December 4, 2020

Microbiology 101 – The Gram Spot

The Gram stain: even many set people have heard of this basic but incredibly important test that serves an integral role in the microbiology laboratory. Here I would like to give a brief summary over the basics of the Gram stain.

The staining procedure for bacteria has been discovered over 100 years ago simply by Hans Christian Gram. Almost all medically important bacteria can be detected by using this stain. Exceptions are few including intracellular bacteria like Chlamydia, bacteria that lack a cell wall structure such as mycoplasma and spirochetes such as Treponema which due to their dimensions can not be resolved by a light microscope.

The Gram stain divide bacteria into two major categories; gram beneficial, which takes up and retains the main stain, and gram negative, which the primary stain can be washed out by a decolorizing acetone-alcohol.

How do you perform the procedure?

First you must fix the specimen to be examined (from a clinical source or from an agar plate) to the microscope slide possibly by heating the slide or even by the use of methanol.

After the specimen is usually fixed, flood the slide with the primary stain, crystal violet (purple). Here the bacterial cells are filled with stain. Rinse the glide with water.

Next flood the particular slide with Gram’s iodine. A crystal violet/iodine complex is produced in the cell. Rinse the slide.

Wash the slide with an natural solvent (acetone-alcohol) decolorizer and finally spot with a counterstain, safranin (red).

For what reason some bacteria stain gram optimistic and others gram negative?

This is because of different cell wall composition using the two bacterial groups. A gram positive organism, e. g. Staphylococcus aureus will retain the crystal purple because of a thick peptidoglycan layer and teichoic acid cross-links (cells appear purple microscopically).
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Gram negative germs, e. g. E. coli lack this cell wall and the very violet/iodine complex is leeched from the bacterial cell during decolorization. The particular acetone-alcohol disrupts the outer membrane of the gram negative cell causing the decolorization. After decolorization of gram negative bacteria, the cells become colorless and are subsequently filled with safranin (red) giving it the gram negative appearance microscopically (pink-red).

A Gram discolored smear is examined using the essential oil immersion objective (1000x). Not only do a person report the Gram reaction (positive or negative) but also the bacterial morphology (cocci or bacilli) and any formations (clusters or stores for example).

How is the Gram stain useful with clinical examples?

Often times treatment decisions can be produced based on the Gram stain result only. One example is in a critical culture site like blood or CSF where time is of the essence, a gram stain of gram optimistic cocci in clusters can allow treating physician know the he/she can be dealing with a staphylococcus species plus an appropriate antibiotic for staph could be started while waiting for the lifestyle result.

The Gram stain is among the most important laboratory procedures in the scientific microbiology laboratory and may appear to be deceptively simple. Instead, a technologist who may be very proficient at reading Gram discolored smears typically has considerable knowledge and training.

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