Doctors often want to determine what type of bacteria is present in a patient who has an infection. Therefore, professionals should perform several tests, and one of those tests is gram staining procedure. Experts use gram staining procedure as one of the techniques to differentiate 2 bacteria groups according to their cell wall characteristics. It involves using a dye to see which bacteria retain either color—violet or red in their cell walls.

When to Apply Gram Staining

• Gram staining procedure is used to determine if bacteria are gram-positive or gram-negative. This is usually the first step in the process of identifying bacteria that are present in cultures.
• This procedure is important in giving a clue to the diagnosis in people with infectious diseases. It also helps in the estimation of the total count of the bacteria.
• Doctors can make an empirical choice on the type of antibiotics they may prescribe to a patient, based on the results of the gram staining procedure.
• The choice of a culture media to be used for inoculation can also be made empirically based on the gram stain report.

How Is the Gram Staining Procedure Performed?

1. Prepare the Glass Microscopic Slide

To make sure that your slides are grease/oil-free, you need to first wash them with soap and water then wipe slides with alcohol. This will remove any fingerprints or dirt from them. Dry the slides and put them on lab towels until they are ready to be used.

2. Label the Slides

Draw a circle under the slides using a marking pen designed for glassware. This will help to designate which area to prepare the smear in the following step. You can also label them with the organism's initials at the edge of each slide. Take care that the labels do not get in contact with the reagentsused forstaining.

3. Prepare the Smear

To make a bacterial suspension in broth, use a cooled sterile loop to place a small amount of broth culture on your slide. Using a circular motion spread the broth with the inoculating loop until it is about 1 cm in diameter. Do not spread excessively to prevent disruption of the cellular arrangement. A suitable smear allows examination of isolated cells and their characteristic cellular arrangement.

To make a bacterial plate culture, use a cooled sterile loop and place a drop of saline solution or sterile water on a slide. After re-sterilizing and cooling the loop, pick up a tiny sample of bacterial colony. Gently stir the colony into the water or saline solution on the slide and make an emulsion.

For swab samples, roll a swab over a clean glass slide

Note: Avoid preparing a thick, dense smear with excess bacterial sample. This will prevent light from passing through and make it difficult to see the cellmorphology. Smears usually require small amounts of bacterial culture only. An ideal smear consists of a whitish thin film after the heat-fixing process.

4. Heat Fixing

This step kills the bacteria on the smear, and makes them firmly adhere to the slide, allowing the sample to take up stains more readily. Air-dry your smear. While holding your slide at one end (smear-side up), pass the slide through flame from a Bunsen burner, 2-3 times. Avoid over-heating to prevent coagulation and distortion of the proteins in the specimen. Now you are ready for the gram staining procedure.

5. Gram Staining

• Place the heat-fixed smear on a staining tray.
• Flood your smear gently with crystal violet. Let it stand for one minute.
• Slightly tilt slide and rinse gently with tap water or distilled water using a wash bottle.
• Now flood the smear gently with Gram's iodine. Let it stand for one minute.
• Slightly tilt slide and rinse gently with tap water or distilled water from a wash bottle. Now your smear will appear purple on your slide.
• Using acetone or 95% ethyl alcohol, decolorize the smear. Slightly tilt slide and apply alcohol by drops for 5-10 seconds until liquid runs clear. Do not over-decolorize.
• Rinse immediately using water.
• Counterstain by gently flooding the smear with safranin. Let stand for about 45 seconds.
• Slightly tilt slide and rinse gently with tap water or distilled water from a wash bottle.
• Blot it dry with bibulous paper.
• Examine the smear under a light-microscope (oil-immersion).

Here is video illustrating how to perform the gram staining procedure in details:

What Results Do Gram Staining Procedure Tell?

Gram staining procedure helps distinguish bacteria with thicker cell walls from those with thinner cell walls, owing to the amount of peptidoglycan in their walls. Cells with thinner walls have more of a lipopolysaccharide layer than peptidoglycans in their walls.

Crystal violet and iodine enter both types of bacterial cell walls and combine to become larger molecules within these walls. However, when washed with alcohol, bacteria with thicker cell walls containing peptidoglycans tend to retain these molecules and remain violet in color, thus they are called Gram-positive bacteria. On the other hand, the dye is washed away from bacteria with thinner cell walls, and these are considered gram-negative. The latter do not become colorless, but pink whencounterstained with safranin.