Culture culture - The care and handling of bacterial strains.

John Roth, 5/3/2000

The lab strain collection
The strain collection is the holy grail of bacterial genetics. It is the reference point for depositing new strains and the source of starting materials for building new strains. Reliable genetics depends on careful cataloguing and care of strains. The strain is part of the data and is essential to repeating any particular experiment. Any strain you build is a temporary, ephemeral entity until it is deposited into the collection and entered in the strain catalogue. The job isn't done until the strain is in the collection and the catalogue book. If you delay entering your strains, they are frequently forgotten and valuable material is lost

Avoid extensive private stock collections.
Even if you think a strain will be useful only to you and no-one else in the lab, the permanent collection is the safest place to keep it. Personal collections may be handy while a construction is in progress, but as soon as you are done, put the final product in the main collection.

Private collections tend to never be entered in main collection or to be entered hastily when the author is ready to leave the lab. This is dangerous because clerical mistakes often happen in the mad rush. Its easy to forget strains especially ones that didn't go on to fame and fortune. The strain is in best condition while you're using it and know its correct; that's the time to freeze it in the collection.

Everyone in the lab will work together to preserve and protect the main collection and its catalogue-- no one else is likely to care much about your private strain collection in a desk drawer or freezer box. You can always get the strain from our collection when you need it and you'll be able to find it easily if its been catalogued. It will be there when you want to set up a collection for your own independent lab.

Start with material from the collection.
Do this whenever building a new strain, rather than borrowing a bit from a neighbor in the lab who may or may not have cared for it or kept records properly. Borrowing a strain that hasn't been entered in the collection cuts you off from all hope of repeating the experiment because you can't ever be sure you can get the same strain again. Be sure your strains are right by taking care of the collection and taking your strains from this, our most reliable reference.

Choose strains that have been worked with most extensively
When selecting a strain from the collection, there are frequently several alternatives. Whenever possible, look through lab publications and choose old friends that have been used before. Alleles that appear in many strains are most likely to be ones that behave correctly and reliably. Many strains that have not been extensively characterized.are deposited in the collection for safe-keeping. For example, a large mutant hunt might yield many strains that are deposited without much detailed characterization. Strains that have been used previously are more likely to be correct and any unadvertised peculiarities are likely to have been encountered.

Place a newly-made strain in the collection immediately and refer to it in your notes by its new strain number and new allele numbers.
This saves later pedigree tracing and avoids clerical errors. When you want to start a new experiment using your strain, take it from the collection (rather than an old plate in the cold room or your private stock.) In this way, all your data is sure to be obtained for the same reference strain. Keeping a private strain collection runs the risk that you forget to enter a valuable strain that is no longer being used. A lost strain means you can't repeat an old experiment.

Enter gift strains promptly (with source indicated)
Strains sent to us by others are valuable gifts that we should treasure. Someone went to the trouble of packing and shipping. Asking for a second copy is an embarassing imposition.

Don't passage strains you are using every day.
It's tempting to streak a strain out and keep a plate of colonies in the cold room, using one as innoculum each time you start a new culture. This is poor practice because the strain is being passaged and perhaps subjected to unconscious selection each time you streak it out. Old colonies are subject to differential killing and selection even in the cold so a single colony doesn't represent a pure clone after sitting for weeks in the cold room. This is particularly bad for Green Plates, which are toxic. It's best to go back to a frozen culture or stab to start each culture.

Depositing and removing strains from the collection
Our strain collection is extremely valuable. It is part of the data for all previous work in this lab and is essential to you and all others in the lab for pursuit of any project. Treat the collection accordingly.
-- Remove only one box at a time and close the freezer while using it.
-- Be sure the box you take out is replaced promptly in the proper position.
-- If you note misplaced boxes, straighten out the problem.
-- Be sure the freezer door closes completely.
-- Note ice accumulation that might prevent the freezer lid from sealing and solve the problem. Don't leave it for someone else.
-- Note temperature of the freezers and to try and spot any mechanical problems before they reach crisis proportions.

When putting a strain in the freezer:
-- be sure it's phage-free
-- grow a fresh culture from a single colony
-- add 1ml to a vial with DMSO and vortex at least a minute to mix.
-- be sure the genotype is accurately entered in the stock book (don't reserve space with the intent of entering the details later-- it's easy to forget and then strain is lost because it can't been located in the collection.

When taking a strain out of the collection:
Streak on rich medium with no selection.

Remember that some strains may be fragile and have problems getting started. There problems are made worse if they are immediately subjected to selection for drug resistance or other properties.

Pick several colonies (5-10) and patch to non-selective medium to make a master that can be printed to several media to check individual phenotypes. Several colonies are picked to avoid the possibility of choosing a contaminant or an individual clone that had enhanced resistance to freezing or was put into the collection as a minority type.

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