of each organism to the corresponding slide.

4. Add a drop of hydrogen peroxide to the smear. Mix with the plastic loop if needed. Do not use a metal loop when using hydrogen peroxide as hydrogen peroxide will give a false positive and degrade the metal.

5. Interpret the results.

a. A positive result is the evolution of oxygen gas evidenced by bubbling or foaming.

b. A negative result is evidenced by no bubbles or only a few scattered bubbles.

Figure 7: Catalase Test Results

6. Record the results.

7. Return the E. coli culture and the S. epidermidis stock culture to the refrigerator for use in future experiments.

8. Clean and disinfect the slides and work area.

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Experiment Methyl Red Voges-Proskauer Test

Questions A. What is meant by the term biochemical profile?

B. What metabolic end product does the MR test for?

C. What does an orange color indicate as a result for an MR test?

D. What metabolic end product does the VP test for?

E. Why do you need to be careful not to jostle the VP tube while waiting for the results to show?

F. Which of the organisms, if any, fermented glucose?

G. Which of the organisms, if any, produced measurable acidic byproducts?

H. What is the cellular role of catalase?

I. Which of the organisms, if any, produced catalase?

J. Which of the organisms, if any, produced acetoin?

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Experiment Methyl Red Voges-Proskauer Test

Methyl red Voges-Proskauer Test Cynthia Alonzo, M.S. Version 42-0246-00-01

lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

observations

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Experiment Methyl Red Voges-Proskauer Test

Questions A. What is meant by the term biochemical profile?

B. What metabolic end product does the MR test for?

C. What does an orange color indicate as a result for an MR test?

D. What metabolic end product does the VP test for?

E. Why do you need to be careful not to jostle the VP tube while waiting for the results to show?

F. Which of the organisms, if any, fermented glucose?

G. Which of the organisms, if any, produced measurable acidic byproducts?

H. What is the cellular role of catalase?

I. Which of the organisms, if any, produced catalase?

J. Which of the organisms, if any, produced acetoin?

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Experiment Methyl Red Voges-Proskauer Test

Antibiotic Sensitivity Cynthia Alonzo, M.S. Version 42-0238-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will use the Kirby-Bauer method to test the sensitivity of Staphylococcus epidermidis to the antibiotics gentamicin, novobiacin, and penicillin. Students will learn about the various types of antibiotics and how they affect bacteria. Students will learn the most common mechanisms through which bacteria become resistant to antimicrobial agents.

ExpErimEnt

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objectives ● Understand the basic principles of antimicrobial therapy

● Become familiar with the phenomenon of antibiotic resistance

● Become familiar with and employ an antibiotic sensitivity test

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Experiment Antibiotic Sensitivity

materials

MATEriAlS QTY iTEM DEScriPTioN Student provides 1 Distilled water

1 10%-bleach Solution 1 Paper towels 1 Stove 1 Incubator 1 Culture: S. epidermidis 1 Prepared nutrient agar dish

LabPaq provides 1 Gloves, disposable (1 pair) 1 Goggles, safety 1 Apron, plastic

1 Ruler, Metric 1 Tweezers, plastic 1 Pencil, marking

1 Antibiotic Disk – Gentamicin in Bag 2″x 3″ 1 Antibiotic Disk – Novobiacin in Bag 2″x 3″ 1 Antibiotic Disk – Penicillin in Bag 2″x 3″

1 Sterile Swabs – 2 per Pack 1 Mask with Ear loops (11) in Bag 5″ x 8″

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment Antibiotic Sensitivity

Discussion and review Antimicrobial therapy is the use of chemicals to inhibit or kill microorganisms in or on the host. Drug therapy is based on selective toxicity, which means the agent used must inhibit or kill the microorganism in question without seriously harming the host.

In order to be selectively toxic, a therapeutic agent must interact with some microbial function or microbial structure that is either not present or is substantially different from that of the host. For example, in treating infections caused by prokaryotic bacteria, the agent may inhibit peptidoglycan synthesis or alter bacterial (prokaryotic) ribosomes. Human cells do not contain peptidoglycan and possess eukaryotic ribosomes. Therefore, the drug shows little if any effect on the host (selective toxicity).

Eukaryotic microorganisms, on the other hand, have structures and functions more closely related to those of the host. As a result, the variety of agents selectively effective against eukaryotic microorganisms such as fungi and protozoans is small when compared to the number available against prokaryotes. Also keep in mind that viruses are not cells and, therefore, lack the structures and functions altered by antibiotics, so antibiotics are not effective against viruses.

There are two general classes of antimicrobial agents based on origin:

● Antibiotics: Substances produced as metabolic products of one microorganism which inhibit or kill other microorganisms.

