INTRODUCTION:

LABORATORY OPERATIONS AND SAFETY

Safety is an important factor in any laboratory environment. In microbiology lab, safety is
essential. We will be using potential pathogens and precautions will be strictly followed.
The following section lists the safety rules appropriate for any laboratory. The procedures
and techniques you will learn here will be useful to you in other laboratory courses, at home
and in other workplace or years to come. Please review and familiarize yourself with these
procedures so that your laboratory experience will be enjoyable and safe.

Safety Rules:
Safety in the microbiology lab

I. If you have any condition which may predispose you to infection, such as pregnancy,
diabetes, CFIDS, HIV, asthma, chemotherapy or steroid treatment, please inform the
instructor.

II. GOOD LAB PRACTICES:

1. Table tops must be disinfected before and after each lab with disinfectant.
2. Hands should be washed before and after every experiment.
3. Dress appropriately, protective eyewear, gloves and lab coats are important barrier
precautions.
4. Do not wear sandals or open-toed shoes.
5. Long hair must be pulled back in a ponytail.
6. Smoking, eating and drinking in the laboratory are not permitted at any time.
7. Mouth pipetting is not permitted under any circumstances. Use the safety pipetting
devices which are provided.
8. Bunsen burners will be used to sterilize microbiological tools and tube tops. Many of
the stains we use contain flammable substances; orient flammable material away from
the burners.
9. The microscope issued to you is both an expensive and delicate instrument--treat it
accordingly. Always, at the end of each laboratory period, carefully clean oil from the
objective and condenser lenses, align the low power dry objective with the condenser
and rack condenser up and body tube down. Use only lens paper to thoroughly clean
prepared slides before putting them away. Q-tips and isopropanol should be used on
the oil immersion lens.

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 10. When finished for the day, dispose of all used glassware and cultures in the
appropriate receptacle, clear workbench and wash the top with a disinfectant. Never
pour a broth culture down the sink, place the used tube in the disposal rack. Wash
hands thoroughly with soap and water before leaving the laboratory.
11. Be sure all burners are turned off at the end of the laboratory period. Double check to
be sure that handles on all gas outlets are in the off position.
12. The inoculating needle should be heated until red hot before and after use. Always
flame needle before you lay it down.
13. Always place culture tubes of broth or slants in an upright position in a rack. Do not
lay them down on the table or lean them on other objects. They may roll onto the
floor and break.
14. All culture containers which are to be incubated should bear the following notations:
i) initials (or group),ii) specimen (name of organism or number of unknown) and iii)
date.

When using Petri plates, these notations should be entered on the bottom half, not the
lid. Unless otherwise directed, all plates are to be inverted, all plugged tubes should
have the plugs firmly set into the tubes, and all screw cap tubes should have the caps
loosened one-half turn to permit gas exchange.

15. The safest and smartest way to conduct a lab experiment is to read it before you begin
and prepare a flow sheet.

III. IN THE EVENT OF AN ACCIDENT:

1. Culture spills require immediate notification of the instructor. Paper towels soaked
with disinfectant are used to prevent aerosol formation.
2. If you burn yourself notify the instructor immediately.
3. Body fluids must be considered as potentially hazardous. If you or your lab partner
bleed for any reason notify the instructor immediately.
4. If a thermometer breaks, stay away from the mercury and notify the instructor
immediately. Mercury is considered a hazardous waste.

IV. SPECIAL PRECAUTIONS:

1. Be aware of the Biosafety and Microbial Risk level of all materials and microbes
used in labs.

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 2. A special precaution with the hood is important. Never turn on the ultraviolet
light. It is damaging to skin and eyes. The UV light is used by the instructor and
technician only.
3. Locate the fire extinguisher, shower, eye wash station, sinks, and disinfectant before
leaving the lab the first day of class.
4. Follow a proper steps in the event of an emergency

Laboratory Skills

A student successfully completing basic microbiology will demonstrate the ability to:

1. Use a bright-field light microscope to view and interpret slides, including

a. correctly setting up and focusing the microscope
b. proper handling, cleaning and storage of the microscope
c. correct use of all lenses
d. recording microscope observations

2. Properly prepare slides for microbiological examination including

a. cleaning and disposing of all slides
b. preparing smears from solid and liquid cultures
c. performing wet mount and/or hanging drop preparations
d. performing Gram stains

3. Properly use aseptic techniques for the transfer and handling of microorganisms and
instruments, including
a. sterilizing and maintaining sterility of transfer instruments
b. performing aseptic transfer
c. obtaining microbial samples

4. Use appropriate microbiological media and test systems, including

a. isolating colonies and/or plaques
b. maintaining pure cultures
c. using biochemical test medias
d. accurately recording macroscopic observations

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5. Estimate the number of microbes in a sample using serial dilution techniques,
including
a. correctly choosing and using pipettes and pipetting devices
b. correctly spreading diluted samples for counting
c. estimating appropriate dilutions
d. extrapolating plate counts to obtain the correct CFU or PFU in the starting sample

6. Use standard microbiology laboratory equipment correctly, including

a. using the standard metric system for weights, lengths, diameters and volumes
b. lighting and adjusting a laboratory burner
c. using an incubator

What to bring in the lab?

1. Lab coat with long sleeves.
2. Permanent marker
3. Two good morning towels
4. Lab manual

STUDENTS WEARING SANDLES OR SLIPPER ARE NOT ALLOWED TO ENTER

THE LAB.

ATTENDANCE IS COMPULSORY!

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