INTRODUCTION TO GRAM STAINING
Grams staining technique is the most important and widely used differential staining technique in Bacteriology. This technique was developed in 1884 by a Danish bacteriologist, Hans Christian Gram. The Gram staining technique differentiates the mixed culture cells into two terms –
One which retains the color of primary stain is known as Gram-Positive Bacteria and the cells which get decolorized and takes the counterstain are known as Gram-Negative Bacteria.
PRINCIPLE / MECHANISM OF GRAMs STAINING
⇒ The exact principle or mechanism of gram’s Staining is still unknown; however certain theories have been proposed to explain the mechanism of Gram’s reaction which is as follows –
- The Acid Protoplasmic Theory – This theory states that As the protoplasm of Gram-Positive Bacteria is more Acidic as compare to Gram-Negative Bacteria, their affinity with basic stain is more and they resists the Decolorization when decolorizer is applied whereas the affinity of Gram-Negative Bacteria with basic dyes is less so they easily get decolorized when decolorizer is applied.
- The Lipid Theory – The lipid content of the cell wall is more in Gram-negative bacteria than Gram-positive bacteria. During Gram’s reaction, there is the formation of Dye-Iodine complex in Gram positive & Gram negative bacteria, when the decolorizer is added to the lipid cell wall dissolves, leading to increases in pore size and the Dye-Iodine complex diffuses out during decolorization.
- The Cell Wall Theory – This theory is most accurate of all the others and widely accepted. It states that:
- The peptidoglycan layer of the gram +ve bacteria is thick while that of gram –ve bacteria is thin. Over the peptidoglycan layer, a Lipopolysaccharide layer is present which is thick in Gram-ve bacteria and thin in Gram +ve bacteria.
- When the primary stain is applied to the mixture of bacteria & fixed (via mordant), it gets fixed in the peptidoglycan layer of Gram +ve bacteria and in Gram-ve bacteria, it gets fixed in the lipopolysaccharide and minutely in peptidoglycan.
- The lipopolysaccharide layer is soluble in organic solvents so when the decolorizer is applied it gets dissolved and removed out from the bacteria. Here, the Gram-ve bacteria lose the Primary stain and got colorless again whereas the Gram +ve bacteria appears violet/blue color because they retain the primary stain as the stain was bonded in the peptidoglycan layer of Gram +ve bacteria which does not get washed away.
- After this, when the counterstain is applied, the colorless gram –ve cells retain it and appears as pink color bodies under the microscope.
REQUIREMENTS FOR GRAMs STAINING
- Glass Slides
- Specimen/Bacterial culture
- Tissue paper
- Inoculating loop
- Spirit lamp/Bunsen burner
- Staining tray
- Wash Bottle
- Crystal Violet
- Gram’s Iodine
- Decolorizer/95% Ethanol
PROCEDURE OF GRAMs STAINING
- Take a Clean, Grease free glass slide.
- Prepare a smear on it from the Clinical specimen or Culture.
- Fix the smear onto the slide by passing over the flame of Spirit lamp or Bunsen burner.
- Gently flood the Smear with Crystal violet & let it stand for 1 minute.
- Wash the smear with distilled water using Wash bottle.
- Cover the smear with Gram’s iodine for 1 minute.
- Wash the smear with distilled water using wash bottle.
- The smear will appear as a blue-black circle on the slide. Allow it to air dry.
- Apply the Decolorizer or 95% Ethanol for 5-10 seconds or until the alcohol runs almost clear. Be careful not to over decolorize it.
- Immediately wash with distilled water using wash bottle and allow it to air dry.
- Cover the smear with Safranine for 45 seconds to 1 minute.
- Wash with distilled water.
- Air dry the smear and observes under the microscope with the 100X objective lens.
INTERPRETATION OF GRAM STAINING
The bacteria that retain the primary stain i.e. Crystal violet and appears Violet/Blue are Gram-Positive Bacteria.
The bacteria that retain the Counter stain i.e. Safranine and appears Pink in color are Gram Negative Bacteria.
LIMITATIONS OF GRAM STAINING TECHNIQUE
⇒ During decolorization, two conditions may arise that results in erroneous results. The Over-decolorization of the smear may result in the identification of false gram-negative results, whereas the under-decolorization of smear may result in the identification of false gram-positive results.
⇒ Due to the Thickness of smear – The Smears which are too thick or viscous may retain the too much of primary stain that may give erroneous results. Also, the Gram-negative organisms may not decolorize properly and retain primary stain and may appear as gram-positive or gram-variable organisms under the microscope.
⇒ Precipitation of Stain particles – As the stain gets old, it may form the precipitate in the stain solution which may cause erroneous results. Using freshly prepared staining solution or filtering the stain solution through gauze will remove excess crystals and produce better results.
⇒ During antibiotic therapy – Gram-stained smears of the specimen taken from the patients on antibiotics or antimicrobial therapy may have altered Gram stain reactivity as many antibiotics alter the bacterial cell wall and cell membranes which results in variable gram staining.
⇒ Variations in staining – Some Gram-negative organisms like Campylobacter sp. and Brucella sp., fainty stain by the Gram-staining technique and for such organisms alternative counterstains, e.g. basic fuchsin can be used for better results.
EXAMPLES OF GRAM-POSITIVE & GRAM-NEGATIVE BACTERIA
Gram-Positive Bacteria: Actinomyces, Bacillus spp., Clostridium spp., Corynebacterium diptheriae, Gardnerella, Lactobacillus, Mycoplasma, Nocardia, Staphylococcus aureus, Staphylococcus epidermidis and other Staphylococcus spp. Streptococcus pneumoniae, Sterptococcus pyogenes, Streptomyces etc.
Gram-Negative Bacteria: Escherichia coli (E. coli), Salmonella typhi, Salmonella paratyphi and other Salmonella species. Shigella dysentriae, Klebsiella pneumoniae, Pseudomonas, Moraxella, Helicobacter pylori, Stenotrophomonas, Legionella etc.