The Gram stain is a rapid procedure used to look for the presence of bacteria in tissue samples and characterize them as Gram positive or Gram negative based on the chemical and physical properties of their cell walls. The Gram stain should almost always be performed as the first step in diagnosing a bacterial infection.
The Gram stain is named for the Danish scientist Hans Christian Gram (1853 - 1938), who developed the technique in 1882 and published it in 1884 as a technique to distinguish between two types of bacteria with similar clinical symptoms: Streptococcus pneumoniae bacteria. (also known as pneumococcus) and Klebsiella pneumoniae.
Part 1 of 3: Prepare the slide
Step 1. Prepare for lab work
Put on gloves and tie your hair if it is long to avoid contaminating the sample of bacteria that you are going to analyze. Sanitize a workspace under the range hood or in another well-ventilated area. Before you begin, check that the Bunsen burner and microscope are operational.
Step 2. Sterilize a glass microscope slide
If the slide is dirty, wash it in soapy water to remove grease and dirt. Disinfect it with ethanol, glass cleaner, or whatever method your lab recommends.
Step 3. Place the sample on the slide
You can use the Gram stain method to help identify bacteria present in medical samples or in bacterial cultures in a Petri dish. To make the Gram stain helpful, layer slim of the sample on the slide. A sample that is less than 24 hours old is recommended, as older bacteria may have damaged cell walls that will respond less predictably to Gram stain.
- If you are using a tissue sample, place 1 to 2 drops of the sample on the glass slide. Spread evenly over the slide to form a thin spot using the edge of a second sterile glass slide. Let it air dry before continuing.
- If you are taking bacteria from a Petri dish, sterilize a bacteriological loop in the flame of a Bunsen burner until it glows, then let it cool. Use it to place a drop of sterile water on the slide and then sterilize and cool the loop again before transferring a small sample of bacteria to the slide and gently swirling it in the water.
- Bacteria in culture medium should be mixed on a vortex shaker and then placed on the slide with a bacteriological loop, as above, but without adding the additional water.
- If you have a sample on a swab, wipe it slightly over the slide.
Step 4. Fix the sample by heat
The heat will fix the bacteria to the slide so that they do not rinse as easily during staining. Quickly run the slide two or three times through the flame of a Bunsen burner or heat it over an electric slide warmer. Do not overheat it or the samples may be distorted. If you are using a Bunsen burner, the flame should be a small, blue cone, not a tall, orange one.
Alternatively, the sample can be fixed with methanol, adding 1 or 2 drops to the dry spot, draining the excess methanol and allowing it to air dry. This method minimizes damage to host cells, giving them a cleaner background
Step 5. Position the slide on a tray for staining
A staining tray is a shallow metal, glass, or plastic tray with a small mesh or wire support across the top. Place the slide on this holder so that liquids to be used can drain onto the tray.
If you don't have a staining tray, the slide can be placed directly on a plastic ice bucket
Part 2 of 3: Perform the Gram stain
Step 1. Soak the sample with crystal violet
Use a pipette to soak the bacteria sample with several drops of crystal violet dye, sometimes called gentian violet. Wait 30 to 60 seconds. In an aqueous solution, crystal violet (CV) dissociates into CV + and chloride ions (Cl-). These ions penetrate through the cell wall and cell membrane of both Gram positive and Gram negative cells. The CV + ion interacts with the negatively charged components of bacterial cells to stain them purple.
Many laboratories use "Hucker's" crystal violet, which adds ammonium oxalate to prevent precipitation
Step 2. Gently rinse the purple crystal
Tilt the slide and use a wash flask to squirt a small stream of distilled or tap water over the top of the slide. The water should run off the surface of the stain but not be pointed directly at it. Don't over-rinse, which can remove the stain from Gram-positive bacteria.
Step 3. Soak the stain with iodine, then rinse
Use a pipette to cover the stain with iodine. Let it sit for at least 60 seconds, then rinse it carefully using the same method. Iodine, in the form of negatively charged ions, interacts with CV + ions to form large crystal violet and iodine compounds (CV-I compounds) within the inner and outer layers of the cell. This traps the color of the crystal violet in the cell, where it was stained.
Iodine is corrosive. Avoid inhalation, ingestion or contact with the skin
Step 4. Add a bleach and then rinse quickly
For this critical step, a mixture of acetone and ethanol in a ratio of 1: 1 is normally used. The completion of this step must be carefully calculated. Hold the slide at an angle, then add the bleach until none of the purple is visible in the runoff. This normally takes less than 10 seconds or even less time if the bleach contains a high concentration of acetone. Stop immediately or the decolorizer will remove the crystal violet stain from both Gram positive and Gram negative cells and the staining will need to be repeated. Immediately rinse off excess bleach using the technique mentioned above.
