3 Ways to Do Gram's Method of Staining

2024 Author: Jason Gerald | [email protected]. Last modified: 2023-12-16 10:50

Gram staining is a rapid technique and is used to see the presence of bacteria in a tissue sample and to classify the bacteria as Gram-positive or Gram-negative, based on the chemical and physical properties of their cell walls. Gram stain is almost always used as the first step in diagnosing a bacterial infection.

This staining technique is named after the Danish scientist Hans Christian Gram (1853 - 1938), who developed this technique in 1882 and published it in 1884 as a technique for distinguishing between two types of bacteria with similar clinical symptoms: Streptococcus pneumoniae (also known as Streptococcus pneumoniae). pneumococci) and the bacteria Klebsiella pneumoniae.

Step

Method 1 of 3: Preparing the Microscope Slide

Step 1. Prepare to work in the laboratory

Wear gloves and a long hair tie to prevent bacterial contamination of the sample you are testing. Clean the workspace under a fume hood, or in another well-ventilated area. Check the Bunsen burner and make sure the microscope is working properly before you start.

Step 2. Sterilize the microscope glass slide

If the glass slide is dirty, wash it with soapy water to remove oil and dirt. Clean the slides with ethanol, glass cleaner, or another method used by your laboratory.

Step 3. Place the sample onto the glass slide

You can use the Gram stain technique to help identify bacteria in a medical sample, or a culture of bacteria growing in a petri dish. For best results, use Gram stain on thin strokes of the sample. It is advisable to use samples that are less than 24 hours old, as older bacteria may have damaged their cell walls and are less responsive to Gram staining.

If using a tissue sample, add 1-2 drops to a glass slide. Spread evenly on the slide to form a thin layer of sample sprinkling, by sliding it using the edge of the other sterile glass slide. Let it dry before doing the next step.
If you are taking bacteria from a petri dish, sterilize the inoculation loop in a Bunsen burner until it glows, then allow it to cool. Use the loop to drip sterile water onto the slide, then sterilize and cool again before using it to collect a small sample of bacteria. After that stir gently.
The bacteria prepared in the broth must be stirred again using a vortexer, then taken with the inoculation loop as above, without adding water.
If you have a swab sample (usually done with a small cotton-tipped handle), touch and gently rotate the swab over the slide.

Step 4. A slightly high temperature can make a good smear

The heat will hold the bacteria on top of the slide, so they don't dissolve easily during the staining process. Tap the slide quickly two to three times over a Bunsen burner, or heat the slide over an electric slide heater. Do not overheat, the sample may be damaged. If you're using a Bunsen burner, it should be a small but blue flame instead of a large orange flame.

Alternatively, a smear can also be made with methanol, by adding 1-2 drops of methanol to a dry smear, drying the remaining methanol on the slide by leaving it in the open air. This technique minimizes cell damage and provides a clean slide image background

Step 5. Position the slide over the coloring tray

Staining trays are made of metal, glass, or shallow plastic plates with a soft mesh lining the top. Place the slides on top of these nets, so that the liquid you use can be drained into the tray.

If you don't have a coloring tray, you can place a slide on a plastic tray to print ice cubes

Method 2 of 3: Gram Stain Process

Step 1. Pour the crystal violet liquid over the smear

Use a pipette to spray a sample of bacteria with a few drops of crystal violet dye, also known as gentian violet. Leave it for 30-60 seconds. Crystal violet (KV) separates in aqueous solution into KV+ and chloride (Cl-) ions. These ions penetrate the cell walls and cell membranes of both gram-positive and gram-negative bacteria. The KV+ ion interacts with the negatively charged components of the bacterial cells, giving the cells a purple color.

Many laboratories use "Hucker" crystal violet, which contains ammonium oxalate to prevent precipitation

Step 2. Rinse the Crystal violet gently

Tilt the slide and use a washer bottle to spray a small stream of distilled or tap water over the slide. Water should run down over the surface of the smear, and should not be sprayed directly on the smear. Do not rinse excessively. It can remove staining in Gram positive bacteria.

Step 3. Flush the smear with iodine, then rinse

Use a dropper to flush the smear with iodine. Leave it for at least 60 seconds, then rinse carefully in the same way as above. Iodine, in the form of a negatively charged ion, interacts with KV+ to form a large compound complex between Crystal violet and iodine (CV-I compound complex) in the inner and outer layers of the cell. This complex of compounds will retain the violet color of the crystal violet inside the cell, at the stained locations.

Iodine is a corrosive substance. Avoid inhalation, ingestion or contact with skin

Step 4. Add color bleach, then rinse off quickly

A 1:1 mixture of acetone and ethanol is usually used for this important step, which must be carefully timed. Position the slide at a certain angle, then add color bleach until no more purple is visible in the water used for rinsing. This usually takes less than 10 seconds, or even less time if the bleach contains a high concentration of acetone. Stop this step immediately, otherwise the dye will also leach crystal violet from the gram-positive and negative cells, and the staining process must be repeated. Immediately rinse off any excess color bleach using the previous technique.

