Staining Procedures

Gram stain

• Make smears of blood, fluid, tissue or bacterial colony from an agar plate, as described in previous sections and air dry. (Heat fix smears but do not heat excessively, i.e., slide should not be too hot to touch.)
• Place slide on a rack over a sink.
• Cover smear with crystal violet dye and let stand for 1 minute.
• Rinse briefly in tap water and drain. Flood smear with Gram's iodine, pour off and flood again. Let stand for 1 minute.
• Wash briefly in tap water and drain. Rinse with decolorizer (95% ethanol or ethanolacetone mixture from a pipet) until no more purple dye comes off the slide (usually 2-5 seconds). Excessive decolorization may give a false Gram-negative reaction.
• Wash briefly with tap water and drain. Flood smear with safranin stain and let stand 1 minute.
• Wash briefly with tap water, drain, and air dry.
• Examine the slide using the oil immersion lens (100x)
• Gram-negative bacteria will be pink to red; Gram-positive bacteria will be purple.

Diff-Quik

• Make smears of blood, fluids, or tissues.
• Dip 5 times in Diff-Quik solution 1, one second each time, and drain.
• Dip 5 times in Diff-Quik solution 2, one second each time, and drain.
• Dip 5 times in Diff-Quik solution 3, one second each time, and drain.
• Rinse in tap water and drain.
• Air dry and examine using 10x, 40x, or 100x.

NOTE: Diff-Quik solution 3 can become weakened with age or use. Check stain intensity on slides periodically. Slides may be re-stained with fresh solution 3 if necessary. Periodically pass solutions 2 and 3 through separate 0.45-μm filters to remove precipitates and contaminating bacteria.

Figure. Demonstration of how to make a thin blood smear.

1. On slide “A” express a drop of blood or hemolymph about one-half inch from the end.
2. The edge of a second slide “B” is placed on the surface of slide “A” at about a 45 angle and is moved to the right until contact with the drop of blood.
3. Contact with the blood will cause the drop to spread along the edge of slide “B” due to capillarity. Slide “B” is then pushed to the left, being careful to keep the edge pressed uniformly against the surface of slide “A”.
4. The size of the drop of blood and acuteness of the angle formed between the slides will determine the thickness of the film. A more acute angle results in a thicker film.
5. The smear is allowed to air dry for transport in a slide box and later staining.