HandsOn 18 - Bacterial Growth

I. Bacterial Growth Preparation

II. Streaking and Inoculating Plates

III. Growth of Bacteria Under Starvation Conditions

IV. Staining Bacterial Colonies

I. Bacterial Growth Preparation

To minimize contamination of your experiment by the bacteria that live on and around us, please:

1. Wash your work area with a disinfectant detergent (e.g., vesphene) before beginning your experiment.


2. Wash your hands and wear gloves for the experiment.


3. Work within 20 cm of a Bunsen burner flame.


4. At the end of your experiment, wash your work area and your hands.

This experiment requires the following supplies and equipment:

• incubator (if available)
• autoclave for sterilization
• Bunsen burner with tubing (gas supply)
• balance (preferably accurate to 1/100 g)
• petri dishes
• laboratory gas lighter (striker)
• scissors
• markers or wax pencils
• nylon gloves
• goggles
• aluminum foil
• autoclave gloves
• beakers
• graduated cylinders
• inoculating needles and loops
• weighing dishes
• glass stirring rods
• parafilm
• autoclave tape (optional)

Chemicals for Nutrient Agar:

• Bacto Agar
• Bacto Peptone
• dibasic potassium phosphate (K₂HPO₄)
• monobasic potassium phosphate (KH₂PO₄)
• Sodium Chloride (NaCl)
• distilled water (H₂0)

Chemicals for an Alternative Nutrient Agar Luria-Bertani (LB):

• Bacto Agar
• Bacto Tryptone
• Sodium Chloride (NaCl)
• Yeast extract
• distilled water (H₂0)
• Bacterial samples such as Bacillus subtilis, Escherichia coli, and it Enterobacter aerogenes.

Bacteria can be purchased from supply companies (e.g., Carolina Biological Supply Company or Difco Laboratories). Specific bacteria can sometimes be obtained from stock centers. B. subtilis is available from the Bacillus Genetic Stock Center at Ohio State University and E. coli is available from the stock center at Yale University. Both stock centers provide the bacteria free to educational institutions. See the "Yellow Pages'' at the end of the book for information on suppliers.

Preparing Nutrient Agar Plates

Bacteria grow on nutrient agar. The formulas in this text are given for one liter solutions; smaller volumes than a liter can be made by scaling down the quantity of each ingredient by the same ratio as the volumes. For instance, if the formula for one liter of nutrient agar calls for 10.0 grams of agar, use 2.5 grams to prepare 250 milliliters of solution. To vary the nutrient levels in this experiment, vary the Bacto Peptone levels in the solution. The formula for the nutrient agar with 1.0 g/L of Bacto Peptone is:

Bacto Peptone Medium:

• 1.0 liter of distilled water
• 10.0 grams/Liter Bacto¨ Agar
• 5.0 g NaCl
• 5.0 g K₂HPO₄
• 2.0 g KH₂PO₄
• 1.0 g Bacto Peptone

For streak plates, you can use a concentration of 1.0 g/L Bacto Peptone. There are many other kinds of nutrient agar that can be used to obtain individual colonies. Since bacteria grow differently on different kinds of nutrient agar, you may find that one works better than another for you. One standard nutrient agar is Luria-Bertani (LB).

Luria-Bertani (LB) Medium:

• 1.0 liter of distilled water
• 15.0 g Bacto Agar
• 10.0 g NaCl
• 10.0 g Bacto¨ Tryptone
• 5.0 g Yeast Extract

For this procedure, you will be using an autoclave. An autoclave is designed to produce temperatures and pressures that will completely sterilize objects. It is important that you use gloves designed for use with the autoclave so that you do not injure yourself.

1. Weigh out the ingredients from either list, and place them in a beaker. Also place in the beaker a glass stirring rod to be sterilized. The stirring rod will be used after autoclaving. Agar will not dissolve into solution until the solution has been heated. Cover the beakers with aluminum foil (shiny side facing the inside of the beaker), and put the nutrient agar in an autoclave. The time necessary to sterilize the solution depends on your particular autoclave. It is a good idea to place autoclave tape on your beakers if it is available. Autoclave tape has stripes on it that are originally light colored but turn black when exposed to the temperature and pressure needed for sterilization. Thus, you can be assured that the sterilization process was successful if the stripes on the autoclave tape change color.


2. After sterilization is complete, remove the beakers wearing autoclave gloves. Remember that the autoclave operates at 121.6^°C (250^°F), and a steam pressure of 15 lbs per square inch (psi). The solution which comes out is very hot. Be careful!

After removal from the autoclave, allow the beakers to cool enough that they can be comfortably handled while wearing vinyl gloves. If the agar is allowed to cool too much, it will begin to solidify. If the nutrient agar is poured while it is too hot, there will be excessive condensation on the cover of the petri dish. Moisture on the cover will adversely effect results of later steps.


3. Stir the nutrient agar using the stirring rod which was autoclaved with the agar. Then pour agar into the petri dish until the agar just covers the bottom of the dish (approximately 20 ml). Place the petri dish cover on the petri dish immediately and allow the agar to solidify (fully gel). After the plate of agar has solidified, turn the plates upside down and let them sit for 48 hours at room temperature to dry. Drying eliminates excess water in the agar and reduces the amount of condensation.