Pure acetone (95%+) can be used as a substitute. The more acetone you use, the faster the bleach will work so you need to pay more attention to the timing.
If you're having trouble keeping track of the time for this step, add color bleach drop by drop.

Step 5. Sprinkle the counter-dye over the smear, then rinse

A counter stain, usually safranin or fuchsin, is used to increase the contrast between gram-negative and gram-positive bacteria, by staining decolorized (decolorized) bacteria, i.e. gram-negative bacteria, pink or red. Leave for at least 45 seconds, then rinse.

Fuchsin will intensively stain many gram-negative bacteria, such as Haemophilus spp and Legionella spp. This method is good for beginners

Step 6. Dry the slide

You can dry them in the open air to dry, or dry them using bibulous paper sold for this purpose. The Gram staining process is complete.

Method 3 of 3: Checking the Coloring Results

Step 1. Prepare the light microscope

Place the slide under the microscope. Bacteria vary widely in size, so the total magnification required will vary from 400x to 1000x. An objective lens with immersion oil is recommended for a sharper image. Drop the immersion oil onto the slide, avoiding movement while dripping to prevent bubbles from forming. Move the microscope lens handle so that the objective lens snaps into place and touches the oil.

Oil immersion can only be used on specially designed lenses, not on "dry" lenses

Step 2. Try to identify gram positive and gram negative bacteria

Examine the slide under a light microscope. Gram-positive bacteria appear purple in color, because the crystal violet is trapped within the thick cell wall. Gram-negative bacteria will be pink or red in color, because the crystal violet is rinsed out through the thin cell walls, where the pink counter-dye enters.

If the sample is too thick, the result may be a false positive. Stain a new sample if it shows all gram positive bacteria, to make sure the result is correct.
If you use too much color bleach, the result can be a false negative. Stain the new sample if it shows all gram negative bacteria, to make sure the result is correct.

Step 3. Compare the results you see with the reference image

If you don't know what bacteria look like, study the collection of reference images, usually sorted by shape and Gram stain. You can also view online databases at [National Microbial Pathogen Database, Bacteria in Photos, and many other online sites. To facilitate identification, below are examples of bacteria that are commonly encountered or important for diagnosis, classified according to their Gram status and shape.

Step 4. Identify gram-positive bacteria based on their shape

Bacteria are further classified according to their shape under the microscope, most commonly as cocci (spherical) or rods (cylindrical). Here are some examples of gram-positive bacteria (purple in color) according to their shape:

Gram positive cocci usually staphylococci (meaning group cocci) or streptococci (meaning chain cocci).
' Gram positive rods such as Bacillus, Clostridium, Corynebacterium, and Listeria. The rod bacteria Actinomyces spp. usually have branches or filaments.

Step 5. Identify gram negative bacteria

Gram-negative bacteria (pink in color) are often classified into three groups. Cocci are spherical in shape, rods are long and thin, while coccoid rods are in between.

Gram negative cocci The most common is Neisseria spp.
Gram-negative rods e.g. E. coli, Enterobacter, Klebsiella, Citrobacter, Serratia, Proteus, Salmonella, Shigella, Pseudomonas, and many others. Vibrio cholerae can be seen as a regular stem or a bent stem.
Gram-negative "coccoid" (or "coccobacilli") rod bacteria e.g. Bordetella, Brucella, Haemophilus and Pasteurella

Step 6. Check carefully if the results are mixed

Some bacteria are difficult to color precisely, due to the brittleness or waxy nature of their cell walls. These bacteria may show a mixture of purple or pink in the same cell, or between different cells in the same smear. Bacterial samples older than 24 hours can show this problem, but there are also some bacterial species that remain difficult to color at any sampling age. These bacteria require more specialized tests for identification, such as acid-fast staining, observation in bacterial culture, TSI culture media, or genetic testing.

Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium spp. all are gram-positive bacteria, but are often not clearly stained.
Small, slender bacteria such as Treponema, Chlamydia, and Rickettsia spp. difficult to stain by the Gram method.

Step 7. Dispose of any remaining consumables and equipment

This waste disposal procedure varies between laboratories and depends on the materials used. Typically, the liquid in the staining tray is disposed of in bottles labeled as hazardous waste. Soak slides in 10% bleach solution, then dispose of in a sharps container.

Tips

Remember that Gram stain results will only be good if the sample is good. It is important to inform patients so that they can provide a good specimen (eg, the difference between spitting versus a deep cough to obtain a sputum sample).
As a color bleach, ethanol reacts more slowly than acetone.
Adhere to standard laboratory regulations to ensure safety.
Use a cheek swab to practice, because it contains both gram-positive and gram-negative bacteria. If you see only one type of bacteria, you may be using too little or too much bleach.
You can use wooden clothespins to hold the slide in place.

Warning

Acetone and ethanol are flammable substances. The acetone will remove the oil from your hands, making it easier for your skin to absorb other chemicals. Wear gloves and use them with care.
Do not allow the smear to dry before you rinse off the Gram stain or counterstain.