LAB 1 Microbiology Lab Safety and General Procedures

Amy Turnbull

LAB 1 Microbiology Lab Safety and General Procedures

This lab has important safety information that will impact you throughout the course. Please read thoroughly, then complete the quiz on FOL to test your understanding.

Learning Objectives

Review the safety precautions taken in a level 2 microbiology lab.
Distinguish biohazard waste disposal practices of different materials.

Lab D3000-1 and -3 are biosafety level 2 labs. Safety procedures for microorganisms associated with human diseases or regarded as opportunistic pathogens are mandatory when working in this laboratory. Bacterial cultures should be handled with caution, and proper aseptic techniques should be followed to prevent contamination. For your laboratory procedures, you are required to come to the laboratory on time with the following:

Your laboratory goggles
Blue laboratory coat. This will be stored in a zip-top plastic bag (handed out during the first lab session) while you are not in lab.
Hardcover lab notebook.

Laboratory Safety and Rules for D3000

Your personal items, along with your partner’s items, will be stored in a locker. Reserve a locker through the Security Office in D1018 . The cost is $12 per term. Please bring a lock and inform your lab partner of the combination.
To protect you and other people working in the laboratory from contamination by microorganisms, stains or chemical reagents, you must wear a buttoned-up laboratory coat, gloves and goggles in the laboratory at all times unless otherwise indicated by the instructor.
Eating, drinking and chewing gum are not allowed in the laboratory. Avoid handling contact lenses, applying make-up in the laboratory, or touching your face. No articles should be placed in your mouth, including pens and pencils.
Telephone calls and texting are not permitted in the lab during laboratory sessions.
You can have your phone on you as we will learn how to take pictures of your results safely.
Be aware of the location of eye wash, shower stations, First Aid kit and Fire extinguisher in the event of an accident that requires the use of any of this equipment.
Shorts, short pants, sandals or open-toe shoes should not be worn in the laboratory. This is to protect you from any accidental dropping of objects that might results in serious injury.
Your work area should be disinfected before and after the experiment with the disinfectant provided for that purpose (70% reagent alcohol or isopropyl alcohol). Ensure your work area is cleaned, tidied-up and all equipment is returned to its appropriate storage cabinet at the end of the experiment. Laboratory coats, gloves and goggles should be worn when cleaning and disinfecting the work surface.
During most of the laboratory experiments, you will be working with open flames/Bunsen burners, it is important to use extreme caution when working with open flames and with alcohol.
Read and familiarize yourself with the weekly protocol before coming to the laboratory session.
Laboratory exercises will be due for submission to the FOL submission folder one week after lab session. Hand it in at the beginning of the next lab. Submissions up to 24 hours late will receive a 25% penalty. Labs handed in after 24 hours late will receive a mark of zero.

Violation of any of these rules at any time during the laboratory session will cause a loss of performance marks and you may be asked to leave the lab.

Good Microbiological Practices and Procedures

Best Practices in the Microbiology Lab

To ensure safe work and control of biological risk.
Never store food or drink, or personal items such as coats and bags in the laboratory.
Activities such as eating, drinking, smoking, and applying cosmetics are only to be performed outside the laboratory.
Never put materials, such as pens, pencils or gum, in the mouth while inside the laboratory, regardless of whether gloves are worn or not.
Wash hands thoroughly, preferably with warm running water and soap, after handling biological material and/or animals, before leaving the laboratory or when hands are known or believed to be contaminated.
Ensure open flames or heat sources are never placed near flammable supplies and are never left unattended.
Ensure that cuts or broken skin are covered before entering the laboratory.
Ensure that supplies are stored safely and according to storage instructions to reduce accidents and incidents such as spills, trips and falls.
Ensure proper labelling of all biological agents.
Protect written documents from contamination using barriers (such as plastic coverings or a physical space on the lab bench that doesn’t have biologicals), particularly those that may need to be removed from the laboratory.
Ensure that the work is performed with care and without hurrying. Avoid working when fatigued.
Keep the work area tidy, clean and free of non-essential objects and materials.
Prohibit the use of earphones, which can distract personnel and prevent equipment or facility alarms from being heard.
Cover or remove any jewelry that could tear gloves, easily become contaminated or become fomites. Cleaning and decontamination of jewelry or spectacles should be considered, if such items are worn regularly.
Refrain from using portable electronic devices (for example, mobile telephones, tablets, laptops, flash drives, memory sticks, cameras when not specifically required for the laboratory procedures being performed.
Keep portable electronic devices in areas where they cannot easily become contaminated or act as fomites that transmit infection. Where close proximity of such devices to biological agents is unavoidable, ensure the devices are either protected by a physical barrier or decontaminated before leaving the laboratory

Technical Procedures

Controlling risk through safe conduct of laboratory techniques.

Avoiding inhalation of biological agents

Use good techniques to minimize the formation of aerosols and droplets when manipulating specimens. This includes refraining from forcibly expelling substances from pipette tips into liquids, over-vigorous mixing, and carelessly flipping open tubes. Where pipette tips are used for mixing, this must be done slowly and with care. Brief centrifuging of mixed tubes before opening can help move any liquid away from the cap.
Avoid introducing loops or similar instruments directly into an open heat source (flame) as this can cause spatter of infectious material. Where possible, use disposable transfer loops, which do not need to be resterilized. Alternatively, an enclosed electric microincinerator to sterilize metal transfer loops can also be effective.

