By way of introduction, Aunt Yersinia is bestowing 8 spores of knowledge garnered from her years at the bench.
I have been an aunt for almost 30 years now and, for a slightly shorter time, a molecular biologist of microorganisms. (No, I am not that old, I just started early in both).
My nephew has grown up and doesn’t need my advice. But people who use microbiological techniques such as molecular biologists, and especially biochemists, didn’t and do. They use E. coli to clone and purify protein but often know very little about basic microbiology and therefore make simple mistakes.
Here is my quick list on how to avoid the most frequent blunders:
The protocols for sterile techniques were developed when the lab was just a dusty room with single pane glass. In a modern lab with air filters in the air conditioning, there is no need to keep the Bunsen burner on all the time. Open flame, especially left unattended, sooner or later equals fire.
Likewise, “flaming the bottle/loop” was designed for glass bottles and metal loops. There is no point, and more than that, it is outright dangerous, to keep your modern plastic universals and single use loops in the flame until they start melting.
The cornerstone of a Good Microbiological Practice (GMP) is keeping your bugs as single colonies, rather than patches. Yes, it takes more time to streak them to single colonies, but then you can be absolutely sure that your cells are clonal (i.e. uniform) and there is no contaminating bugs lurking in your culture. Every time you do a “patch,” or don’t streak your bugs to single colonies after a transformation, bacteriology founder Robert Koch rolls in his grave.
If you open a sterile bottle of media, don’t put it back into the communal pool; it is yours forever. Before re-using a bottle of media, swirl it around. If it’s not crystal clear, ditch it – it is not worth possible contamination of your experiment.
If you are unsure of the sterility of your medium, do not re-autoclave or filter sterilize it – pour it off and make a fresh batch. You don’t know what the bugs living in it secreted into the medium.
Do not leave your old Petri dishes on the bench until they grow furry things, which tend to produce spores. Spores will fly around and contaminate yours and others’ cultures.
Do not grow your E. coli in a half-full flask. You will not get more cells, but fewer. Under low volume to surface ratio, air does not go deep into the culture and the nutrients are not well distributed. In this case, less is more – use 1/5 of the flask volume.
As soon as you break cells open, there is absolutely no need for sterile technique for any biochemistry or molecular biology work – save yourself the time and sterile plastic.
Happy bug growing.
Awaiting your microbiology and classic molecular biology questions, I remain faithfully yours.
Friends and colleagues are excellent sources of scientific information. The Internet is too, because it is a virtual library at your fingertips. If you are looking for a great solution to a common problem (i.e. how to eliminate RNAse contamination in a laboratory), the information is waiting for you. For help with more difficult problems […]
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