Contaminations and what to do about them

Good microbiological techniques

Contaminations can happen, but the best way to deal with them is to avoid them. Always use good microbiological techniques.

Aseptical techniques

• Work with sterilized materials. This includes all consumables, toothpicks, media, pipettes, supplements, etc. For sterilization see autoclaving and filter sterilization.
• Sanitize before starting: clean everything well with Bacillol AF or 70% ethanol.
• Clean hands and pipettes.
• Clear the clutter! Work in a clean space!

Culturing techniques

• Work in a hood or next to a flame
• Think about every step: what is sterile should stay sterile!
• Pour plates and tubes using sterile methods
• use proper techniques such as streaking, patching, spreading (with beads or spatula) or replica plating in the appropriate ways (ask if you don't know!)

Storage

• Store plates in a clean place, upside-down and
• Freeze strains at -80°C in your personal box. Things won't get contaminated there.
• Trash what is no longer needed. Old plates are mold magnets and yeast will die at some moment anyways.

Preventing contaminations

When performing less sterile methods, such as cell sorting using FACS antibiotics can be added preventively to avoid bacterial contaminations. We have experienced that the cell sorters are not sterile, and will cause bacterial growth.

Penicillin-Streptomycin

The most commonly used broad spectrum antibiotic for cell culture is Penicillin-Streptomycin. This antibiotic does not greatly affect yeast growth or reproduction.

For use, add the antibiotic in 1:100 dilution from the stock solution (Thermofisher 10.000 units/ml Penicillin & 10.000 µg/ml Streptomycin) to the medium (when using agar, assure it has cooled down to below 60°C). Aliquots are available in the top drawer of freezer 7.

Ampicillin

You can use ampicillin, however there is more natural resistance in bacteria present, so this might not work that well.

For use, add the antibiotic in 1:1000 dilution from the stock solution (100 mg/mL) to the medium (when using agar, assure it has cooled down to below 60°C). Aliquots are available in the top drawer of freezer 7.

Kanamycin

The story for kanamycin is similar to that for ampicillin.

For use, add the antibiotic in 1:1000 dilution from the stock solution (100 mg/mL) to the medium (when using agar, assure it has cooled down to below 60°C). Aliquots are available in the top drawer of freezer 7.

Contaminations in strains

If you have strains that are essential and cannot be recreated, you need to figure out a way to get rid of the contamination. If this is in liquid, I think you don't have to try, but when grown on a plate, there are some possibilities.

Bacterial contaminations

When your sample is contaminated with bacteria (check under the microscope), you have a few options.

• Streak out your cells on a plate with a broad spectrum antibiotic (see above), incubate them, pick a single colony, and repeat if needed.
• If the contamination is very severe, you can first 'wash' away some of the bacteria, which might inhibit growth, by resupending the yeast in PBS or sterile water, shortly spinning down the cells in the tiny table top centrifuge, pipetting off the water/PBS (the bacteria will not pellet down as quickly as the yeast) and streaking out the yeast on a plate with antibiotic.

Yeastie contaminations

Sometimes, contaminations with some random budding yeast (common skin commensals) can occur. These are much more difficult to get rid off, so best to not get contaminations at all.

Some possible solutions:

• Streak out the yeast, and hope you can morphologically identify the pombe colonies. Pombe forms colonies that are much taller and more dome shaped than most other yeast species. Check under the stereo microscope for the right type. Repeat and hopefully you will only see pombe colonies. Verify by making a slide and checking under the microscope. Confirm by checking genetic markers if available.
• If your strain happens to have a resistance marker in its genome, you might be in luck! You can grow your cells on a plate with the corresponding antibiotic and kill other strains like that. Continue and verify as described above.

Moldy contaminations

If at all possible, do not open your plates when you have molds growing on it, especially not when it is sporulating. If sporulating, parafilm it close and dispose for autoclaving. Spores in a lab are a pain to get rid off, so try to not let them escape!

If you really need to open the plate, do this in the down flow. Potential spores will be caught and should end up in the hepa filter. Sterilize the hood after use with ethanol and/or UV. Carefully take cells and plate them on a new plate. If you have a resistance marker in the yeast, see above for using antibiotics.

If the mold is small and not sporulating, and you need the plate (e.g. transformation or spores germinating), you can cut out a piece of agar in a wide circle around the mold and hope for the best. Individually wrap the plate (to avoid spreading the contamination), and place the plate back in the incubator... fingers crossed.