● Antimicrobial chemicals: Chemicals synthesized in the laboratory which can be used therapeutically on microorganisms.

Today the distinction between the two classes is not as clear, because many antibiotics are extensively modified in the laboratory (semi-synthetic) or even synthesized without the help of microorganisms.

Most of the major groups of antibiotics were discovered prior to 1955, and most antibiotic advances since then have come about by modifying the older forms. In fact, only three major groups of microorganisms have yielded useful antibiotics: the actinomycetes (filamentous, branching soil bacteria such as Streptomyces), bacteria of the genus Bacillus, and the saprophytic molds Penicillium and Cephalosporium.

To produce antibiotics, manufacturers inoculate large quantities of medium with carefully selected strains of the appropriate species of antibiotic-producing microorganism. After incubation, the drug is extracted from the medium and purified. Its activity is standardized, and it is put into a form suitable for administration.

Some antimicrobial agents (penicillins, cephalosporins, streptomycin, and neomycin) are cidal in action: they kill microorganisms. Other antimicrobial agents (tetracyclines, gentamicin, and sulfonamides) are static in action: they inhibit microbial growth long enough for the body’s own defenses to remove the organisms.

Antimicrobial agents also vary in their spectrum. Drugs that are effective against a variety of both Gram-positive and Gram-negative bacteria are said to be broad spectrum (tetracycline, streptomycin, cephalosporins, ampicillin, and sulfonamides). Those effective against just Gram-

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Experiment Antibiotic Sensitivity

positive bacteria, just Gram-negative bacteria, or only a few species are termed narrow spectrum (penicillin G, clindamycin, and gentamicin).

If a choice is available, a narrow spectrum is preferable since it will cause less destruction to the body’s normal flora. In fact, indiscriminate use of broad spectrum antibiotics can lead to superinfection by opportunistic microorganisms, such as Candida (yeast infections) and Clostridium difficile (antibiotic-associated ulcerative colitis), when the body’s normal flora is destroyed. Other dangers from indiscriminate use of antimicrobial chemotherapeutic agents include drug toxicity, allergic reactions to the drug, and selection for resistant strains of microorganisms.

Following are examples of commonly used antimicrobial agents arranged according to their modes of action:

● Antimicrobial agents that inhibit peptidoglycan synthesis: Inhibition of peptidoglycan synthesis in actively-dividing bacteria results in osmotic lysis. These include penicillins, cephalosporins, carbapenems, monobactems, carbacephem, vancomycin, and bacitracin.

● Antimicrobial agents that alter the cytoplasmic membrane: Alteration of the cytoplasmic membrane of microorganisms results in leakage of cellular materials. These include polymyxin B, amphotericin B, nystatin, and imidazoles.

● Antimicrobial agents that inhibit protein synthesis: These agents prevent bacteria from synthesizing structural proteins and enzymes. These include rifampins, streptomycin, kanamycin, tetracycline, minocycline, doxycycline, and gentamicin.

● Antimicrobial agents that interfere with DNA synthesis: These agents inhibit one or more enzymes in the DNA synthesis pathway. These include norfloxacin, ciprofloxacin, sulfonamides, and metronidazole.

A common problem in antimicrobial therapy is the development of resistant strains of bacteria. Most bacteria become resistant to antimicrobial agents by one or more of the following mechanisms:

● Producing enzymes which detoxify or inactivate the antibiotic such as penicillinase and other beta-lactamases.

● Altering the target site in the bacterium to reduce or block binding of the antibiotic, which produces a slightly altered ribosomal subunit that still functions but to which the drug cannot bind.

● Preventing transport of the antimicrobial agent into the bacterium, which produces an altered cytoplasmic membrane or outer membrane.

● Developing an alternate metabolic pathway to bypass the metabolic step being blocked by the antimicrobial agent and overcome drugs that resemble substrates and tie up bacterial enzymes.

● Increasing the production of a certain bacterial enzyme, which overcomes drugs that resemble substrates and ties up bacterial selection of antibiotic resistant pathogens at the site of infection – indirect selection enzymes.

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Experiment Antibiotic Sensitivity

These changes in the bacterium that enable it to resist the antimicrobial agent occur naturally as a result of mutation or genetic recombination of the DNA in the nucleoid, or as a result of obtaining plasmids from other bacteria. Exposure to the antimicrobial agent then selects for these resistant strains of organism.

