The Gram stain (Clinical laboratory exercise)

The Gram stain (Clinical laboratory exercise)

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Individual bacterial cells exhibit morphology typical of their species: size, shape, and arrangement of cells. These can be demonstrated by making a smear on a glass slide, then staining the smear with a suitable dye. The use of a stained smear permits microscopic examination of the smear with the oil immersion lens, which gives the greatest magnification, revealing the size, shape, and arrangement. The study of individual bacterial cells is thus frequently one of the first steps in the identification of bacteria.

Gram stain is called a differential stain, because it not only shows bacterial morphology but allows differentiation of different bacterial types since different species react differently to the stain. The differential Gram stain gives information about the bacterial cell wall, which may be grampositive or gram-negative.

Gram-positive bacteria will appear purple, the color of the primary stain, crystal violet.

Gram-negative bacteria will appear pink-red, the color of the counterstain, safranin.

The Gram stain is especially useful as one of the first steps in the identification of a bacterial species, since it reveals both the morphology and the Gram reaction of the bacteria.

The bacteria may show the following shapes: coccus/cocci(spherical), bacillus/bacilli(rod-shaped), or spirillum/spirilli(curved or spiral). The cells may assume a characteristic arrangement: some occur singly, others appear in pairs (diplo-), chains (strepto-), or clusters (staphylo-).

Materials needed

1. Slides of Staphylococcus epidermidis (Gram +) and Escherichia coli (Gram -) (Already prepared in the smear preparation exercise)

2. Wash bottle (with tap water)

3. Rinse bucket

4. Clothespins (slide holders)

5. Absorbent mat

6. Glass marker

7. Reagents used in the Gram stain: crystal violet, Gram’s iodine, 95% ethyl alcohol, safranin

The Gram Stain Procedure

1. Add crystal violet stain until the slide is completely covered. Stain for one minute.

2. Do not drain the stain off of the slide before rinsing, because the crystal violet will form dye crystals on the slide. Dilute the crystal violet stain on the slide with a gentle stream of water from a wash bottle. Then tip the slide and drain off the stain, and continue rinsing until all the purple color has washed off of the slide. Drain off excess rinse water. If viewed under the microscope at this point, all bacterial cells will appear purple.


3. Cover the slide with Gram’s iodine solution and let it stand for one minute. This step will not change the color of the cells; the iodine forms a complex with the crystal violet in the cell wall. Rinse with water, using the wash bottle.

4. Decolorize the smear by letting 95% ethyl alcohol run down over the slide, which should be held at an angle with the clothespin until the purple stain no longer is being visibly removed from the slide. This step should only take a few seconds. (NOTE: a thick smear will take longer to decolorize than a thin one.)

5. Quickly rinse the slide with water. At this stage, if viewed under the microscope, gram-positive bacteria will still appear purple and gram-negative bacteria will appear colorless.

6. Add safranin, the counterstain, to cover the slide. Stain for two minutes. At this stage, if viewed under the microscope, gram-positive bacteria will still appear purple, and gram-negative bacteria will appear the color of the counterstain, pink-red.

7. Rinse with water, and let the slide air-dry or blot gently (DO NOT RUB) with bibulous paper. The slide must be completely dry before adding oil for observation under the oil-immersion lens.


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