In this video, we will demonstrate how to streak bacteria on LB agar plates so that the bacteria
form monoclonal colonies, that can be used in later experiments. Though simple in concept, the procedure
that you follow can make this process go very right, or very wrong.
When growing bacteria from
glycerol stocks or bacterial stabs, it's very important to start liquid cultures from single
colonies struck on plates. Why? While the majority of bacterial cells in the stock or stab
should be genetically identical, there may be the occasional mutant hidden among the population. If you
were to simply take a glob of bacteria from this mixed population and grow it in a liquid culture,
one of the mutants could potentially take over the culture and affect your experiment. We solve
this problem by streaking for single colonies.
Starting liquid cultures from these colonies, will
generally lead to expected plasmid yields and more consistent results in later experiments.
Before we begin, we'll need to gather the necessary materials. You will need:
Bacterial glycerol stock or stab, with your strain of interest.
LB Agar plate, with the appropriate antibiotic.
Sterile toothpicks or sterile loop.
Bunsen burner or other flame.
Dry incubator at 30 or 37 degrees Celsius.
And, good sterile technique.
First, we take out our LB agar plate with the appropriate antibiotic, making sure
that there is no contamination on the plate and that it is dry. If the plate has condensation, you
can place it next to a flame with the lid open for about 10 minutes to dry it out. Next, we take
a look at our stab to make sure that there's visible bacterial growth.
If nothing has grown on the stab, it's unlikely that anything will grow on the plate.
Now, we take a sterile toothpick and
insert it into the stabbed culture dabbing at regions with bacterial growth
This could also be done with a glycerol stock
by immersing a sterile toothpick into the semi-frozen solution.
Remember that you don't need a giant blob of cells.
A toothpick that looks clean is likely sufficiently covered in bacteria.
With the tip of the toothpick now covered in bacteria, we can begin streaking.
Be sure to be very gentle, and not to stab the plate with the toothpick, or you'll dig up some agar during the streaking process.
With the first toothpick, we gently streak back and forth across roughly 1/3 of the plate.
Taking care to avoid the edges. We then dispose of the first toothpick, turn the plate, and use a new, sterile toothpick
to start streaking a new path from where we left off. Again, we streak over 1/3 of the plate and end the path near the edge.
We then repeat this process one final time, with a third, sterile toothpick. Although we must be careful,
the whole process can be performed quite quickly.
Remember to place the plate upside down so that condensation
doesn't drip onto the colonies and disturb them. Now, that we've completed our streaks,
we can close the plate and place it in the incubator overnight or for at least 12 hours.
As you can see, our careful streaking has resulted in individual colonies that can be picked for
a wide variety of downstream experiments. For optimal bacterial streaks with single colonies,
always remember these key points:
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