The spread of antibiotic resistance in pathogenic bacteria is due to both direct selection and indirect selection. Direct selection refers to the selection of antibiotic-resistant normal floras within an individual any time an antibiotic is given. At a later date, these resistant normal floras may transfer resistance genes to pathogens that enter the body. In addition, these resistant normal flora may be transmitted from person to person through such means as the fecal-oral route or through respiratory secretions. The direct selection process can be significantly accelerated by both the improper use and overuse of antibiotics.

For some microorganisms, susceptibility to antimicrobial agents is predictable. However, for many microorganisms there is no reliable way of predicting which antimicrobial agent will be effective in a given case. This is especially true with the emergence of many antibiotic-resistant strains of bacteria. Consequently, antibiotic susceptibility testing is often essential in order to determine which antimicrobial agent to use against a specific strain of bacterium.

Several tests may be used to tell a physician which antimicrobial agent is most likely to combat a specific pathogen.

● Tube dilution test: In this test, a series of culture tubes are prepared, each containing a liquid medium and a different concentration of an antimicrobial agent. The tubes are inoculated with the test organism and incubated. After incubation, the tubes are examined for turbidity (growth). The lowest concentration of antimicrobial agent capable of preventing growth of the test organism is the Minimum Inhibitory Concentration (MIC).

● The Minimum Bactericidal Concentration (MBC) is determined by subculturing tubes showing no turbidity into tubes containing medium but no antimicrobial agent. MBC is the lowest concentration of the antimicrobial agent that results in no growth (turbidity) of the subcultures. These tests, however, are rather time-consuming and expensive to perform.

● The Kirby-Bauer test (agar diffusion test): The Kirby-Bauer disc diffusion method is commonly used in clinical labs to determine antimicrobial susceptibility. In this test, the in vitro response of bacteria to a standardized antibiotic-containing disc is correlated with the clinical response of patients given that drug.

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Experiment Antibiotic Sensitivity

Figure 1: Agar Diffusion Test

In the development of this method, a single high-potency disc of each chosen chemotherapeutic agent was used. Zones of growth inhibition surrounding each type of disc were correlated with the minimum inhibitory concentrations of each antimicrobial agent (as determined by the tube dilution test). The MIC for each agent was then compared to the usually-attained blood level in the patient with adequate dosage. As a result, the categories of Resistant, Intermediate, and Sensitive were established.

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Experiment Antibiotic Sensitivity

Exercise 1: Antibiotic Sensitivity Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic. Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter-free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

procedure Pre-Experiment Preparation: Place the stock culture of S. epidermidis in an incubator 12–24 hours prior to the start of the experiment.

Part i: Kirby-Bauer Test

1. Disinfect the work area.

2. Use the extra nutrient agar dish prepared in the Isolation of Individual Colonies experiment. Using a sterile swab, thoroughly coat the surface of the agar with liquid S. epidermidis. Do not leave any un-swabbed areas on the agar dish.

3. After swabbing the dish, turn it 90o and repeat the swabbing process. It is not necessary to re-moisten the swab.

4. Run the swab around the circumference of the dish. Then soak the swab in the 10%-bleach solution and discard it. Let the dish dry upright for 5 minutes to allow the S. epidermidis culture to absorb completely.

5. Using a marking pencil, divide the outside bottom surface of the dish into three triangular segments similar to Figure 2.

Figure 2: Petri Dish Segments

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Experiment Antibiotic Sensitivity

6. Label the first section novobiocin; the second penicillin, and the third gentamicin.

7. Wash the tweezers with detergent, rinse well, and shake dry. Use the tweezers to transfer the gentamicin antibiotic disk to its corresponding section on the surface of the agar. Transfer the novobiocin and penicillin disks to their appropriate sections on the agar.

8. Lightly touch each disc with the tweezers to ensure each is in good contact with the agar surface.

9. Incubate the agar dish upside down at 35oC–37oC for 24–48 hours.

10. You will not need the S. epidermidis stock culture for future experiments. Mix 1 tablespoon of bleach into the stock culture and let it stand for at least 30 minutes to ensure all organisms have been destroyed. Then discard the contents.

Part II: Data Interpretation

To interpret the results:

1. Place the metric ruler across the zone of inhibition at the widest diameter and measure from one edge of the zone to the other. Note: Holding the dish up to the light may help.

a. The disc diameter will be part of the measurement.

b. If there is no zone at all, record the measurement as 0, even though the disc itself is approximately 7 mm.

Figure 3: Zone of Inhibition 2. Record the zone diameter in millimeters.

3. Locate the zone on the following Antibiotic Susceptibility Zone: Diameter Interpretation chart to determine if S. epidermidis is sensitive, resistant, or intermediate.

4. Soak the dish in 10%-bleach solution for 1 hour and then discard it.

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Experiment Antibiotic Sensitivity

Questions A. Define the term selectively toxic. Why is it an important feature of antimicrobial agents?

