Working with microbes: Safety and aseptic technique
All the microbial
experiments in this lab will involve one species of bacteria: Escherichia coli. E. coli
K12 is a Risk Group 1 organism that can be worked with in an open lab, as long
as proper aseptic technique is followed.
Disposal of waste
Disposable materials (e.g. micropipette tips, toothpicks, disposable plastic inoculation
loops, gloves, plastic cuvettes) contaminated
with Risk Group 1 organisms can be disposed of in the buckets containing clear plastic bags on your bench. Do not
put any glass in the buckets on the bench.
Contaminated pipettes
should be disposed of in the rectangular
white boxes on your bench. Culture supernatants contaminated with
microbes should be poured or pipetted into an
Erlenmeyer flask. Contaminated flasks, tubes, and plates that have had microbes in
them should be discarded in the appropriate containers located near the door of
the lab. Tape labels should be removed from glassware before disposal. Do not pour contaminated media, buffers, etc.
out of flasks or tubes unless it is necessary for the experiment.
Aseptic Technique
Aseptic technique is a set
of procedures that must be followed when working with microbes to prevent the
contamination of the experimenter, the environment, or the experimental
cultures. Bacteria worked with in a lab should always be treated as potential
pathogens, so it is important not to expose yourself accidentally to these bacteria. It is also
important to avoid the release of experimental cultures into the lab
environment. These strains may contain
genetic markers such as antibiotic resistance, which should not be released
into the environment. Finally, it is important
for the experimental cultures themselves not to become contaminated with
microbes from the environment, which will result in confusing and invalid
experimental results. Microbes are
widely distributed in dust, skin, dandruff, and nasal secretions, so it is very
easy for cultures to become contaminated unless steps are taken to minimise the
risks of these agents coming in contact with experimental materials.
Procedures
1. Lab coats must be worn.
2.
3.
Avoid touching your face or mouth while in the lab. This will reduce the risk of
infecting yourself, and also the risk of contaminating your cultures with
dandruff.
4.
Do not touch any contaminated materials or the contaminated parts of
equipment. Also avoid touching any
materials or equipment parts that are supposed to be sterile or that will come
into contact with your experimental cultures.
5.
If you have any cuts, scrapes, burns etc. let the TAs know about it
before or during the lab.
6. Wipe down bench with disinfectant (ethanol)
before and after lab work.
7.
Wipe up any spills or drips
(including drips on the lips or sides of a flask or tube resulting from
pouring) using a paper towel freshly
soaked in ethanol. Soak any
contaminated broken glassware with ethanol, and notify one of the TAs about it.
8.
Avoid creating aerosols by spraying liquid out of a pipette. The pipette tip should be placed against the
side of the flask or tube while liquid is being ejected.
9. When inoculating, pipetting,
or pouring material from a glass tube or flask, reduce the risk of
contamination by passing the rim of the flask or tube briefly through a Bunsen
burner flame before and after the
transfer. If you are pouring from a
tube or flask, allow the rim to cool for a second before letting liquid come
into contact with it. If a drop remains
on the rim of the tube or flask after pouring, wipe it off using a paper towel freshly soaked in ethanol before
flaming the rim. This avoids the
creation of aerosols by the splattering of boiling liquid. Do not
flame pipettes, plastic tubes, or other plastic equipment.
10. Glass or metal spreading rods are used
to spread liquid cultures evenly over the surface of agar plates. These rods are sterilized by dipping in
ethanol, tapping off the excess, and then igniting the ethanol in a Bunsen
burner flame. Let the rod cool for a
couple of seconds before touching the agar surface. This will avoid creating aerosols from the
liquid cultures that the plate has been inoculated with. Do not hold the burning rod over the ethanol
stock, and avoid lifting the burning end above the end you are holding. (This may allow burning ethanol to run down
onto your fingers.) If you do ignite the
ethanol stock, quickly cap it to smother the flames.
11. Pour
plates are prepared using 2-3 ml top agar overlays. These can be prepared in glass tubes and
sterilized ahead of time. When needed,
the tubes are melted in a microwave or boiling water bath, and then cooled to
45-50 °C
before use.
Add bacterial cells to tubes containing 2-3 ml top agar. Gently mix the tubes, and immediately pour onto prewarmed LB plates. Spread overlay across the plate by tilting and rotating the plate until overlay is evenly distributed. The rim of the sample tube can also be used to spread overlay and pop any bubbles that are present. Do not attempt to spread overlay further once it starts to set. This will produce a grainy, opaque overlay, which will make colonies difficult to see.
12.
The longer that experimental cultures or equipment are in contact with
the open air, the greater the chance that they will become contaminated by dust
or aerosols. Because of this, transfers
between tubes and/or plates should be carried out as quickly as safety and
accuracy allow. This can be accomplished
by understanding what you have to do
before opening the tube or plate, and by not leaving the tube or plate open between transfers. In addition, sterile equipment should be kept
sterile until right before use. The best
way to do this is to leave the equipment in its package (e.g. wrapper or closed micropipette tip box) until you
are ready to carry out the transfer. Do not set sterile or contaminated
equipment down on your lab bench or other surfaces. This
includes caps or plugs for culture tubes or flasks, which should be held
with the little finger of your transfer hand during transfers. (The transfer hand is the one holding the pipettor or inoculating loop.)