Student Procedures

Purpose:  is to determine the Gram stain of your bacterial sample.

Introduction:  Gram staining is a method commonly used to determine the chemical make up of the cell wall of bacteria. The cell wall can stain either positive or negative, depending on its chemistry. Knowing the chemical make up makes it easier to manipulate the bacteria for various purposes. The medical, research, developmental, and biotechnical fields are just a few occupations whose greatest scientific advancements were due to knowing the chemical composition of the bacteria cell wall. If the bacteria stains positive it will retain a purple/blue color. If the bacteria stains negative, the bacteria will not retain the purple/blue color, but rather have a pinkish/red color.

Procedure:

1. Smear and heat fix a clean microscope slide with your bacterial culture.

2. Put 10 to 15 drops of Crystal Violet stain on your bacteria smear and leave on for one minute.

3. Rinse the Crystal Violet stain off with water (from the washing bottle).

4. Put the Grams' stain on the smear and leave it on for one minute, then rinse it off with water from the washing bottle.

5. Add 10 to 15 drops of 95% ethanol and leave on for 10 to 15 seconds.  Rinse off with water for the washing bottle.

6. Place the Safranin on the slide and leave on for 45 seconds. Rinse off with water from the washing bottle. Add a cover slip.

7. Look under the microscope. Determine whether or not your bacterial culture stained positive or negative.

Questions

1. Why is it important to follow sterile technique?

2. What can happen to cause gram-positive bacteria to appear gram-negative?

3. Name three gram-positive bacteria and the diseases they cause.

4. Name three gram-negative bacteria and the diseases they casue.

For Further Study

Go to this site on Microbiology, select "The Cell" and name 4 functions of the cell wall. Select "Gram-negative bacteria" and explain why gram-negative bacteria will not hold the crystal violet stain. At the bottom of the page there are 6 categories of bacteria. Choose one bacteria from each category, describe it, and tell what disease it causes.

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