B. What are broad and narrow spectrum antimicrobials? What are the pros and cons of each?

C. What is direct selection?

D. What is the difference between an antibiotic and an antimicrobial chemical?

E. What is the mode of action for each of the following:

a. bacitracin:

b. nystatin:

c. tetracycline:

d. ciprofloxin:

F. Describe three mechanisms by which microbes might become resistant to the action of an antimicrobial drug?

G. Why do you think neglecting to finish a prescribed course of antibiotics might contribute to the rise of antibiotic resistance?

H. What is a tube dilution test? How is it used to determine susceptibility?

I. Define the following:

a. Minimum Inhibitory Concentration (MIC):

b. Zone of Inhibition:

J. What were the results of the Kirby-Bauer test for S. epidermidis?

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Experiment Antibiotic Sensitivity

Antibiotic Sensitivity Cynthia Alonzo, M.S. Version 42-0238-00-01

lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

observations

Table 1: Antibiotic Susceptibility Zone: Diameter Interpretation

Zone Diameter Standards (mm) Control Zone Diameter Limits (mm)

Antibiotic Name Antibiotic code resistant intermediate Susceptible E. coli 25922)

S. ureus (25923) other

Amikacin AN-30 <14 15-16 >16 19-26 20-26 P. aerugi-nosa 18-26

Ampicillin AM-10 16-22 27.35 H. influen-zae for gram-enterics <13 14-16 >17 for staphylococci <28 >29 for enterococci <16 >17

for Listeria monocytogenes <19 >20

for Haemophilus species <18 19-21 >22 13-21

Erythromycin E-I5 22-30 S. pneu-moniae for S. pneumoniae <15 16-20 >21 25-30

for other organisms <13 14-22 >23

gentamicin

GM-120 GM-10

H. influen- zaefor testing

enterococci with high level resistance

<6 7-9 >10

<12 13-14 >15 19-26 19-27 16-21 for other organisms

Kanamycin K-30 <13 14-17 >18 17-25 19-26

lincomycin

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Experiment Antibiotic Sensitivity

Neomycin N-30 <12 13-16 >17 17-23 18-26

Novobiocin NB-30 <17 18-21 >22 22-31

oxacillin OFX-5 18-24 S. pneu-moniae

for staphylococci <10 11-12 >13 8-12

for S. pneumoniae >20

Penicillin P-10 26-37 N. gonor-rhoeae for staphylococci <28 >29 26-34 for enterococci <14 >15 for L. monocytogenes <19 >20

for N. gonorrhoeae <26 27-46 >47

Polymyxin B PB-300 <8 9-11 >12 12-16 –

Streptomycin

S-300 S-10

for testing enterococci for high level resistance

<6 7-9 >10

for other organisms <11 12-14 >15 12-20 14-22

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Experiment Antibiotic Sensitivity

F. Describe three mechanisms by which microbes might become resistant to the action of an antimicrobial drug?

Bacitracin: Nystatin: Tetracycline: Ciprofloxin:

Questions A. Define the term selectively toxic. Why is it an important feature of antimicrobial agents?

B. What are broad and narrow spectrum antimicrobials? What are the pros and cons of each?

C. What is direct selection?

D. What is the difference between an antibiotic and an antimicrobial chemical?

E. What is the mode of action for each of the following:

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Experiment Antibiotic Sensitivity

G. Why do you think neglecting to finish a prescribed course of antibiotics might contribute to the rise of antibiotic resistance?

H. What is a tube dilution test? How is it used to determine susceptibility?

I. Define the following:

J. What were the results of the Kirby-Bauer test for S. epidermidis?

Minimum Inhibitory Concentration (MIC) –

Zone of Inhibition –

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Experiment Antibiotic Sensitivity

Microbes in the Environment Cynthia Alonzo, M.S. Version 42-0247-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will identify environmental sources of microbes, learn about microbial adaptability and scientific significance, and classify microorganisms. Students will grow microorganisms obtained from soil, water, and air in order to view the vast variety of microorganisms found within these environments.

ExpErimEnt

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objectives The student will have the opportunity to:

● Gain an appreciation for the adaptability and importance of microbes.

● Identify environmental sources of microbes.

