Variables Influencing Results, and the Precise Definition of Steps in Gram Staining as a Means of Standardizing the Results Obtained

Abstract

Results of a Gram staining procedure varied with modifications of each of the steps involved. The best Gram differentiation was obtained when crystal violet and iodine solutions of high concentrations were used, and when n-propyl alcohol was used as the decolorizer. The decolorization step must be carefully quantitated, and one of the most important variables observed was whether a slide was brought into the decolorizer wet, or dry. Dry slides took 6 to 12 times as long to decolorize as wet. Wash steps, following crystal violet, and following the decolorizer, also greatly influence results by causing Gram-positive organisms to appear to be Gram-negative. The results indicated that Gram-stain procedures should not be varied to suit the whims of individual operators, and that each step could be specifically defined both as to the reagent used, and the procedure to be followed. The followng Gram procedure is recommended for heat-fixed bacterial smears on glass slides. Flood the slide with Hucker's crystal violet for 1 ruin. Wash for 5 sec by dipping into tap water running into a 250 ml beaker at a rate of 30 ml per sec Rinse off the excess water with Burke's iodine, flood the slide with this solution for 1 min, then wash 5 sec in tap water as above. Decolorize by passing the wet slide through 3 (75 × 25 mm) Coplin dishes containing n-propyl alcohol, decolorize 1 min in each dish for a total of 3 min. Wash 5 sec in tap water as above, rinse off the excess water with 0.25% safranin, then flood the slide with this solution for 1 min. Wash as above, blot dry, and examine. An alternate procedure for decolorization would be to use either 95% n-propyl alcohol or 95% ethyl alcohol, but shorten the decolorization time to 30 sec per dish for a total of 1.5 min. After 10 slides, the decolorizer in the first dish should be replaced by fresh. This dish is then placed last in the sequence, with dish No. 2 moved to the No. 1 position.