HandsOn 18 - Bacterial Growth

I. Bacterial Growth Preparation

II. Streaking and Inoculating Plates

III. Growth of Bacteria Under Starvation Conditions

IV. Staining Bacterial Colonies

IV. Staining Bacterial Colonies

If you want to preserve your bacterial colony, you can stain it. Staining the colony kills the bacteria to prevent further growth, and makes the colony easier to look at both with the naked eye and under a microscope.

Note of Caution: Please note that the methanol and acetic acid used in the staining solution are hazardous. To prevent damage to the mucus membrane, a fume hood should be used. Gloves should be worn to prevent skin irritation and neither chemical should be ingested. Be sure to keep the Material Safety Data Sheets provided by the chemical supply company. You will also be working with a strong staining agent. Be sure to take precautions regarding skin and clothing.

You will need:

• stirring rod
• beaker
• graduated cylinder
• 0.1% Coomassie Brilliant Blue R stain (available from Sigma)
• methanol
• acetic acid (or vinegar)
• distilled water
• Staining Solution
• Rinsing Solution

To prepare 1 liter of staining solution (be sure to follow the exact order of these steps):

1. To a 1000 ml beaker add 400 ml distilled water.


2. Add 500 ml methanol.


3. Add 100 ml acetic acid. If acetic acid is not available, vinegar can be used in its place.


4. Add 1 gram of 0.1% Coomassie Brilliant R stain powder. Mix thoroughly until the solution becomes a uniform blue.

The ingredients can be scaled proportionally for smaller amounts of staining solution. Use the base proportions of (4:5:1) distilled water:methanol:acetic acid.

The rinse solution can be made by following Steps 1-3 for making the staining solution. Be sure not to add the stain powder to your rinse solution.

1. Pour staining solution onto plate. Solution should just cover the surface of the nutrient agar. Let the staining solution stand on the nutrient agar for approximately 45 seconds. This may vary depending on the size and color of the colony that you are staining.


2. Pour solution off the nutrient agar. Pour on rinse solution. Agitate rinse solution for approximately ten seconds. Let the rinse solution stand on the nutrient agar for approximately 50 more seconds.


3. Pour off the rinse solution. If any rinse solution remains, you can invert the plate onto a paper towel until it is dry.

Note of 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.

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