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Experiment Microbes in the Environment

materials

MATEriAlS QTY iTEM DEScriPTioN Student provides 1 Distilled water

1 10%-bleach solution 1 Paper towels 1 Marker, black, permanent 1 Samples: Soil and water from the bottom of a ditch or

pond LabPaq provides 1 Cup, Plastic, 9 oz Tall 1 Gloves, Disposable

3 Petri dish, 60 mm 2 Pipet, Long Thin Stem 2 Agar, Nutrient – 18 mL in Glass Tube 1 Mask, Face with earloops

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment Microbes in the Environment

Discussion and review The many and varied metabolic activities of microbes assure they participate in chemical reactions in almost every environment on earth. They require an energy producing system to sustain life and nutrients, including liquid water, in order to grow and reproduce. Since microbes have been present on Earth longer than other organisms, they have evolved the ability to thrive in almost any environment that meets these minimal criteria.

Microorganisms are classified as either heterotrophs which derive energy from preexisting organic matter; or autotrophs which derive energy from one of two sources, light (photosynthesis) or the oxidation of reduced molecules. Oxidizable molecules may be organic or a variety of inorganic molecules such as sulfur, iron, hydrogen, carbon monoxide, ammonia, or even a combination of organic/inorganic molecules. Autotrophs and heterotrophs can be further divided into the following four subcategories:

● Photoautotrophs: Use light as an energy source and CO2 as a carbon source

● Photoheterotrophs: Use light as an energy source and reduced organic compounds as a carbon source

● chemoautotrophs: Use inorganic chemicals as an energy source and CO2 as a principal carbon source

● chemoheterotrophs: Use organic compounds as an energy source as well as a principal carbon source

Microorganisms can reproduce by doubling in approximately 20 minutes or by dividing only once in 100 years. In most natural environments, such as soil or lakes, the average generation time is approximately one day. Microbes are estimated to comprise one-third or more of Earth’s biomass. On average, bacteria are found in concentrations of up to 106 cells/mL of surface water, and up to 109 cells/mL of soil or sediment.

Microbes have a significant impact on the natural world including:

● Production of oxygen: Almost all of the production of oxygen by bacteria on Earth today occurs in the oceans by the cyanobacteria (blue-green algae).

● Soil fertility maintenance: Decomposition releases mineral nutrients such as potassium and nitrogen from dead organic matter, making it available for primary producers to use. Primary production of organic material would not be possible without the recycling of mineral nutrients. Decomposition also produces CO2 and CH4 that is released into the atmosphere.

● Nitrogen fixation: Bacteria are the only organisms capable of removing N2 gas from the atmosphere and fixing it into a useable nitrogen form (NH3).

● Base of ocean food chain: Plankton are the most numerous organisms in Earth’s oceans and include the protists, algae, and phytoplankton that comprise the basis of the marine food chain.

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Experiment Microbes in the Environment

Exercise 1: Microbes in the Environment Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic. Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter-free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

procedure Pre-Experiment Preparation: Prepare four Petri dishes with agar prior to the start of the experiment. Refer to the Preparation of Solid Media section in the Introduction for further instruction.

Part i: Microbes in the Air

1. Label the bottom of three prepared dishes: air, water, and soil. Set aside the dishes labeled soil and water for Parts II and III. You will have an extra dish if you wish to test an additional environmental source!

Figure 1: Labeled Petri Dishes

2. Choose a location in your home and leave the agar dish labeled air uncovered for 1–2 hours.

3. Close the dish and incubate it upside down at room temperature for 24–72 hours.

4. Observe the dish and count the number and types of colonies. Record the results in Data Table 1.

Data Table 1: Environmental Colony Formation

Location Number of colonies Number of types Description of Colony Morphology

Air Water Soil

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Experiment Microbes in the Environment

5. Soak the dish in a 10%-bleach solution for 1 hour and then discard it.

Part ii: Microbes in the Water

1. Choose an environmental site to collect a water sample, such as a pond, puddle, birdbath, or stream.

2. Use a sample cup to collect the water sample. Stir the water to mix any sediment or edge bacteria before sampling.

3. With a pipet, inoculate the agar dish labeled water with the water sample. Use only enough water to cover the top surface of the dish (approximately 4 drops).

4. Cover the dish and let it sit for 30 minutes to ensure the water soaks into the agar.

5. Incubate the dish upside down at room temperature for 24–72 hours.

6. Observe the dish and count the number and types of colonies. Record the results in Data Table 1.

7. Soak the dish in a 10%-bleach solution for 1 hour and then discard it.

Part iii: Microbes in the Soil

1. Choose an environmental site to collect a soil sample. Then use a new sample cup to collect a soil sample.

2. Pour distilled water into the cup, so the water sits just above the soil level. Mix the water and soil well.

3. Let the sample sit until the soil settles to the bottom of the cup.

4. With a pipet, collect a sample of the water layer on top of the soil and inoculate the agar dish labeled soil. Use only enough water to cover the top surface of the dish (approximately 4 drops).

