GRAM TECHNIQUE

 Gram technique

The Gram staining method was first described in 1884 by the Danish bacteriologist Hans Christian Gram, after  whom the test was named.  The Gram staining test for bacteria is one of the most important tests in microbiology and is often  one of the first tests performed in the identification of bacteria. 

The Gram staining reaction is used to help identify pathogens in specimens and cultures by their Gram reaction (Gram positive or Gram negative) and morphology. Pus cells can also be identified in Gram

smears.

Gram positive bacteria: Stain dark purple with crystal violet (or methyl violet) and are not decolorized

by acetone or ethanol. Examples includes species of:Staphylococcus, Actinomyces ,streptococcus

Clostridium,Corynebacterium

Gram negative bacteria: Stain red because after being stained with crystal violet (or methyl violet)

they are decolorized by acetone or ethanol and take up the red counterstain (e.g. neutral red, safranin, or

dilute carbol fuchsin). Examples include species of:

Neisseria Klebsiella

Haemophilus Brucella

Salmonella Yersinia

Shigella Coliforms

Vibrio

Gram reaction

Differences in Gram reaction between bacteria is thought to be due to differences in the permeability of the cell wall of

Gram positive and Gram negative organisms during the staining process. Following staining with a triphenyl methane

basic dye such as crystal violet and treatment with iodine, the dye–iodine complex is easily removed from the more permeable

cell wall of Gram negative bacteria but not from the less permeable cell wall of Gram positive bacteria. Retention of

crystal violet by Gram positive organisms may also be due in part to the more acidic protoplasm of these organisms binding

to the basic dye (helped by the iodine).

Gram staining technique

Required

– Crystal violet stain

– Lugol’s iodine

– Acetone–alcohol decolorizerb

– Neutral red, 1 g/l (0.1% w/v)

Notes

A Gentian violet or methyl violet can also be used. Some workers prefer to use acetone by itself, ethanol 95%

v/v, or ethanol–iodine as the decolorizing solution. A mixture of acetone and alcohol is recommended because it decolorizes

more rapidly than ethanol 95% v/v, and is less likely to over decolorize smears than acetone without alcohol added.

neutral red is selected as the counterstain because it stains well gonococci and meningococci. Safranin can also be used.

The use of dilute carbol fuchsin (1 in 10) is recommended for staining Vincents’ organisms, Yersinia, Haemophilus, Campylobacter,

and Vibrio species.

Method

1 Fix the dried smear as explained in subunit Note: When the smear is for the detection of

gonococci or meningococci, it should be fixed with methanol for 2 minutes (avoids damaging

pus cells).

2 Cover the fixed smear with crystal violet stain for 30–60 seconds.

3 Rapidly wash off the stain with clean water.

Note: When the tap water is not clean, use

filtered water or clean boiled rainwater.

4 Tip off all the water, and cover the smear wit  Lugol’s iodine for 30–60 seconds.

5 Wash off the iodine with clean water.

6 Decolorize rapidly (few seconds) with acetone–alcohol. Wash immediately with clean

water.

Caution: Acetone–alcohol is highly flammable, therefore use it well away from an open flame.

7 Cover the smear with neutral red stain for 2 minutes.

8 Wash off the stain with clean water.

9 Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry.

10 Examine the smear microscopically, first with the 40_ objective to check the staining and to

see the distribution of material, and then with the oil immersion objective to report the

bacteria and cells

Results

Gram positive bacteria . . . . . . . . . . . . . Dark purple

Yeast cells . . . . . . . . . . . . . . . . . . . . . . . Dark purple

Gram negative bacteria . . . . . . . . . . Pale to dark red

Nuclei of pus cells . . . . . . . . . . . . . . . . . . . . . . . Red

Epithelial cells . . . . . . . . . . . . . . . . . . . . . . . . Pale red

Reporting Gram smears

The report should include the following information:

● Numbers of bacteria present, whether many, moderate, few, or scanty

● Gram reaction of the bacteria, whether Gram positive or Gram negative

● Morphology of the bacteria, whether cocci, diplococci, streptococci, rods, or coccobacilli. Also,

whether the organisms are intracellular.

● Presence and number of pus cells

● Presence of yeast cells and epithelial cells.

1. Numerical

a. 1+ (< 1 per oil immersion field, 100x)

b. 2+ (1 per oil immersion field)

c. 3+ (2 to 10 per oil immersion field)

d. 4+ (predominant or >10 per oil immersion field)

2. Descriptive

a. Rare (<1 per oil immersion field)

b. Few (1 to 5 per oil immersion field)

c. Moderate (5 to 10 per oil immersion field)

d. Many (>10 per oil immersion field)

Example

A urethral smear report might read: ‘Moderate numbers Gram negative intracellular

diplococci and many pus cells.’

Variations in Gram reactions

_ Gram positive organisms may lose their ability to retain crystal violet and stain Gram negatively for the

following reasons:

– Cell wall damage due to antibiotic therapy or excessive heat-fixation of the smear.

– Over-decolorization of the smear.

– Use of an iodine solution which is too old, i.e. yellow instead of brown in colour (always

store in a brown glass or other light opaque container).

– Smear has been prepared from an old culture.

_ Gram negative organisms may not be fully decolorized and appear as Gram positive when a

smear is too thick.

Control: Always check new batches of stain and reagents for correct staining reactions using a smear

containing known Gram positive and Gram negative organisms

C. Gram stain review

a. After interpretation of smears, hold slides long enough to allow a

 confirmatory review, especially if culture or other lab test results are inconsistent.

i. Drain or gently blot excess oil

ii. For slide libraries and teaching collections that will be stored for longer periods, immersion oil can be removed with xylene solution and the slides can be coverslipped using Permount to prevent fading.

iii. When discarding stained smears, handle as biological waste and use biohazard containers as required by local/state regulations. Slides may puncture biohazard bags and should be treated as “sharps.” Cardboard boxes or other protective containers may be used.

b. If a repeat Gram stain or a special stain to confirm findings suggested by a Gram smear interpretation is required when extra unstained smears are not available, a Gram-stained smear may be destained.

i. Remove immersion oil on the slide with xylene solution.

ii. Flood slide with acetone-alcohol until smear appears

colorless.

i.              Re stain.

VII. LIMITATIONS OF THE PROCEDURE

A. Use results of Gram stains in conjunction with other clinical and

laboratory findings. Use additional procedures (e.g., special stains, direct antigen tests, inclusion of selective media, etc.) to confirm findings suggested by Gram-stained smears.

B. Careful adherence to procedure and interpretative criteria is required for accurate results. Accuracy is highly dependent on the training and

skill of microscopists.

C. Additional staining procedures are recommended for purulent clinical specimens in which no organisms are observed by the Gram stain

method.

D. Gram stain-positive, culture-negative specimens may be the result of

contamination of reagents and other supplies, presence of

antimicrobial agents, or failure of organisms to grow under usual

culture conditions (media, atmosphere, etc.)

E. False Gram stain results may be related to inadequately collected

specimens.