TECHNICAL DATA SHEET #905 REV.

GRAM STAINS AND REAGENTS

PRODUCT:

Bottle:a
Crystal Violet, item no. R5815, R5916, R5817, R5818, R5820
Gram Iodine, 50x Concentrate (unstabilized), item no. R5836, R5837, R5838, R5840
Gram Iodine, Stabilized, item no. R5841, R5842, R5843, R5844
Gram Decolorizer, item no. R5825, R5826, R5827, R5828
Safranin, item no. R5830, R5831, R5832, R5833
Basic Fuchsin, item no. R5819, R5824, R5829, R5834
Gram Stain Set w/ Safranin, item no. R5814
Gram Stain Set w/ B Fuchsin, item no. R5812
Gram Stain Set w/Stablized Iodine, item no. R5813
see catalog for ordering options

PURPOSE:

The Gram stain is used to determine the Gram reactions of microorganisms, thus aiding in their identification.

PRINCIPLE:

Gram2 first described the Gram reaction using an aniline-gentian violet and iodine method for staining bacteria, thereby separating organisms into groups-gram-positive and gram-negative. Hucker3, utilizing crystal violet and ammonium oxalate, later modified the method. The principle is not clearly understood, but it is postulated that the Gram reaction is related to the structure of
the cell wall. Crystal violet, the primary stain, stains the bacterial cell. Iodine serves as a mordant to bind the stain. Because
inorganic iodine is rapidly oxidized and loses its effectiveness as a mordant, the use of an organic iodine was employed, which
offers a more stable organic iodine complex (Gram Iodine, Stabilized).7 Certain bacteria, by nature of their cell wall, retain the
crystal violet after treatment with a decolorizer. Some bacteria have a high lipid content in their cell wall and do not retain the
crystal violet and are stained by the counterstain. The Gram reaction combined with the morphology (cocci or bacilli) and the
arrangement of the bacteria, make it useful in the presumptive identification of bacteria.
Some species of bacteria, such as Campylobacter and Legionella species, do not readily counterstain with Safranin. Basic
Fuchsin may be substituted for Safranin for organisms that are hard to stain.

FORMULAS:

Approximate ingredients per liter.

(1)

Crystal Violet Stain:

Water, Deionized ........................................ 800.0 ml
Ethanol, Denatured ..................................... 200.0
Ammonium Oxalate ....................................... 8.0 g
Crystal Violet ................................................. 20.0

(2)

Gram Iodine Concentrate, 50x

Water, Deionized ...................................... 1000.0 ml
Iodine ............................................................. 3.3 g
Potassium Iodide ........................................... 6.6

(3)

Gram Iodine, Stabilized:

Water, Deionized ...................................... 1000.0 ml
Polyvinylpyrrolidone (PVP) .......................... 110.6 g
Iodine ............................................................ 18.0
Potassium Iodide, Anhydrous ....................... 19.0

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TECHNICAL DATA SHEET #905 REV. 2

(4)

Gram Decolorizer:
Ethanol, Denatured ..................................... 750.0 ml
Acetone ....................................................... 250.0

(5)

Safranin Stain:
Water, Deionized ........................................ 900.0 ml
Ethanol, Denatured ..................................... 100.0
Safranin O (Certified) ...................................... 2.5 g

(6)

Basic Fuchsin:
Water, Deionized ........................................ 900.0 ml
Ethanol, Denatured ..................................... 100.0
Basic Fuchsin ................................................. 1.0 g

PRECAUTIONS:*

For in vitro diagnostic use. Observe all safety precautions consistent with the hazard(s) stated on the product label and/or
Material Safety Data Sheet.
Storage: Upon receipt store according to the instructions on the product label. Reagents should not be used if there are signs
of deterioration or if the expiration date has passed.
Limitations: Care must be taken for proper decolorization in order to obtain an accurate Gram reaction. Stain with a known
control slide. If the background is purple, the decolorization was not done properly.
The procedure should be used on young (18- to 24-hour cultures), since older cultures may be gram-variable. Many gram-positive
organisms readily decolorize with age, especially spore-formers. A smear from an acidic medium or from a patient receiving
antimicrobial therapy may cause gram-positive organisms to appear as gram-negative.
The Crystal Violet stain may become precipitated with age. Filter remaining stain through gauze to remove excess crystals.
Care should be taken not to introduce environmental organisms into the stock stains. These organisms can ultimately be
transferred to a test slide during the staining process and alter the interpretation of the smear.
After the Gram Iodine Concentrate has been diluted (see Method of Use) the resulting stain may be used for approximately one
month and then discarded.

