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.

1. Light a Bunsen burner in your bench space. To maintain sterile conditions, inoculation should occur within 20 cm of the flame. Wait 20 seconds before opening the petri dish and inoculating. This gives the flame time to sterilize the local air. Remember that you want to achieve sterile conditions. Do not work with the plate close to your face. This will violate the sterile environment.


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.

Figure 5.2: Draw a "T'' on the bottom of your petri dish as shown.


3. Sterilize the inoculating loop (Figure ), by holding its tip in the flame until it turns red.

Figure 5.3: Wire innoculating loop and needle used in this experiment.


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.

Figure 5.4: Touch the inoculating loop to the upper left hand corner and then move it across the agar from left to right as shown.


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 ).

Figure 5.5: Touch the loop to the area previously streaked and then move the loop across the agar as shown.


8. Repeat Step 7 as shown in Figure .

Figure 5.6: Touch the loop on the previously streaked area. Then move the loop across the agar onto the third area as shown.


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 .

Figure 5.7: Incubate the streak plate until you can see individual colonies. Make sure to keep an open beaker of water in the incubator.

1. As in plate streaking, light a Bunsen burner in your bench space and work within 20 cm of the flame to maintain sterile conditions. Again, wait 20 seconds before opening the petri dish and inoculating.


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.

Caution: When disposing of unwanted bacterial colony plates, you must first autoclave them. This will kill the bacteria, and make the plates safe for disposal in a regular trash bag.

Previous: I. Bacterial Growth Preparation