Gram staining is a quick procedure used to check for the presence of bacteria in tissue samples and to identify them as gram-positive or gram-negative, based on the chemical and physical properties of their cell walls. Gram staining should always be the first step in diagnosing a bacterial infection.
This practice is named after the Danish scientist Hans Christian Gram (1853-1938), who developed it in 1882 and published it in 1884, as a technique for distinguishing two types of bacteria with similar clinical symptoms: Streptococcus pneumoniae (known also as pneumococcus) and Klebsiella pneumoniae
Steps
Part 1 of 3: Prepare the Slide
Step 1. Prepare for laboratory work
Wear gloves and tie your long hair to avoid contaminating the sample of bacteria under test. Disinfect the work surface under a fume hood or in another well-ventilated area. Check that the microscope and Bunsen burner are in perfect working order before proceeding.
Step 2. Sterilize the slide
If it's dirty, wash it with soap and water to get rid of the grease and grime. Then disinfect it with ethanol, a glass cleaner or any other method recommended by the procedures of the laboratory where you work.
Step 3. Put a simple sample on the slide
You can use the Gram stain technique to identify bacteria on a medical sample or culture from a Petri dish. For a Gram stain to be useful, you need to add a subtle sample layer on the dye. It is best to work on a sample that is no older than 24 hours because, after this time, there is a greater chance that the cell membranes of the bacteria are damaged and therefore the staining is less effective and accurate.
- If using a tissue sample, pour 1-2 drops onto a slide. Spread it evenly on the slide to form a thin film. You can do this by pressing another slide over the first one that contains the sample. Let it air dry.
- If the bacteria came from a culture in a Petri dish, sterilize an inoculation stick over the flame of a Bunsen burner until it glows. Wait for it to cool and use it to drop a drop of sterilized water on the slide; sterilizes and cools the stick once again before transferring a thin sample of bacteria into the water. Mix them gently.
- The bacteria present in cultures (the bacterial “broth”) should be mixed in a centrifuge and then added to the slide via the stick without adding water.
- If the sample was collected with a swab, roll the tip of the cotton swab along the surface of the slide.
Step 4. Heat the specimen to fix the smear
The heat allows the specimen to adhere to the slide so that it is not lost during the stain rinses. Quickly slide the slide two or three times over the flame of a Bunsen burner or use a special electric heater. However, try not to overheat the smear to prevent it from being distorted. If you are using the Bunsen burner, adjust the flame to be a small blue cone, not a long orange one.
Alternatively, use methanol by adding 1-2 drops to the dry smear. Eliminate excess methanol and let the slide dry in the air. This method minimizes damage to host cells while leaving a sharper background
Step 5. Place the slide on the staining tray
It is a small shallow dish made of plastic, glass or metal with a grid or wire mesh on top. Place the slide on top of this mesh so that the liquids used can drain into the dish below.
If you don't have a coloring tray, use an ice cube tray instead
Part 2 of 3: Performing the Gram Stain
Step 1. Wash the slide with crystal violet
Use a pipette to wet the smear with several drops of this dye (sometimes called gentian violet). Wait 30-60 seconds. Crystal violet dissociates in an aqueous solution (CV +) and in chloride ions (Cl-). Ions penetrate through the membrane of both gram-positive and gram-negative cells. CV + ions interact with negatively charged components of bacterial cell walls by staining them purple.
Many laboratories use "Hucker's" gentian violet which is enriched with diammonium oxalate to prevent precipitation
Step 2. Rinse the crystal violet gently
Tilt the slide and use a spray bottle to wash it with a gentle stream of distilled or tap water. The water should flow over the smear without the flow hitting it directly. Do not overdo this as it may remove the stain from gram-positive bacterial cells.
Step 3. Rinse the sample with iodine and then with water
Again, use a pipette and coat the smear with iodine. Wait 60 seconds and then rinse with the same method described above. Iodine, in the form of negative ions, interacts with CV + cations to form larger complexes of crystal violet and iodine (CV-I) between the inner and outer layers of cells. This phase allows the purple color to settle on the cells where it has been absorbed.
Iodine is corrosive, avoid inhaling it, ingesting it and coming into contact with bare skin
Step 4. Now decolor the sample and perform a quick rinse
For this phase, a mixture of equal parts of acetone and ethanol is usually used. The bleaching time must be monitored very carefully. Grab the slide and tilt it, add the bleach mixture until you no longer notice the purple color in the rinse stream. It usually takes 10 seconds of flushing with the bleach (or even less if the concentration of acetone in the mixture is high). As soon as all the gentian violet has been removed from both gram positive and negative cells, stop or you will have to repeat all over again. Immediately rinse the excess bleaching mixture with the method described above.
