The overall goal of the experiments this week is to remember/learn some of the
fundamental microbiological techniques that we will be using this semester.
Experiment 1 - Streaking for isolated colonies using toothpicks:
The goal here is to adapt the methods for streaking a plate to obtain isolated
bacterial colonies, to the use of toothpicks.
When performing bacterial genetics, one
must often streak out large numbers of colonies at a time.
Using individual sterile
toothpicks to perform the streaking greatly speeds this process compared with using
an inoculating loop that must be flamed and cooled at each step.
Instead, each time
a sterile tool is needed, a new toothpick is simply taken from the beaker.
attached procedure for “streaking a plate with toothpicks”.)
We use flat toothpicks
that have been sorted so all the “fat” ends point down in a 50 ml beaker.
sorting process is time consuming, and the toothpicks actually improve with age
(they accumulate bacterial cells and agar in them, and thereby become smoother,
and easier to streak with), we will routinely return the used toothpicks to a non-
sterile beaker for re-autoclaving.
You will first streak a bacterial strain onto a full
plate using toothpicks, to become acquainted with the technique.
As I mentioned
earlier, bacterial geneticists often need to streak out relatively large numbers of
colonies at a time.
In order to save plates, we often will streak up to 8 different
isolates on the same plate.
It is important to become proficient at obtaining
isolated colonies even when streaking in a small area of a plate (such as a 1/8th
We will practice this by streaking the same bacterial strain onto 6
individual sectors of a single plate - attempting to obtain isolated colonies in each
Experiment 2 - Patching:
Another very commonly used technique in bacterial genetics is “patching”.
involves drawing a small line of each of up to 100 different bacterial isolates onto
the surface of a single plate.
In this way, hundreds or thousands of different isolates
can be tested for a particular phenotype (such as, can they grow on a given type of
media, what is their color on a particular type of indicator media, etc.) relatively
quickly, and using a small number of plates.
In this technique, the goal is not to
obtain isolated colonies, but simply to grow a small sample of the strain in a defined
location on a plate.
In order to define the position of the individual isolates, a
template with 25, 50 or 100 evenly sized, numbered squares is placed under the
plate(s) onto which you wish to patch.
If you need to test the same isolates on more
than one type of media at a time (either to test multiple phenotypes, or to include a
control), then the same isolate is simply patched into the same numbered square on
each of the plates to be tested (i.e. - isolate #1 is patched into square 1 on each plate,
isolate #2 is patched into square 2 on each plate, etc.).
To make this process much