5. Cover the dish and let it sit for 30 minutes to ensure the water soaks into the agar.

6. Incubate the dish upside down at room temperature for 24–72 hours.

7. Observe the dish and count the number and types of colonies. Record the results in Data Table 1.

8. Soak the dish in a 10%-bleach solution for 1 hour and then discard it.

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Experiment Microbes in the Environment

Questions A. List five environments in which you are likely to find microbial life.

B. What is the difference between an autotroph and a heterotroph?

C. Define the following:

● photoautotroph:

● photoheterotroph:

● chemoautotroph:

● chemoheterotroph:

D. How plentiful are bacteria in water? In soil?

E. What is nitrogen fixation? What role do microbes play?

F. How do microbes contribute to soil fertility?

G. Describe what type of growth you observed in the air dish (i.e., number of colonies, shape, color, defining characteristics, etc.).

H. Describe what type of growth you observed in the soil dish (i.e., number of colonies, shape, color, defining characteristics, etc.).

I. Describe what type of growth you observed in the water dish (i.e., number of colonies, shape, color, defining characteristics, etc.).

J. Did you see the same or different types of microbes in each dish? Explain your answer.

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Experiment Microbes in the Environment

Microbes in the Environment Cynthia Alonzo, M.S. Version 42-0247-00-01

lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

observations

Data Table 1: Environmental Colony Formation Location Number of colonies

Number of types Description of Colony Morphology

Air Water Soil

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Experiment Microbes in the Environment

Questions A. List five environments in which you are likely to find microbial life.

B. What is the difference between an autotroph and a heterotroph?

C. Define the following:

1. Photoautotroph

2. Photoheterotroph

3. Chemoautotroph

4. Chemoheterotroph

D. How plentiful are bacteria in water? In soil?

E. What is nitrogen fixation? What role do microbes play?

F. How do microbes contribute to soil fertility?

G. Describe what type of growth you observed in the air dish (number of colonies, shape, color, defining characteristics, etc.).

H. Describe what type of growth you observed in the soil dish (number of colonies, shape, color, defining characteristics, etc.).

I. Describe what type of growth you observed in the water dish (number of colonies, shape, color, defining characteristics, etc.).

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Experiment Microbes in the Environment

J. Did you see the same or different types of microbes in each dish? Explain your answer.

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Experiment Microbes in the Environment

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AppenDix

Preparation of Cultures Culture tubes should remain lidded while incubating. Do not open them once inoculated unless under aseptic conditions and to perform a necessary experimental step.

1. Saccharomyces cerevisiae: Add 1/2 teaspoon dry Saccharomyces cerevisiae (active dry yeast envelope) to 1/8 cup warm water (you can use a sample cup or any household cup) and gently swirl to mix. Set the culture aside to activate for at least 10 minutes. Stir to mix prior to using.

2. Escherichia coli:

a. Remove the tube labeled: Broth, Nutrient – 5 mL in Glass tube, from culture media bag #2 from the refrigerator and allow it to come to room temperature.

b. Moisten a paper towel with a small amount of alcohol and wipe the work area down.

c. Once the nutrient broth media is at room temperature:

i. Remove the numbered E. coli culture tube from the cultures bag and remove its cap. Set the cap upside down to avoid contamination.

ii. Uncap the nutrient broth; set its cap upside down to avoid contaminating it while the broth is open.

iii. Use sterile techniques and draw 0.25 mL of the nutrient broth into a sterile pipet. NOTE: To sterilize the pipet draw a small amount of 70% alcohol into the bulb and then expel it into a sink. Remove any excess alcohol by forcefully swinging the pipet in a downward arch several times to ensure that the pipet is dry before drawing up the nutri- ent broth. Add the broth to the vial containing the lyophilized E. coli pellet. Recap the E. coli vial and shake to mix until the pellet has dissolved in the broth. Note that the vial should be about one-half full to allow for shaking and mixing the pellet.

iv. Once the pellet has dissolved, use the same sterile pipet to draw up the E. coli solu- tion and expel it into the original tube of nutrient broth. Recap the broth. NOTE: If the pipet has become contaminated, simply draw a small amount of 70% alcohol into the bulb, and then expel it into a sink. Remove any excess alcohol by forcefully swinging the pipet in a downward arch several times to ensure that the pipet is dry before drawing up the E. coli solution.

d. Recap the nutrient broth and incubate the now E. coli inoculated tube of nutrient broth at 37°C. The culture should show active growth between 24 to 48 hours; it can be left as a liquid culture or plated out. Most freeze dried cultures will grow within a few days however some may exhibit a prolonged lag period and should be given twice the normal incubation period before discarding as non-viable.