Avoiding ingestion of biological agents and contact with skin and eyes

Wear disposable gloves at all times when handling specimens known or reasonably expected to contain biological agents. Disposable gloves must not be reused.
Avoid contact of gloved hands with the face.
Remove gloves aseptically after use and wash hands as outlined below.
Shield or otherwise protect the mouth, eyes and face during any operation where splashes may occur, such as during the mixing of disinfectant solutions.
Secure hair to prevent contamination.
Cover any broken skin with a suitable dressing.
Prohibit pipetting by mouth.

Avoiding injection of biological agents

Wherever possible, replace any glassware with plastic-ware.
If required, use scissors with blunt or rounded ends rather than pointed ends.
If glassware must be used, check it on a regular basis for integrity and discard it if anything is broken, cracked or chipped.
Never use syringes with needles as an alternative to pipetting devices.
Never re-cap, clip or remove needles from disposable syringes.
Dispose of any sharps materials (for example, needles, needles combined with syringes, blades, broken glass) in puncture-proof or puncture-resistant containers fitted with sealed covers. Disposal containers must be puncture-proof/-resistant, must not be filled to capacity (three-quarters full at most), must be never reused and must not be discarded in landfills.

Preventing dispersal of biological agents

Discard specimens and cultures for disposal in leak-proof containers with tops appropriately secured before disposal in dedicated waste containers.
Waste containers are present at every workstation.
Regularly empty waste containers and securely dispose of waste. Ensure all waste is properly labelled.
Consider opening tubes with disinfectant-soaked pad/gauze.
Decontaminate work surfaces with a suitable disinfectant at the end of the work procedures and if any material is spilled.
When disinfectants are used, ensure the disinfectant is active against the agents being handled and is left in contact with waste materials for the appropriate time, according to the disinfectant being used.

Personal Protection

1. Laboratory coats must be worn at all times for work in the laboratory.

When putting on lab coats, care should be taken to minimize contact with the outer/exposed side of the material in case the material is contaminated from the previous use.
When removing lab coats, gloves should be removed first followed by the lab coat. Place the lab coat in the plastic bag then wash your hands.

2. Appropriate gloves must be worn for all procedures.

After use, gloves should be removed aseptically and hands must then be washed.
Disposable gloves should not be disinfected, for example, with ethanol, before starting work and they should not be reused as exposure to disinfectants and prolonged wear will reduce the integrity of the glove material. If disposable gloves become noticeably contaminated, they should be removed immediately and disposed in the red bin to prevent further contamination of other PPE, equipment and specimens.
To remove disposable gloves correctly, pinch the thumb and forefinger together on one hand. With the other hand, pinch the material just below the top of the cuff of the glove, and pull the glove down towards the closed thumb and forefinger, turning the glove inside out in the process. Stop once you have reached the thumb and forefinger so that the glove is only partially removed. Repeat on the other hand. At this point the gloves are both partially removed with the clean undersides of the gloves now forming the outer surface. The gloves can now be removed by touching only the clean underside of the material.
Used disposable gloves should be discarded in the red bin. Once gloves are removed hands must be washed. See Figure 8.1 on page 35 of the Personal Protective Equipment pdf posted on FOL for a visual of this process.

3. Eye protection

Lab glasses should be put on with clean hands (for example, not after handling microorganisms) to avoid contamination of the face and the eye protection itself.
Eye protection should be removed after taking off your lab coat with clean hands to avoid contamination of the head.

4. Hand washing

Personnel must wash their hands after handling infectious materials and animals, and before they leave the laboratory working areas.

5. It is prohibited to wear protective laboratory clothing outside the laboratory, e.g. in the hallway or washrooms.

You could contaminate non-lab environments with microorganisms.
Even if your coat is sterile, your appearance will scare others in the college!

6. Protective laboratory clothing that has been used in the laboratory must be stored in the plastic bag when not in use.

Disposal procedures in the lab

We make lots of waste in the lab and it is treated based on if the item is going to be reused and if it is contaminated.

Table 1.1. Waste disposal in the microbiology lab
Waste	Contaminated Y / N	Disposal location	Notes
Test tubes, flasks with culture	Y	In tube rack on side bench	Will be autoclaved by the lab tech. Do not rinse in sink.
Agar plates with culture, other plastics	Y	Orange biohazard bags on benchtop	Will be autoclaved by the lab tech. Pipette tips can go into small waste container first.
Paper towels from drying hands	N	Green bin
Liquid waste from Gram staining	N	Mixed inorganic waste in fumehood	If staining procedure kills microorganisms, waste will not be contaminated.
Liquid culture waste	Y	Biohazard waste in fumehood
Slides	Y	Glass tray by carboy	If live-stained or wet mount, it is contaminated
Gloves	Y	Red bin

Adapted from Laboratory Biosafety Manual. 2020. by World Health Organization, licensed under Creative Commons Attribution Non-Commercial ShareAlike 4.0 International License except where otherwise noted.

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