Aim of the Experiment: - To Understand the Basic Microbiology Laboratory Rules

A microbiology laboratory is a unique environment that requires special practices and

containment facilities in order to properly protect persons working with microorganisms. Safety

in the laboratory is the primary concern. In addition to microorganisms, there are some chemicals

used in this laboratory that are potentially harmful. Many procedures involve glassware, open

flames, and sharp objects that can cause trauma/damage if used improperly. The three main

elements of safe containment of microorganisms are:-

1. Good Laboratory Practices and Technique,

2. Safety Equipment

3. Facility Design.

Microbiology Lab Practices and Safety Rules: - “All microorganisms should be treated as

potential pathogens (organisms capable of producing disease)”. The following basic steps should

be observed at all times to reduce the ever-present microbial flora of the laboratory environment.

1. Upon entering the laboratory, place coats, books, and other paraphernalia in specified

locations—never on bench tops.

2. Wash your hands with disinfectant soap when you arrive at the laboratory and again

before you leave.

3. Do not smoke, chewing gum, eat, or drink in the laboratory. Do not put anything in your

mouth such as pencils, pens, labels, or fingers. These activities are absolutely prohibited.

4. You must wear closed shoes at all times in the laboratory setting. Sandals or open toed or

canvas shoes are not permitted because of the constant danger of cuts and infections from

broken glass found on the lab floors and the possibility of chemical spills.
5. A laboratory coat or apron may be necessary while working in the laboratory. Lab coats

protect clothing from contamination or accidental discoloration by staining solutions.

6. Do not place contaminated instruments, such as inoculating loops, needles, and pipettes,

on bench tops. Loops and needles should be sterilized by incineration, and pipettes

should be disposed of in designated receptacles.

7. Keep hands and other objects away from your face, nose, eyes, ears, and mouth. The

application of cosmetics in the laboratory is prohibited in the laboratory.

8. Wear a paper cap or tie back long hair to minimize its exposure to open flames.

9. Work areas/surfaces must be disinfected before and after use with 70% ethanol or fresh

10% bleach.

10. Laboratory equipment and work surfaces should be decontaminated with an appropriate

disinfectant on a routine basis, and especially after spills, splashes, or other

contamination.

11. Carry cultures in a test-tube rack when moving around the laboratory. Likewise, keep

cultures in a test-tube rack on the bench tops when not in use. This serves a dual purpose:

to prevent accidents and to avoid contamination of yourself and the environment.

12. Protective eyewear must be worn when performing any exercise or procedure in the

laboratory.

13. Wear disposable gloves when working with potentially infectious microbes or samples

(e.g., sewage). If you are working with a sample that may contain a pathogen, then be

extremely careful to use good bacteriological technique.

14. Label all materials with your name, date, and any other applicable information (e.g.,

media, organism, etc.).

dishes in the lab unless absolutely necessary.

16. Inoculating loops and needles should be flame sterilized in a Bunsen burner before you

lay them down.

17. Turn off Bunsen burners when not in use. Long hair must be restrained if Bunsen burners

are in use.

18. Treat all microorganisms as potential pathogens. Use appropriate care and do not take

cultures out of the laboratory.

19. Never pipette by mouth. Use a pipetting aid or adjustable volume pipettors. Consider

everything a biohazard.

20. Do not pour chemicals and bio-hazardous fluids down the sink.

21. Autoclave liquids and broth cultures to sterilize them before discarding.

22. Dispose of all solid waste material in a biohazard bag and autoclave it before discarding

in the regular trash.

23. Dispose of broken glass in the broken glass container.

24. Dispose of razor blades, syringe needles, and sharp metal objects in the “sharps”

container.

25. Report spills and accidents immediately to your instructor.

26. When handling chemicals, note the hazard code on the bottle and take the appropriate

precautions indicated.

27. Return all chemicals, reagents, cultures, and glassware to their appropriate places.

28. Glassware should be washed with soap and water, then rinsed with distilled water.
 29. Flame transfer loops, wires, or needles before and immediately after use to transfer

biological material.

30. Do not walk about the laboratory with transfer loops, wires, needles, or pipettes

containing infectious material.

31. Be careful around Bunsen burners. Flames cannot always been seen.

32. Report any broken equipment, immediately, report any broken glass, especially those

containing infectious materials.

33. Report all injuries or accidents immediately to the instructor.

34. Always wipe and clean the lenses of your microscope before putting it away. Use the

appropriate tissue paper and cleaning solution for this purpose.

35. Do not remove any materials from the laboratory without the written permission of the

course instructor or TA.

Laboratory Safety Equipment

Biological Safety Cabinet or Laminar Air Flow: - A biological safety cabinet (BSC) is used as

a primary barrier against exposure to infectious biological agents. A BSC has High Efficiency

Particulate Air (HEPA) filters. The airflow in a BSC is laminar, i.e. the air moves with uniform

velocity in one direction along parallel flow lines. Depending on the design, a BSC may be

vented to the outside or the air may be exhausted into the room. BSCs are not chemical fume

hoods. A percentage of the air is recirculated in most types of BSCs. HEPA filters only trap

particulates, allowing any contaminant in non-particulate form to pass through the filter.

Use of Laminar Air Flow: -

purge airborne contaminants.

b) Before and after use, wipe the surface of the BSC (Biological Safety Cabinet) with a

suitable disinfectant, e.g., 70% alcohol or a 10% bleach solution.

c) Place everything you will need inside the cabinet before beginning work, including a

waste container. You should not have to penetrate the air barrier of the cabinet once work

has begun.

d) Do not place anything on the air intake grills, as this will block the air supply.

e) You should prevent unnecessary opening and closing of door because this will disrupt the

air flow of the cabinet.

f) Always wear a lab coat while using the cabinet and conduct your work at least four

inches inside the cabinet.

g) Place burners to the rear of the cabinet to reduce air turbulence.

h) Do not work in the BSC while the ultraviolet light is on. Ultraviolet light can quickly

injure the eye.

i) When finished with your work procedure, decontaminate the surfaces of any equipment.

j) Remove the equipment from the cabinet and decontaminate the work surface.

k) Thoroughly wash your hands.

Biohazard Waste Disposal

Dispose of items in the special receptacles as indicated below. If you have a question regarding

the proper disposal of an item, please ask your course instructor or TA.

Material Method of Disposal

Agar slants with biological material. Place tube upright in indicated test tube rack,
 but place caps in basket as indicated.
Leave in container with closed cap and/or
Biological liquids (not in test tubes) follow specific instructions given by the TA
or course instructor.
Place tube upright in indicated test tube rack
Biological liquids in test tubes
with caps left on the tube
Broken glass (contaminated) Sharps container
Broken Glass (not contaminated) Broken glass container
Cotton Swabs (contaminated) Benchtop disinfectant/discard can
Needles, glass slides, syringes, pipettes, other
Sharps container
types of sharps
Non-contaminated paper Regular trash
Petri dishes and contaminated solids (other
Biohazard “orange/red bag” container
than pipettes or swabs
Transfer pipettes (contaminated) Bench-top disinfectant/discard can