PROCEDURE:

Specimen Collection: Information on specimen collection and transport is found in standard reference material on the subject.
In general, specimens should be delivered to the laboratory without delay. Consult appropriate references for the details of
preparing a primary stain.1,4,5,6 Procedures may vary according to the type of specimen.
Method of Use: Prior to use, the Gram Iodine Concentrate (unstabilized) should be diluted according to the label instructions;
add 5 ml of concentrate to 245 ml of deionized water or 76 ml of concentrate to 3724 ml of deionized water and mix.
A smear of the organism is made directly from a specimen or from an 18- to 24-hour culture. In preparing a smear, use a clean,
dry glass slide. For specimens on a swab, gently roll over the surface of the slide, or in the case of a liquid specimen or broth,
place a small portion in the center of the slide. For preparing a colony for Gram stain, emulsify the colony in a drop of deionized
water on the slide. Heat-fix, by passing through a flame 2 or 3 times, then cool. Alternately, the slide may be placed on a slide
warmer at 65-75C for 30 minutes, or the slide may be flooded with methanol for 2 minutes. Methanol fixing preserves the
integrity of the human cells.
The slide is then flooded with Crystal Violet stain for 1 minute. Rinse the smear briefly with water and drain off excess. Flood the
smear with Gram Iodine and let stand 1 minute. Rinse with water and decolorize until the solvent flows colorlessly from the slide.
This is a critical step. Do not overdecolorize. Rinse briefly with tap water and counter stain with Safranin or Basic Fuchsin for 10
seconds. Rinse briefly with water, blot dry, and examine microscopically.

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TECHNICAL DATA SHEET #905 REV. 2

Interpretation: Gram-positive organisms appear violet; gram-negative organisms appear red. Consult appropriate references
for information regarding morphology and arrangements.1,4,5,6
Materials Required but Not Provided: Standard microbiological supplies and equipment such as loops, needles, pipettes,
glass slides, and slide warmers are not provided.

QUALITY CONTROL:

Using prepared smears of known gram-positive and gram-negative organisms controls the effectiveness of the stains and the
staining techniques. Using control slides, stain simultaneously with test slides. Any inconsistencies in the control slides are an
indication that the technique was incorrect or that the stains themselves are defective.
User Quality Control: Check for signs of contamination and deterioration. Check the performance of the stain weekly with
known quality control slides.

BIBLIOGRAPHY:
1.
2.
3.
4.
5.
6.
7.
8.

Finegold, S. M., and E. J. Baron, Bailey and Scotts Diagnostic Microbiology, 7th ed., C. V. Mosby, St. Louis, 1986.
Gram, H. C., Fortschritte der Medicin, 2:185, 1884.
Hucker, M., et al., Agr. Exp. Sta. Tech. Bull., 93:1-37, 1923.
Koneman, E. W., et al., Color Atlas and Textbook of Diagnostic Microbiology, 3rd ed., J. B. Lippincott, Philadelphia, 1988.
Lennette, E. H., et al, Manual of Clinical Microbiology, 4th ed., American Society for Microbiology, Washington, D.C., 1985.
Lillie, R. D., H. J. Conns Biological Stains, 9th ed., Williams and Wilkins, Baltimore, 1977.
Magee, C. M., et al., Am. J. of Surg., 130:341-346, 1975.
Salton, M., The Bacterial Cell Wall, Elsevier Publishing, Amsterdam, 1964.

*For more detailed information, consult appropriate references and/or details in the preface of the PML Technical Manual.
PML MICROBIOLOGICALS, INC.
Data #905
Copyright 1989 by PML Microbiologicals, Inc.
Revision Date: January 2001

27120 SW 95th Avenue Wilsonville, OR 97070 800.547.0659

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