HandsOn 18 - Bacterial Growth
I. Bacterial Growth Preparation
II. Streaking and Innoculating Plates
III. Growth of Bacteria Under Starvation Conditions
IV. Staining Bacterial Colonies
II. Streaking and Innoculating Plates
Cultures ordered from a supply company or stock center will probably not consist of genetically identical bacteria. The bacteria will all be of the same species, and available as a single strain. However, random mutations may still exist due to the large number of bacteria present. To obtain a source of genetically identical bacteria, streak plates are used. Streaking a plate allows the bacteria to be spread out so that a single bacterium can be isolated from all other bacteria. This technique is called streaking for individual colonies. Since bacteria are so small, you will not be able to see that isolated bacterium. However, that bacterium will reproduce itself by binary fission (typical division time is on the order of 20 minutes), resulting in bacteria which are genetically identical to the original bacterium and to each other. These bacteria are visible as a small round colony growing where there had been one isolated bacterium. This method allows you to use the individual colony repeatedly and expect similar results.
There are several acceptable streak plate methods. The method described here is called the "T'' streak and is one of the easiest.
2. Use a marker or wax pencil to draw a T on the bottom of a plate
of nutrient agar. This divides the plate into three sections (Figure
. One section covers one half the plate. The other half is divided
into two quarters.
3. Sterilize the inoculating loop (Figure ), by holding its tip
in the flame until it turns red.
4. Lift up the lid of the plate you will be inoculating and poke
the inoculating loop through the agar close to the side of the
petri dish to cool it. This prevents the heat from killing the
bacteria sample you want to use. The heat will not harm the agar.
Try to lift the lid of the plate up only as much as is necessary
to put the loop inside. If you completely remove the lid, it can
become contaminated with bacteria from the environment.
5. Touch the loop to the edge of the colony growing on the plate.
Then take the loop and place the lid securely back on the plate.
6. Set the plate you will be streaking so that its bottom is sitting on the
bench top and you can see the T clearly. The largest section should be at
the top. Carefully lift up the lid and touch the inoculating loop to the upper
left hand corner of the largest section of the plate. Move the loop from left
to right, back and forth, across the surface of the agar. See Figure . Since
nutrient agar is a gel with properties similar to Jell-O, do not push down
with the loop or you will gouge the agar.
7. Replace the lid of the petri dish and flame the loop again
to kill any remaining bacteria on it. Rotate the plate 90 degrees
counterclockwise. Carefully lift the lid slightly and touch the
loop into the left side of the plate which contains the area you
streaked in the previous step. Move the loop across the surface
of the agar until it is in the smaller section in the upper right
of the plate. Within that quarter of the plate, move the loop
back and forth across the agar surface (Figure ).
8. Repeat Step 7 as shown in Figure .
9. Replace the lid of the petri dish and flame the loop again
to kill any remaining bacteria on it.
10. Seal the petri dish with a layer of parafilm around the edge.
This keeps the agar from drying out while it is in the incubator.
Incubate the streak plate at 37°C until you can see
individual colonies. Make sure to keep an open beaker of water
in the incubator. Periodically check that the beaker has water
in it-do not let it run dry. The water will maintain a constant
level of humidity (100%) in the incubator. See Figure .
2. Use a marker or wax pencil to place a dot in the center of
the outside bottom of the petri dish of nutrient agar. Now turn
the plate over so that the bottom is sitting on the bench top.
Notice that you can see the dot you just made through the agar.
This will help you place the bacteria on the surface of the nutrient
agar's center.
3. Sterilize the inoculating needle by placing the tip of the
needle in the flame. Keep the tip there until the metal turns
red.
4. Choose a streak plate containing an individual colony. Lift
up the lid of the streak plate you will be inoculating from, and
poke the inoculating needle into the agar close to the side of
the plate to cool it. This prevents the heat from killing the
bacteria sample you want to use. The heat will not harm the agar.
Lift the lid of the plate up only as much as is necessary to put
the needle inside. If you completely remove the lid, it may become
contaminated with bacteria from the environment.
5. Touch the inoculating needle to an individual colony growing
on the plate. Take care not to stab the inoculating needle down
into the agar. Then remove the inoculating needle from the plate
and place the lid securely back on the plate.
6. Carefully lift up the lid of the plate you are inoculating
onto. Touch the inoculating needle to the very center of the surface
of the nutrient agar. The dot you drew on the bottom should make
it easier to locate. Be careful that you do not stab the inoculating
needle into the nutrient agar.
7. Place the lid on the plate, and flame the inoculating needle
to kill any remaining bacteria.
8. Seal the plate with a layer of parafilm around the edges. This
keeps the nutrient agar from drying out while it is in the incubator.
Make sure to keep an open beaker of water in the incubator. Periodically
check that the beaker has water in it - do not let it run dry.
The water will maintain a constant level of humidity (100%) in
the incubator.
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