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3. Lactobacillus acidopholis: Remove a tube of MRS broth from the refrigerator and allow it to come to room temperature. Aseptically transfer a portion of a capsule of L. acidopholis into the tube of media. To do so, sterilize your work area with alcohol and allow to dry. Carefully open the capsule and divide the contents between the two capsule halves. Set one half aside on the sterile work area and get the test tube of MRS broth. Open the tube and flame the top. Allow the tube to cool for a few seconds before transferring the contents of half of the capsule to the test tube. Carefully swirl the tube to remove any powder from the sides, then flame the top and close the tube. Close the capsule and set aside in case you need to start a new culture. Allow the tube to set, swirling periodically, as the powder dissolves. There will be a significant amount of sediment in the bottom of the tube. Mark the level of the sediment with a marker pencil or pen. Incubate the inoculated tube at 37°C. The culture should show active growth between 24 to 48 hours. Refer to Experiment 3 for a description of indicators of growth. L. acidopholis often sediments as it grows. An increase (above the sediment line you marked on the tube) in the sediment is an indication of growth. Swirl the tube to mix the organisms back into the broth prior to use.

4. Staphylococcus epidermidis: You can culture S. epidermidis as a liquid or solid culture. Because you are inoculating from an environmental source (your skin) your sample may contain bacteria other than S. epidermidis. Thus, broth cultures derived directly from sampling may not be pure cultures of S. epidermidis. With the exception of Experiments 3 and 4 (#3 establishes a broth culture and #4 uses it to establish a pure culture), use the dish culture method to ensure you are using a pure sample for your experiment.

5. Broth cultures of S. epidermidis: Without contaminating the cotton tip, cut the length of swab such that it will fit entirely into a capped test tube. Dampen the cotton tip sterile swab with distilled water and rub it vigorously on your skin. Do not try to obtain a bacterial culture soon after washing your skin. Additionally, choose an area that is not as likely to have been scrubbed recently (the inside of the elbow or back of the knee is generally a good site). Do not obtain a sample from any bodily orifice (mouth, nose, etc.) as you are not likely to culture the desired microbe (Staphyloccocus epidermidis). Using aseptic technique, place the swab into a tube of nutrient media, label the tube accordingly. Incubate the inoculated tube at 37°C. The culture should show active growth between 24 to 48 hours. Refer to Experiment 3 for a description of indicators of growth.

6. Dish cultures of S. epidermidis: Use a sterile swab to obtain a sample of S. epidermidis from your skin described in the generation of a broth culture. Rub the swab lightly on the surface of one dish of nutrient agar to inoculate it with S. epidermidis. As the swab may not contain a high number of bacteria, be sure to rub all sides of the swab on the dish to transfer as many individual bacterium as possible. Incubate the dish at 37°C for 24 to 48 hours. The S. epidermidis culture was not a pure culture (derived from a single organism) and will most likely contain colonies from several different organisms. You will need to identify and select a colony. Staphylococci produce round, raised, opaque colonies, 1 – 2 mm in diameter. S. epidermidis colonies are white in color. Below is a picture of S. epidermidis grown on blood agar.

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As the sample is of human origin, it potentially contains bacteria that can act as opportunistic pathogens. Do not select or use any colony that does not appear to be S. epidermidis. If your dish contains colonies other than S. epidermidis, soak it in a 10%-bleach solution and discard. Do not attempt to save the dish for use in future experiments!

You can either use the S. epidermidis colonies directly or amplify growth in a broth culture. If you choose to amplify into nutrient broth, 24 hours beginning the experiment, choose a S. epidermidis colony from the incubated dish and aseptically transfer the colony using an inoculation loop into a tube of nutrient media. Be sure to mix the broth gently to disburse the clumped bacteria into the broth. Incubate the tube at 37°C for an additional 24 hours.

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Preparation of Disinfecting Solution When working with live organisms, always disinfect your work area and tools prior to use. Soak experiment tools in disinfectant for 30 minutes, and then rinse the tools with distilled water to remove any chemical residue. Both alcohol and bleach are good choices for disinfectants. However, due to the dilution factor when disinfectant solution is added to broth cultures, use undiluted bleach when disposing of cultures.

When mixed with water, alcohol is an effective disinfectant. The water prevents organism cells from dehydrating and allows the alcohol component to enter the cell and denature the cellular proteins. 70% alcohol mixtures are capable of killing most bacteria within 5 minutes of exposure. The primary disadvantages of using 70% alcohol as a disinfectant are that it is ineffective against spores and has limited effectiveness against many viruses. Alcohol is also flammable and should not be used near a flame source. Rubbing alcohol, which is a 70% isopropyl alcohol solution, is readily available at most drug stores and is safe for contact with the skin.

