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. Alternatively, use a sterile pipette and transfer 1-2 drops onto the slide. Spread to obtain a thin, even smear.
- From plated media: Using a sterile pipette, place a drop of sterile water or saline on the slide. Select the isolated colony to be stained and pick a small amount using a sterile loop. Be careful to touch only the top and center of the isolated colony being Gram-stained. Use the loop to create an emulsion by mixing it into the drop of sterile water or saline using circular motions. Mix evenly to make a thin smear.
After the prepared slide has air dried, the smear should be methanol-fixed before performing the Gram stain. Heat fixation is acceptable, but not encouraged. (Microorganisms may lyse or become distorted with heat.)
The thickness of the smear is what determines the degree of decolorization that is necessary and will ultimately affect the Gram stain result. Slides for Gram stain should be thinly prepared, without areas of clumping or inconsistency. The image demonstrates a macroscopic view of an adequate smear.
Decolorization is the most crucial step affecting the outcome of the stained smear, potentially leading to erroneous results. When staining a thinly prepared slide, a short decolorization time should be used, 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.