- Pure acetone (95% +) can be used instead. The more acetone there is, the faster the bleach will work, which will require more accurate timing.
- If you are having trouble calculating this step, consider adding the bleach drop by drop.
Step 5. Soak the stain with a secondary stain, then rinse
A secondary dye, usually safranin or fuchsin, is used to add additional contrast between Gram positive and Gram negative bacteria, staining discolored (Gram negative) bacteria pink or red. Leave the dye in the sample for at least 45 seconds, then rinse it off.
Fuchsin will stain many Gram negative bacteria more intensely, such as Haemophilus and Legionella species. This can make it a better option for beginners
Step 6. Dry the slide
You can either let the slide air dry or dry it using absorbent paper that is bandaged for this purpose. The Gram stain is complete.
Part 3 of 3: Examine the result
Step 1. Prepare the light microscope
Place the slide under the light microscope. Bacteria vary greatly in size, so the total magnification required will vary from 400x to 1000x. At the higher end of these magnifications, it is recommended to use an oil immersion objective lens for clarity. Place a drop of immersion oil on the slide, avoiding movement during application to avoid bubbles. Move the microscope nosepiece so that the objective lens clicks into place, touching the oil.
Immersion oil can only be used on specially designed lenses, not on a "dry" lens
Step 2. Identify Gram positive and Gram negative bacteria
Examine the slide under the light microscope. Gram positive bacteria will appear purple due to the purple crystal trapped in their thick cell walls. Gram negative bacteria will appear pink or red as the violet was rinsed through the thin cell walls and then the pink secondary dye entered them.
- If the sample is too thick, you can get false positives. Stain a new sample if all of your results are Gram positive, to make sure the result is correct.
- If the bleach has run off for a long time, you can get false negatives. Stain a new sample if all your results are Gram negative to double check the results.
Step 3. Look for reference images
If you are not sure what a bacterium is, search collections of reference images classified by shape and result of Gram stain. You can find databases online at the US National Microbial Pathogens Database, Bacteria in Photos, and many other sites. To further facilitate identification, common or diagnostic examples are listed below by Gram status and form.
Step 4. Identify Gram positive bacteria by their shape
Bacteria are further classified by their shape under the microscope, most commonly as cocci (spherical) or rods (cylindrical). Here are some common Gram positive (violet stained) bacteria classified by shape:
- Gram positive cocci they are generally either staphylococci (which means cocci in groups) or streptococci (which means cocci in chains).
- Gram positive rods they include the bacilli and the bacteria Clostridium, Corynebacterium and Listeria. Rods of the Actinomyces species often have branches or filaments.
Step 5. Identify Gram negative bacteria
Gram negative bacteria (stained pink) are often classified into three groups. The cocci are the spherical bacteria, the rods are the long, thin bacteria, and the coccoid rods are somewhere in between.
- Gram negative cocci they are most commonly the Neisseria species.
- Gram negative rods They include the bacteria E. coli, Enterobacter, Klebsiella, Citrobacter, Serratia, Proteus, Salmonella, Shigella, Pseudomonas, and many others. Vibrio cholerae bacteria can appear as normal rods or curved rods.
- Gram negative "coccoid" rods (or "coccobacilli") They include Bordetella, Brucella, Haemophilus, and Pasteurella.
Step 6. Evaluate the mixed results
Some bacteria are difficult to stain accurately due to the fragility of their cell walls or how waxy they are. They can have a mixture of a purple or pink stain in the same cell or between different cells in the same sample. Any sample of bacteria that is older than 24 hours can have this problem, but some species are difficult to stain at any age. They may require more specialized tests to narrow down the number of possibilities for their identification, such as Ziehl-Neelsen stain, observation of culture growth, TSI agar, or genetic testing.
- Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium species are considered Gram-positive bacteria, but they often appear inconclusively stained.
- Small, thin bacteria, such as Treponema, Chlamydia, and Rickettsia species, are difficult to stain correctly.
Step 7. Discard the materials
Procedures for waste disposal vary between laboratories and depending on the materials used. Normally, the liquid in the staining tray is disposed of as hazardous waste in sealed bottles. Dip the slides in a 10% bleach solution, then dispose of them in containers for sharp instruments.
- Remember that the performance of the Gram stain is only as good as the specimen. It is important to teach patients to provide good specimens (for example, teach them the difference between a spit and a deep cough for sputum samples).
- As a bleach, ethanol works slower than acetone.
- Follow standard laboratory precautions.
- Use a buccal swab for practice, as the sample must contain both Gram positive and Gram negative bacteria. If you only see one or the other type of bacteria, you have probably used too little or too much bleach.
- You can use a spring-loaded wooden clothespin to hold the slide.
- Acetone and ethanol are flammable and acetone will dry out your hands. Wear gloves and handle them with caution.
- Don't let the stain dry before rinsing off the stain or secondary stain.