Bleach is also a strong and effective disinfectant. Its active ingredient, sodium hypochlorite, denatures protein in micro-organisms and is effective in killing bacteria, fungus, and viruses. Household bleach works quickly and is widely available at a low cost. Exercise caution when using bleach as bleach irritates mucous membranes, the skin, and the airway. Bleach also decomposes under heat or light and reacts readily with other chemicals. Improper use of bleach can reduce its effectiveness in disinfection and can be harmful to your health. Bleach solutions begin to lose effectiveness after 2 hours, so you will need to make a fresh solution for each experiment.

Diluted Bleach Solution Preparation

● A 10% bleach solution is one part bleach to every nine parts water. For a spray bottle that holds 100 mL, add 10 mL liquid bleach to 90 mL of water.

● Keep windows open when using bleach to ensure good ventilation.

● Take care not to splash or inhale fumes when using bleach. The fumes irritate mucous membranes, the skin, and the airways. Wear gloves and an apron to protect your skin and clothes when preparing and using bleach solutions.

● Use cold water for dilution. Hot water can release some of the chlorine in the bleach as a gas, and the fumes can irritate your respiratory system.

● Spray your work surface thoroughly with the bleach solution and wipe it down with paper towels before and after every experiment.

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Precautions

● Avoid using bleach on metals, wool, nylon, silk, dyed fabric, and painted surfaces.

● Avoid touching the eyes. If bleach gets into the eyes, immediately rinse the eyes with water and continue rinsing for at least 15 minutes. Consult a doctor if needed.

● Bleach should not be used or mixed with other household detergents. Mixtures reduce the bleach’s effectiveness in disinfection and may cause harmful chemical reactions (e.g., a toxic gas is produced when bleach is mixed with ammonia or acidic detergents). Chemical reactions could result in accidents and injuries. If necessary for disinfection, use detergents first and then rinse thoroughly with water before rinsing with diluted bleach.

● Undiluted bleach liberates a toxic gas when exposed to sunlight, so it should be stored in a cool and shaded place out of reach of children and pets.

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Final Cleanup Instructions Congratulations on completing your science course’s lab assignments! We hope you had a great science learning experience and that what you have learned in this course will serve you well in the future. Studying science at a distance and performing laboratory experiments independently are certainly not easy tasks, so you should be very proud of your accomplishments.

Since LabPaqs often contain potentially dangerous items, it is important that you perform a final cleanup to properly dispose of any leftover chemicals, specimens, and unused materials. Please take a few minutes to protect others from possible harm and yourself from future liability by complying with these final cleanup instructions.

While you may wish to sell your used LabPaq, this is not advisable and would be unfair to a potential purchaser. It is unlikely that a new student trying to utilize a used LabPaq would have adequate quantities or sufficiently fresh chemicals and supplies to properly perform all the experiments and to have an effective learning experience. Further, it is doubtful that adequate safety information would be passed on to a new student in the same way it was presented to you. This is a significant concern and one of the reasons why a new user would not be covered by LabPaq’s insurance. Instead, you would be responsible for any problems experienced by a new user.

chemical Disposal

● Due to the minute quantities, low concentrations, and diluted and/or neutralized chemicals used in LabPaqs, it is generally sufficient to blot up any remaining chemicals with paper towels and dispose of them in a trash bin or flush remaining chemicals down a drain with copious amounts of water. Empty dispensing pipets and bottles can be placed in a normal trash bin.

● These disposal methods are well within acceptable levels of the waste disposal guidelines defined for the vast majority of state and community solid and wastewater regulations. However, since regulations can vary in some communities, if you have any doubts or concerns, you should check with your area authorities to confirm compliance with local regulations and/or if assistance with disposal is desired.

Specimen and Supply Disposal

● To prepare any used dissection specimens for normal garbage disposal, wrap them in news or waste paper and seal them in a plastic bag before placing them in a securely covered trash container that will prevent children and animals from accessing the contents.

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● Non chemical supplies can also be discarded with household garbage, but should first be wrapped in news or waste paper. Place such items in a securely covered trash container that will prevent children and animals from accessing the contents.

Lab Equipment

● Many students choose to keep the durable science equipment included with their LabPaq as most of these items may have future utility or be used for future science exploration. However, take care to store any dangerous items, especially dissection knives and breakable glass, out of the reach of children.

● Please do not return items to LabPaq as we are unable to resell items or issue any refunds.

Best wishes for a happy and successful future!

The LabPaq Team

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