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Summary Safe Microbiological Techniques
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This is an extensive summary of the course Safe Microbiological Techniques. The summary is complete and written in English.
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Samenvatting Safe Microbiological Techniques (SMT)PRACTICALS
General
Experiments:
- Skin flora
- Disinfection of surfaces
- Survival of E.Coli in cloth
- Isolation of bacteria in a pure culture
- Heat sterilization using an autoclave
Objective
Becoming aware of the microbial world around us, possible routes of contamination, how to
prevent contamination routes and how to eradicate contamination directly.
- Reproduce the rules for working according to SMT (Safe Microbiological Techniques) and
shows that he/she can observe these SMT rules while working in the laboratory.
- Explain how micro-organisms spread
- Explain why certain actions in the lab entail biological risks and how these risks can be
mitigated
- Explain how micro-organisms can be killed or removed and understand the potential pitfalls of
disinfection and sterilization.
- Practice aseptically and purifying techniques
Background information - SMT
Techniques that keep unwanted bacteria out of the sample
SMT: Safet Microbiological Techniques
Basic techniques: “working sterile”, as customary in microbiology
Now also prevent that genetically modified (micro-)organisms (GMOs) escape from the laboratory.
So: sterilize all biological waste, disinfect contaminated glassware and equipment.
Background information ML1
Experiments specifically related to SMT and work at
ML-1 level throughout the course.
ML-1: Microbes not known to consistently cause disease
in immunocompetent adult humans, of minimal potential
hazard to laboratory personnel and the environment.
Example: skin bacteria, yeast
ML-2: Microbes pose moderate potential hazard to
personnel and the environment
Examples: herpes simplex, common cld viruses (RSV,
rhinoviruses), salmonella
ML-3: Indigenous or exotic microbes that may cause serious or potentially lethal disease via
inhalation.
Examples: tuberculosis, SARS-CoV-2, highly pathogenic avian influenza, plague (Yersinia pestis)
ML-4: Exotic agents that pose a high risk of aerosol-transmitted laboratory infections and life-
threatening disease that is frequently fatal, for which there are no vaccines or treatments.
Examples: Ebola virus, smallpox virus
Pagina 1 van 25
,Background information - Ten commandments for safe microbiological techniques
1. (a) The activities may be carried out exclusively by individuals who have been given
permission by the Biological Safety Officer (Dutch: Biologische veiligheidsfunctionaris (BVF).
(b) Always follow the rules, even if you think there is no apparent risk.
2. Whilst working, all doors and windows must be closed. Verify that there are no insects and
other bugs in the work area.
3. (a) Wear a closed laboratory coat
(b) You may only wear this lab coat outside the work areas where parts of the experiments are
conducted. If the lab coat becomes contaminated due to spillage or an accident, it must be
decontaminated or sterilized before being washed
4. Decontaminate any spills immediately; serious contaminations should be reported
immediately to the Biological Safety Officer (sterilize by paper tissue and 70% ethanol)
5. It is absolutely prohibited to eat, drink or smoke in the lab, this includes taking cups, plates or
silverware with food or drink inside the work area
6. (a) Pipetting by mouth is prohibited
(b) Used pipettes are collected in a disinfecting liquid or autoclaved
7. Prevent aerosol formation. These may be created by: splashing drops, pouring of liquids,
discharging pipettes, opening of moist plugs, using inoculation loops that are too hot
(incorrect use). Use hypodermic needles only if no alternative is available.
8. (a) Glassware and instruments that have been in contact with GMOs have to be sterilized or
disinfected prior to being washed, reused or discarded
(b) Biological waste has to be collected in autoclavable waste bags, which are autoclaved
before discarding (use indicator tape to demonstrate that the bag was autoclaved)
Autoclave uses high pressure and temperature to kill off bacteria.
9. (a) Wash hands with soap and water after taking off your lab coat, work and before leaving the
room
(b) Daily clean and decontaminate work areas. Keep work area clean and organized
10. Record the general nature of the work clearly in a lab notebook
Skin flora
- Explain how micro-organisms spread
- Explain why certain actions in the lab entail biological risks and ow these risks can be mitigated
- Learn about the different nature of the bacteria that live in and on your skin.
- Learn about the efficacy of washing and disinfecting your hands.
Background information - skin microbiota
Distinguish “own” and “alien” microorganisms.
- Own (resident): e.g. Staphylococci (S.aureus, S.epidermidis)
- Alien (transient): many possibilities, cocci but also rod-shaped bacteria, yeasts and fungi.
Transfer by fingerprint
Analyse colonies
Pagina 2 van 25
, Background information - agar plates
Medium: “Bouillon” agar plates - mimics our skin
Bouillon —> Meat extract
Agar in a”plate is not the source of growth of the cells (inert)! —> Consist of agarose
- Only few bacteria can metabolize agar
- Dissolves at temperature of at least 95 ºC (gelatin at 30 ºC)
- Solidifies only at 45 ºC easy to pour plates without burning your hands
Procedure
Take a fingerprint 4 times only need to lightly press into agar
1. Before washing hands
2. After washing hands (2 minutes) and having airdrie fingers
3. After using disinfectant (take notes of how long it took to dry etc.)
4. After having done other activities for several hours
Reporting —> Lab journal (Take notes at home)
Diversion from protocol -> write it down
Include what materials you used e.g. brand/type of soap and disinfectant, Times etc.
Include notes in lab journal + formulate hypothesis
Results + Conclusion
Disinfection of surfaces
- Explain how micro-organisms can be killed or removed and understands the potential pitfalls of
disinfection and sterilization
- Learning the importance of disinfection of work surfaces
- Learn about the difference in efficacy of a soup solution and a disinfectant with different
bacteria
Background information - Gram character
Gram positive and Gram negative differs in cell
wall and cell membrane and can be detected by
using different dyes as indicators —> Crystal
Violet, Iodine, Alcohol or Safranin.
Gram positive bacteria have a single cell
membrane covered by a very thick layer of
peptidoglycan (cell wall).
Gram negative bacteria hav two membranes around the cell a plasma membrane and an outer
membrane with only a relative thin layer of peptidoglycan located in between the two membranes
Procedure
Make sure you have all the materials you require (check manual) because of the time points
Prepare the “working area” before handling the bacterial suspension
Purposely contaminate the lab bench with a bacterial culture in a controlled manner
Bacillus subtilis or Escherichia coli.
Mix the soap solution or disinfectant with the bacterial culture (sterile cotton swabs).
Take a sample of the mixture at t(time) = 0, 150 and 300 sec
Swipe the cotton swabs on a plate
Negative control (check for contamination)
Positive control (check if medium/culture work)
Leave the (easily identifiable) plates (upside down) for collection by the assistant (they will transfer
to 30 ºC incubator)
Disinfect bench surface with 70% ethanol
Pagina 3 van 25
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