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How to do a Gram's Stain Test

Gram’s Stain is one of the most frequently used processes in identifying bacteria and is used daily in hospitals. It is a primary test which quickly and cost effectively divides bacteria into one of two types: Gram-Positive or Gram-Negative.

Gram-Positive Bacteria have thicker cell walls with less fatty substances (lipids) than Gram-Negative bacteria. When washed with solvent, the cell pores close becoming less permeable and are able to retain the stain, in this case, purple.

Gram-Negative Bacteria have thinner cell walls with more lipids. The solvent dissolves the lipids, which combined with thinner cell walls, washes out or decolorizes the stain. In order to see the decolorized bacteria, a counter stain (Safranine O) is added to exaggerate the contrast with Gram-Negative cells.

Principle of Gram's Stain
The crystal violet stain is the primary stain, which stains everything in the smear purple- blue. The Gram's iodine acts as a mordant that causes the crystal violet to penetrate and adhere to the gram-positive cell. The acetone-alcohol mixture acts as the decolorizer that washes the stain away from everything in the smear except the gram-positive organisms. The safranine is the counter-stain that stains everything in the smear that has been decolorized in gram-negative organisms.

DIRECTIONS
There are six basic steps:

  1. Apply a smear of bacteria on to a slide. Air dry and then heat fix by passing it through a flame a few times. Make sure you air dry the bacteria before heat fixing.

  2. Add about 5 drops of Hucker’s Crystal Violet to the culture. Let stand for one minute. Bacteria will stain purple. Wash briefly with water and shake off excess.

  3. Add about 5 drops of iodine solution to the culture. Let stand for 30 seconds, wash briefly with water and shake off excess.

  4. 4.    Tilt slide and decolorize with solvent (acetone-alcohol solution) until purple color stops running. Be careful not to over-decolorize. Wash immediately (within 5 seconds) with water and shake off excess.

  5. Add about 5 drops of Safranine O. Let stand for one minute, wash briefly with water and shake off excess.

  6. Examine under microscope at both 400x and 1,000x oil immersion.

Examples:

Gram-Positive Bacteria
: Staphylococcus (Mostly harmless sphere-shaped, skin bacteria); Streptococcus (rod shaped bacteria: Rheumatic fever, Scarlet fever).

Gram-Negative Bacteria: Escherichia, Salmonella, Shigella (Meningitis, Gonorrhea, Flu and various Respiratory Diseases)

Interpretation:

  1. How does your stain look? In most cases, you will need a “control” image to ensure the gram stain works as it should. You can test your reagents against a known negative sample and a known positive sample. If you don’t get a solid red/purple stain, you will need to alter either the staining method or select new reagents.

 

As an alternative, if you don’t have known samples available, is to Gram stain a swab of the inside of your mouth. The oral bacteria present will show positive and negative in a good stain. If only one result shows up, either your staining method was flawed or there was a problem with the reagent used.

 

If everything shows negative (red) you can shorten the decolorization time. If everything shows positive (purple), try a slightly longer decolorization period.

 

  1. Is your Gram Stain positive or negative? See if the organisms show Grams-Negative (red) or Grams-Positive (purple). Try to evaluate a number of different areas in the slide, ideally where the organisms are spread out. Compacted areas will often show purple even with negative species.

 

  1. How big are the cells? In microscopy, size variation can be enormous. For instance, microbes are only 10 nanometers or less, while common debris on the slide can be much larger. Even cells taken from your body can be 10 times larger, or more. If you swabbed and stained a sample of your cheek, you may notice a faint pink (cell cytoplasm) along with a light purple (the cell nucleus). These cells are usually bigger than 10 microns in size.

 

You may also notice large positive-stained filaments, bigger than bacteria, indicating the presence of mold and fungi.

 

  1. What is the origin of the bacteria? Oval bacteria are called rods, while circular organisms are called cocci. Slightly oblong bacteria are named ‘coccobacilli’. You may run across fibrous or filamentous cells similar to fungi, but not quite as wide. These are commonly found in soils. You could find cells with areas of red and purple, often referred to as “Gram-variable”, provided your Gram test was sufficient. This may happen when variations in the lipids of a cell wall repel some of the stain.