Day 1: Syllabus, Safety, and Movie introduction to the microbiological laboratory




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GENERAL MICROBIOLOGY LAB MANUAL


Joe Corsini

With contributions from Mike Bynum and Kitty Canaday


Helpful Information for the Students


There are 18 scheduled laboratory sessions for this semester. Each session will have a background, exercise, and questions to be completed. The lab exercises will be available to you on-line. Read through your lab exercise before the lab session begins. Review your past labs often for quizzes.

When taking notes for this class, be sure to write down all of your observations. These observations should include the microbe(s) you are studying, how it was prepared, its characteristics (including size, shape, color, and smell), and any other information that would help you study for quizzes and test. When using a microscope, it is helpful to write down the magnification used next to any drawings you made of the microbes.

There are texts available to you in the laboratory. Familiarize yourself with the Bergey’s and Difco Manuals. These texts give specific information about the microbes you will be studying, including their distinguishing characteristics. This information, if learned alongside your experiments, will make the search for your “unknown” at the end of the semester much easier.

In the microbiology lab, many experiments will continue on through more than one lab session. Keep good records of each experiment separate from other experiments to avoid confusion, because the experiments may not be mentioned again, but you are still responsible for the results on quizzes and final exam.


Day 1: Syllabus, Safety, and Movie




INTRODUCTION TO THE MICROBIOLOGICAL LABORATORY


You will need a notebook in this lab. Record in detail all observations, as they will be helpful when attempting to identify the organisms in your unknown sample.

This lab has a Biosafety Level 2 designation that you will better understand as we proceed through the semester (for more information on this topic, visit the website for the Centers for Disease Control and Prevention at www.cdc.gov.) Many of the techniques that you will learn in this laboratory, you may use within your future career--you will finish this semester with the basics to begin work in any microbiology laboratory in the world.




SAFETY – READ THROUGH LIST


In the microbiology laboratory, safety is extremely important. There is a great potential for fire, spills, and other accidents. Around the laboratory are various safety devices available to you, such as a fire extinguisher, fire blanket, first aid kit, eye wash solution, and more. Take time to familiarize yourself with the laboratory so that if an accident does happen, you will be able to utilize the safety features.


The organisms you will be working with are either non-pathogenic or pathogenenic only in unusual circumstances. However, all specimens need to be handled as if they were pathogenic. By learning these procedures, referred to as sterile technique, you can protect yourself from exposure to disease agents in the clinic.


How do you protect yourself and others (including your instructor!!) from microbes in the laboratory?

a) Wash your hands when you enter and exit the laboratory, and immediately after your hands have been contaminated with microbes.

b) Wear your lab coat at all times within the lab. There may be times that you will need to wear your goggles, too.

c) Do not eat, drink, or chew gum in the laboratory; keep any object, including pencils and pipettes, out of your mouth.

d) Avoid touching your hair, cheek, lips, eyes, or any other part of your body within the laboratory.

e) Be CAREFUL!!! Don’t talk to your neighbor while performing inoculations.

f) No mouth pipetting.


How do you protect yourself from injury?

a) Do not wear sandals or open-toed shoes in the laboratory.

b) Tie long hair back so that it will not touch the counter surface or be set on fire from the Bunsen Burners in this lab.

c) Some of the equipment you will be using will need to be autoclaved. The professor will inform you when this should be performed.

d) If there is broken glass in the lab, inform instructor. DO NOT TOUCH BROKEN GLASS WITH YOUR HANDS. The glass should be swept up and if it contained microbes, it should be autoclaved before thrown away in the glass disposal bucket.

e) If you spill microbial culture on the counter, squirt 10% bleach onto spill and then cover with paper towels; finish cleaning up after about 20 minutes (this time lapse ensures that most of the microbes have been successfully killed). Inform instructor about any spills

f) Avoid inhaling the chemicals you will be using in the lab. Some of these chemicals are cancerous; the professor will point out the dangerous ones.

g) Do not make off with the 95% ethanol with the idea of concocting jungle juice for a party. It has been denatured with methanol and is very toxic.

h) Other important considerations: If you have allergies to penicillin or any other compound, notify your professor immediately; we may be working with a potential allergen or its derivative and you need to be aware of this. Also, if you are pregnant, notify the professor to ensure that you are not exposed to any microbes or reagents that might put you or fetus at risk. If you have any medical conditions that might endanger yourself or others in the laboratory, please discuss the situation with your instructor. Along that same line, in the interests of your safety and that of your neighbors, if you have a history of seizures, fainting, or blacking out, let the professor know. This information will be kept confidential.




EXERCISE - Watch video ‘The domains of Life’ and ‘Ebola: The Plague Fighters’.



Lab Prep

1) 36 Nutrient agar plates, 20 Trypticase Soy Agar plates, 20 YPD agar plates

2)Streak plate of Saccharomyces cerevisiae (YPD agar),Cosmarium species, Chlorella vulgaris in Alga-Gro liquid medium

3) Initiate cultures of all bugs to be used this semester: Rhodospirillum rubrum grows on Trypticase Soy Agar, Saccharomyces cerevisiae grows on YPD, Lactobacillus species grow on Tomato Juice Agar, Cosmarium and Chlorella vulgaris are propagated in liquid Alga Gro, and the remainder on Nutrient agar plates: Staphylococcus epidermidis,Staphylococcus aureus, Enterococcus (Streptococcus) faecalis, Saccharomyces cerevisiae, Chlorella vulgaris Rhodospirillum rubrum, Bacillus thuringiensis, Escherichia coli, Micrococcus luteus, Lactobacillus caesei,

4)Squirt bottle with 70% ethanol every table

5)Squirt bottle with distilled water at each table

6)Squirt bottle with 95% ethanol at each table

7)Squirt bottle with 10% bleach under the sink

8)Lens paper each cubby

9)Test tube racks to stay in lab for the term – 12

10)20 sterile test tubes, empty

11)20 test tubes each with 5 mls of dI water, autoclaved.

12)autoclave all pipettman tips and microcentrifuge tubes.

13)Bench top autoclave bags

14)Check the Gram’s reagents, fill if necessary. Crystal violet with ammonium oxalate is OK to use (actually better than without).



REFERNECES


Laboratory Experiments in Microbiology, 6th Edition, Johnson & Case, Benjamin Cummings (Addison Wesley Longman, Inc.), San Francisco, © 2001, pages 2-4.


Laboratory Manual and Workbook in Microbiology: Applications to Patient Care, 6th Edition, Morello, Mizer, & Wilson, McGraw-Hill Companies, Inc., Boston, © 1998, pages 3-5.


Microbiological Applications, 6th Edition, Harold J. Benson, Wm. C. Brown Publishers, Dubuque, © 1994, pages ix-x.


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