Microscopic slides used for Gram stain must remain free of any oils and should be labeled according to facility procedures. Preparation of the smear is as follows:
- From broth: Using a cooled, sterile loop, place a loopful of broth on the slide and spread in a circular motion to about 1 cm in diameter.
- From plated media: Place a drop of sterile water or saline on the slide. Select the isolated colony to be stained. Be careful to touch only the top and center of the isolated colony being Gram stained. Use the loop to create an emulsion.
After the prepared slide has air dried, the smear should be heat fixed prior to Gram staining.
The thickness of the smear is what determines the degree of decolorization that is necessary and will ultimately affect the Gram stain result. It is preferable that slides for Gram stain be thinly prepared, without areas of clumping or inconsistency. The image demonstrates a macroscopic view of an adequate smear.
Decolorizing is the MOST crucial step affecting the outcome of the stained smear and leading to potentially erroneous results. When staining a thinly prepared slide, a short decolorization time should be used, which is typically less than 15 seconds.
- Over-decolorizing: Gram-positive organisms may stain pink to red, leading to an erroneous gram-negative result.
- Under-decolorizing: Gram-negative organisms may stain blue to purple, leading to an erroneous gram-positive result.