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The Gram Stain


               The Gram stain is an empirical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. The method is named after its inventor, the Danish scientist Hans Christian Joachim Gram (1853 1938), who formatted the technique in 1884.

               Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell wall), which stain purple and Gram-negative bacteria have a thinner layer (10% of cell wall), which stain pink.

               Gram-negative bacteria also have an additional outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space. There are four basic steps of the Gram stain, which include applying a primary stain crystal violet to a heat-fixed smear of a bacterial culture or specimen, followed by the addition of a mordant (Gram's iodine), rapid decolorization with alcohol or acetone and counterstaining with safranin. The Gram stain is the most common staining procedure in clinical microbiology. This tool can aid the physician in determining if infection is present, a possible etiology and a guide to choosing the appropriate antibiotic in just 15 minutes.


The Gram Positive Cell Wall

              The Gram positive cell wall The Gram positive cell wall is characterized by the presence of a very thick peptidoglycan layer, which is responsible for the retention of the crystal violet dyes during the Gram staining procedure. Imbedded in the Gram positive cell wall are polyalcohols called teichoic acids which are lipid-linked to form lipoteichoic acids. Because lipoteichoic acids are covalently linked to lipids within the cytoplasmic membrane they are responsible for linking the peptidoglycan to the cytoplasmic membrane. Teichoic acids give the Gram positive cell wall an overall negative charge due to the presence of phosphodiester bonds between teichoic acids monomers.

The Gram Negative Cell Wall

              The Gram negative cell wall Unlike the Gram positive cell wall, the Gram negative cell wall contains a thin peptidoglycan layer adjacent to the cytoplasmic membrane, which is responsible for the cell wall's inability to retain the crystal violet stain upon decolorization with ethanol during Gram staining. In addition to the peptidoglycan layer, the Gram negative cell wall also contains an additional outer membrane composed by phospholipids and lipopolysaccharides.

Gram Staining Tips

By Jack D. Rihs

1. Slide preparation

               Purulent material should be selected whenever possible.

               Apply the sample evenly and thinly to the slide. Smears that are too thick will be difficult to decolorize and imposable to read.

               Do not cover the entire slide with the sample. This will make handling difficult and areas may be missed during decolorization. An area the size of a nickel usually is adequate.

2. Slide fixation

               The material must be fixed to the slide to prevent it from washing off during staining. This can be done by quickly passing the slide over a gentle flame (the slide should not become to hot to touch) or on a slide warmer. Overheating may alter cell morphology or cause organisms to decolorize more quickly.

               An alternative and superior method of fixation is to flood the slide with methanol for 1 minute. Methanol fixation prevents liquid specimens from washing off the slide better than heat fixing, preserves blood cell morphology and results in a clearer background.

3. Staining

               Flood the entire slide when crystal violet, iodine and safranin are applied. This will ensure that all areas are stained evenly.

4. Decolorization

               The critical step of the Gram staining procedure is the decolorization step. Hold the slide in a tilted downward position and allow the decolorizer to flow over the smear. Be careful not to miss any portion of the smear. Usually a few seconds will suffice.

               95% ethanol will decolorize slower than acetone/alcohol, than does acetone.

5. Reading the Gram stain

               Begin reviewing the slide using the 10x objective. This will allow you to focus quickly and to look for areas of purulence. The 100x oil immersion lens is essential for viewing individual bacteria.

               When reviewing a Gram stain, look at many microscopic fields and in different areas of the slide.

               The presence of many squamous epithelial cells in the smear usually indicates a poorly collected sample that will contain normal flora, whereas, the presence of polymorphonuclear leukocytes and no squamous epithelial cells indicates a good sample and an inflammatory process.

               Some specimens on preparation will have areas on the slide that are thicker than others. This can result in areas that are under decolorized, thus organisms that are truly Gram-negative will appear Gram-positive. Microscopic fields that contain crystal violet precipitant, or PMNs, macrophages or epithelial cells that are staining purple are areas that are under decolorized.

               Gram positive bacteria will usually stand out easily against the pinkish background. Gram negative organisms because of their lower contrast can be missed, particularly in thicker smears. Haemophilus, Bacteroides and Fusobacterium are Gram-negative bacteria that are often overlooked due to their size, pleomorphic morphology or faintly staining qualities.