MICROBIOLOGY LAB EXPERIMENT # 2

STAINING: GRAM STAIN

Unstained bacteria are difficult to see under the microscope because they are almost as transparent as
the slide itself. Therefore, for successful observation, the bacteria must be stained with a dye that has an
affinity for bacteria protoplasm. Preparation of a stain mount consists of preparing a smear of bacteria on a
microscope slide, fixing it with heat, and staining using a dye. A good smear preparation is the key to a high-
quality stain.
In 1883, Christian Gram, a Danish bacteriologist working at the municipal hospital in Berlin, discovered
the most important differential stain, the Gram stain. Gram was attempting to find a combination of dyes that
would stain tissues one color and bacteria another. His early work was with lung tissues obtained from
patients with fatal case of pneumococcus pneumonia. The particular combination of dyes that he used, and of
which we will use a modified version, stained the bacteria purple-black and the tissues a brownish color. Later,
workers established that certain bacteria retain the initial stain and the others show the color of the second
counter stain. Those bacteria that retain the stain even after washing with ethyl alcohol or acetone are now
designated gram-positive organisms, while those that lose the stain and are colored with counter stain are
termed gram-negative. With this staining procedure the bacteria that appear purple to purple-black are
stained by the second stain, safranin, and will be reddish or a reddish pink. They are termed gram-negative.
This method is useful for separating bacteria into two groups and has proved most valuable in the diagnosis of
certain diseases.

MATERIALS:

1. Agar cultures of Staphylococcus aureus, and Escherichia coli ( and Bacillus subtilis for the second gram
stain lab).
2. Gram-staining reagent
a. Hucker’s Crystal violet
b. Gram’s iodine solution
c. Decolorizer
d. Safranin
3. Microscopic slides
4. And instrumentation

PROCEDURE:

Note: Complete Gram stain of one organism before beginning the next organism. Time and order of stains
applied is critical. Follow instructions.

1. Prepare the bacterial smear:

(a) Label your slides with the name of the bacteria and draw a circle in the middle of about one square
inch using a wax pencil. Flip the slide for the next step.
(b) Squeeze a drop or two of distilled water on to the slide in the middle of the circle.
 (c) Sterilize the loop and transfer a bit of growth from the agar plate to the water droplet. Spread over the
circle area. (NOTE: care must be used to prevent the smear from becoming too thick for satisfactory
examination).
(d) Dry the slide in the warm air about one foot above the flame. Hold slide with cloths pin. This is the
hardest part of this lab. Too little flaming and the microbes get washed off of the slide. Too much
flaming and they are burned to a cinder.

2. Heat fixation:
(a) Heat fix bacteria to the slide by passing it three times through the flame with the smeared side
uppermost.
(b) This procedure simultaneously kills and attaches the bacteria to the slide.

3. Gram Staining:
The slide now should have a smear of bacteria. Stain it as follows:
a. Cover the smear with Hucker’s crystal violet for 1 minute, and then wash with water.
b. Cover the smear with Gran’s iodine solution for 1 minute, and then wash with water.
c. Decolorize with acid-alcohol. Hold the slide at 45⁰ angle and add decolorizer drop wise from
above the stained area. Watch the drops as they come off the slide. When they are clear or just
slightly tinged with purple you have completed the decolorization. Wash with water.
d. Apply safranin (as counter stain) for 30 seconds, wash with water and dry.
2. Examine under oil immersion.
3. Draw and label the bacteria examined, indicated color, total magnification, and shape.
Draw and label the bacteria examined, indicated color, total magnification, and shape.

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