DIYBIO: Streaking Plates to Isolate Colonies

Isolated colonies. Also note initial thick streaks in to the top of the plate, and then the less continuous sreaks in the bottom right, and the thinnest concentration of isolated colonies on the bottom left

So, you want to isolate that plastic degrading, or bioluminescent, or plasmid-bearing microbe from the rest of a mixed sample.  Or maybe you want to grow up a colony from a single bacteria for a clean PCR sample, or to inoculate a liquid culture.  To do this you will need to streak a plate!  There are many techniques for this, but there are really two key ideas:

  • Work cleanly; don’t contaminate your sample.  Use an open flame
  • You want to thin out the amount of bacteria on your loop

My technique for this is to heat my loop up to orange-hot, then cool it in the agar of the sample I am taking the colony from, being careful not to touch any of the colonies on the plate.  Once quenched (So I don’t heat-kill the bacteria),  I rub it on the target colony or area on the sample plate.  Then I close the sample plate and open the target plate.  I like to do 3 streaks.  The first one is to spread out the bacteria I picked up on the top 1/3 of the plate.  Once this is done, I sometimes flame and cool the loop in the target agar, although it is ok to skip this step.  Either way, I then make another zig-zag, starting in the area that I spread the bacteria in, and then moving out onto an unused third of agar.  The last streak takes up the last third, and starts in the last area I covered.

Good, isolated colonies

If everything works out, you should have isolated colonies like in the picture above.  A few common pitfalls are:

Clear streaking pattern, but there are too many bacteria, and it looks like I overlapped my first zig-zag with my last zig-zag! oops.

  1. TOO MUCH bacteria.  Bacteria are very small, and if you pick up too many, you will end up with a bacterial “lawn”, which is useless for isolating colonies.
  2. Accidentally killing your sample bacteria by sticking your red-hot loop into them (doh!).
  3. Streaking back into an already streaked area.  This defeats the purpose of streaking, which is to spread out the bacteria.  If you go back from a low concentration area to a high concentration area, and then back to the low concentration area, you risk bringing extra concentrated bacteria that would form a lawn into an area where you want single colonies.

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