The Gram-Stain is the single most common and cost effective staining technique used in identifying bacterial organisms. The method was originally devised in 1884 by a Danish bacteriologist, Hans Christian Gram.

PRINCIPLE OF THE METHOD
Gram staining is based on the ability of the bacterial cell wall to retain crystal violet dye during decolorizing treatment with a decolorizing agent. The cell walls of gram-positive bacteria have a higher peptidoglycan and lower lipid content than gram-negative bacteria. The bacterial cell walls are initially stained with crystal violet. Iodine is added as a fixative to form a complex so that the dye cannot be easily removed. This step is commonly referred to as fixing the dye. Subsequent treatment with a decolouriser, a mixture of ethanol and acetone, dissolves the lipid layer from the gram-negative cells. The removal of the lipid layer allows the leaching of the crystal violet stain from the cells. When the counter stain of safranin is added the decolorized gram-negative bacteria stain pink. In contrast, the solvent dehydrates the thicker gram-positive cell walls, closing the pores as the cell wall shrinks. As a result, the violet-iodine complex is retained and the cells remain purple.


THE BASIC STAINING PROCEDURE
1) Make a thin layer of bacterial cells on a clean glass slide from a fresh (18-24 hours of growth), single isolated colony off a nutrient agar growth plate. Allow to air dry.
2) Fix with 95% methanol for 2 minutes, air dry. This is the current recommended fixing method as it does not distort cells. Heating the slide (which is the traditional method) may cause artifacts and does not fix the specimen to the slide well.
3) Flood the slide with crystal (or gentian) violet staining reagent for thirty seconds.
4) Wash the slide with a gentle and indirect stream of tap water for two seconds. Tilt the slide to drain off the excess water.
5) Flood the slide with iodine staining reagent for thirty seconds.
6) Repeat step 4.
7) Add the decolouriser to the slide with it slanted until no colour washes out, about 10 – 30 seconds.
8) Repeat step 4.
9) Flood the slide with safranin (pink colour) counter staining reagent for thirty seconds.
10) Wash the slide in a gentle, indirect stream of tap water until no colour washes out. Air dry or blot the slide dry with absorbent paper.
11) View the slide with the light microscope under oil-immersion. Gram-positive bacteria appear blue or violet and gram-negative bacteria appear pinkish red.


TROUBLESHOOTING THE GRAM STAIN

Smear Preparation

PROBLEM: If the smear is too thick, the cells can appear Gram-positive in very thick area. You may see Gram-variability from the thick to the thin areas.
SOLUTIONS: Try to prepare a single cell layer of organism. Running positive and negative controls can help (pre-prepared slides with controls are available for purchase).

PROBLEM: If cells are prepared in hyper or hypotonic solutions, morphology may be disturbed.
SOLUTIONS: Smear the cells onto the slide dry with a sterile toothpick.

PROBLEM: Over warming the smear (this happens most often when smears are warmed prior to being completely air dried, or when flaming too much to fix the slides) will cause all cells to appear Gram-negative.
SOLUTIONS: Dry slide thoroughly prior to “heat fixing”, be extremely careful when using flames.

PROBLEM: Old culture smears can cause cells to appear Gram-negative (weak walls).
SOLUTIONS: Prepare smears only from fresh, log-phase growth (usually overnight culture).

PROBLEM: The medium from which the colonies are taken is important. Often, if you take colonies from liquid media or from selective media, cells will appear Gram-negative and more coccoid.
SOLUTIONS: Take sample from fresh colonies from nutrient agar medium.

Staining

PROBLEM: Over-decolourisation: This is an extremely common problem, often caused by using a strong decolouriser or by leaving the decolouriser on the slide for too long.
SOLUTIONS:
> Use decolouriser manufactured by the same company as your stains, and follow their protocol.
> Run positive and negative controls with your daily stains
> Test your decolouriser solution using different times (e.g. 5, 10, 15 seconds) with positive, negative and some weakly positive cultures; until you find a method that works well for you.

PROBLEM: Gram variability: This can be due to the organism itself, and not to the staining method.
SOLUTIONS: The vast majority of Gram-variable organisms are Gram-positive. Characteristically Gram-variable organisms (e.g. Corynebacterium variabilis) or those whose membranes alter with age and appear Gram-variable (e.g. Arthrobacterium spp.) are grouped with the Gram-positive organisms. Therefore, they are treated as Gram-positive organisms.


GRAM STAINING OPTIONS
The conventional preparation for Gram’s iodine is relatively unstable and may lose up to 60% of the available iodine in 30 days when stored at 25°C. Gram-positive organisms may stain poorly when 40% of the available iodine is lost. The iodine solution is stabilized by some manufactures by the addition of sodium bicarbonate to the solution, or creating stabilised iodine-polyvinylpyrrolidone iodine.


ADDITIONAL METHODS TO DISTINGUISH BETWEEN GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA
Gram stain results are not always a conclusive test to indicate the structure of the cell wall of bacteria. Certain gram-positive bacteria lose some of their cell wall properties with age or exposure to harsh or bacterial static/ bactericidal agents causing them to appear gram-negative or gram-variable. Also many Bacillus sp. and Clostridium sp. stain falsely gram-negative. Several additional tests can be performed to aid in the determination of gram reactivity, although none are foolproof.

1. L-alanine-4-nitroanilide (LANA) can distinguish aerobic and facultative anaerobes. The reagent is sold commercially impregnated in cotton swabs, which turn yellow when touched to the colony of a gram-negative bacterium.

2. Potassium hydroxide (KOH) test also detect gram-negative bacteria. A loop of growth from a colony is emulsified on the surface of a glass slide in a suspension of 3% KOH and stirred continuously for 60 seconds. Gram-negative cell walls break down and when the loop is gently pulled from the suspension it will be thick and stringy.

3. Vancomycin is an antimicrobial agent that acts on the wall of most gram-positive bacteria. Heavily inoculate the organism onto the surface of a sheep blood agar plate and place a 5-ug vancomycin disk on the inoculated area. Any zone of inhibition of growth after overnight incubation usually indicates a gram-positive bacterium. Many lactic acid bacteria are typically resistant to vancomycin. Some gram-negative organisms, especially Moraxella and Acinetobacter, may be sensitive to vancomycin. Conversely, most gram-negative bacteria are susceptible to polymyxin at 10ug/ml.

4. Maconkey agar is selective for the growth of gram-negative bacteria. Gram-positive organisms are inhibited by the crystal violet and bile salts.

5. Columbia colistin-nalidixic acid agar (CNA) is selective for the growth of gram-positive cocci.

 

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