- To decolourise the smear, you can also use pure acetone (concentration greater than 95%). The faster the bleaching is the higher the acetone content in the bleaching mixture; precisely for this reason you must be even more precise in calculating the timing.
- If you have trouble tracking the discoloration, add the mixture drop by drop.
Step 5. Wet the smear with the background stain and then rinse it off
The background coloring, mostly safranin or fuchsin, is used to increase the contrast between gram-positive and gram-negative bacteria by coloring the latter (which have been discolored by acetone) pink or red. Leave the second dye on for at least 45 seconds and then rinse it off.
Fuchsin stains gram-negative bacteria more intensely, such as haemophilus and legionella. These two types of bacteria are great as an exercise for beginners
Step 6. Dry the slide
You can wait for it to dry in the air or use absorbent paper specially designed for this purpose. The Gram stain is now complete.
Part 3 of 3: Review the Results
Step 1. Prepare the light microscope
Insert the slide under the objective and check for bacteria. These can vary a lot in size, so the total magnification needed varies from 400x to 1000x. If you use a very high magnification, it is better to use an objective lens in an oil bath to get clearer images. Put a drop of oil (for microscope) on the slide, avoiding moving it so as not to form bubbles. Move the microscope turret to select the immersion objective and then put it in contact with the oil.
The oil bath should only be used with specific lenses and not with normal "dry" lenses
Step 2. Recognize Gram Positive and Gram Negative Bacteria
Examine the slide with the light microscope; positive bacteria will be purple due to crystal violet trapped in their thick cell membranes. Gram negatives will be pink or red, because the gentian violet has been "washed away" from their thin cell walls and the background dye has penetrated.
- If the smear is too thick you may have false positive results. Stain a new sample if all bacteria are gram positive to make sure there are no errors.
- If the bleach flow has gone on too long, then you may have false negatives. Stain a new sample if all bacteria are gram negative to be sure of the results.
Step 3. Check the reference images
Step 4. Recognize Gram Positive Bacteria by Shape
Bacteria, in addition to coloring, are also grouped according to the shape that appears under the microscope. The most common are the “cocci” (spherical) or the rods (cylindrical). Here are some examples of gram-positive (purple-colored) bacteria categorized by shape:
- The gram-positive cocci they are generally Staphylococci (meaning cocci in groups) or Streptococci (meaning cocci in chains).
- The gram-positive rods they include Bacillus, Clostridium, Corynebacterium, and Listeria. Actinomyces often have filaments or branches.
Step 5. Identify Gram Negative Bacteria
These are colored pink and are mostly classified into three groups. The spherical cocci, the elongated and thin rods and finally the coccoids which are somewhere in between.
- The gram-negative cocci they are most commonly Neisseria.
- The gram-negative rods they include E. coli, Enterobacter, Klebsiella, Citrobacter, Serratia, Proteus, Salmonella, Shigella, Pseudomonas and much more. Vibrio cholerae can take the form of a normal rod or a curved rod.
- Gram-negative coccoid rods (or coccobacilli) include Bordetella, Brucella, Haemophilus, Pasteurella.
Step 6. Evaluate the mixed results
Some bacteria are difficult to accurately assess due to their fragile or impermeable cell walls. They may show a mixed purple and pink color or different colors for bacteria of the same type present in the same smear. All samples older than 24 hours have this problem, but there are strains of bacteria that are difficult to detect at each stage. In this case, specific tests are required to reduce the range of possibilities and reach their identification, such as Ziehl-Neelsen staining, observation in culture, genetic tests and TSI agar.
- Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium are all considered to be gram-positive bacteria, although sometimes they do not appear unambiguously colored.
- Small, fine bacteria such as Treponema, Chlamydia and Rickettsia are difficult to stain with the Gram technique.
Step 7. Dispose of the material
Procedures for disposing of material vary from laboratory to laboratory based on the type of material itself. Liquids in the staining tray are usually disposed of as hazardous waste after being sealed in special bottles. The slides are sterilized in a 10% bleach solution and then discarded as sharp instruments.
Advice
- Remember that the Gram stain result depends on the quality of the sample. It is important to teach patients how to provide good quality samples (such as indicating the difference between a spit and a deep cough for a phlegm sample).
- Ethanol is a slower bleaching agent than acetone.
- Follow all the prevention measures provided for the analysis laboratories.
- Use a swab from the inside of your cheeks to exercise, it should contain both gram positive and gram negative bacteria. If you only see one type of bacteria, you have probably used the bleach in the wrong amount.
- You can use a wooden clothespin (such as a clothespin) to grab the slide.
Warnings
- Acetone and ethanol are flammable. Acetone removes the sebum from the skin making it more permeable to other chemical agents. Use them with caution and wear gloves.
- Do not let the smear dry before rinsing off the main or basic stain.
