How to Do a Gram Stain Step by Step for Accurate Results

How to Do a Gram Stain Step by Step for Accurate Results

Gram staining is a rapid method for characterizing the presence of bacteria in tissue samples and distinguishing Gram-positive from Gram-negative bacteria based on the chemical and physical properties of their cell walls. Gram staining is almost always the first step in diagnosing a bacterial infection. Gram staining is named after the Danish scientist Hans Christian Gram (1853–1938), who developed the technique in 1882 and published it in 1884 to distinguish between two types of bacteria with similar clinical symptoms: Streptococcus pneumoniae (also known as pneumococcus) and Klebsiella pneumoniae

Prepare the blade


 Put on gloves and tie back your long hair to limit the risk of contaminating the sample you are testing. Disinfect a work area under a fume hood or in a well-ventilated area. Make sure the Bunsen burner and microscope are operational before beginning.

 If it's dirty, wash it in soapy water to remove any visible traces. Then disinfect the slide with ethanol, glass cleaner, or any other method used in your lab.

 You can use Gram staining to identify bacteria from medical specimens or bacteria grown in Petri dishes . For Gram staining to be usable, use a "thin" layer of the sample. A recent sample (less than 24 hours old) is recommended; older bacteria may have damaged cell walls, which can distort Gram stain results.
  • If using a biological specimen, add one or two drops to the slide. Spread them in a thin layer over the entire surface of the slide with the edge of another sterilized slide. Allow to air dry before proceeding.
  • If using bacteria grown in a Petri dish, heat an inoculating loop in a Bunsen burner flame to sterilize it. Once cooled, use it to place a drop of sterile distilled water on the slide, then sterilize and cool the inoculating loop again to take a small sample of bacteria and mix it gently with the drop of water on the slide   .
  • Bacteria already in solution should be passed through a magnetic mixer and then placed on the slide using the inoculation loop, without the need to add water  .
  • If your sample is on a cotton ball (like a cotton swab), roll it gently over the slide 

 Heat will fix the bacteria to the slide so that they are not removed during staining. Quickly pass the slide through the Bunsen burner flame two or three times or heat it on top of an electric slide warmer. Do not overheat, or the samples may be skewed. If using a Bunsen burner, the flame should be small and blue, not a tall orange flame .
  • Bacteria can also be fixed in methanol, by adding one or two drops of methanol to the dry sample and allowing the preparation to dry on the slide. This method minimizes damage to host cells and allows for a clearer view of the bacteria that have grown within them.


 A staining tray is a shallow metal, glass, or plastic tray with a small rack or holder on top to hold the slide while allowing the staining liquid to drain into the tray.
  • If you do not have a staining tray, the slide can be placed on a plastic ice cube tray.

Carry out the coloring


 Using a pipette, place a few drops of crystal violet (a dye sometimes called gentian violet). Wait 30 to 60 seconds. Crystal violet (CV) in an aqueous solution dissociates into CV+ and chloride (Cl-) ions. These ions penetrate the cell wall and membrane of both Gram-positive and Gram-negative cells. The CV+ ions interact with negatively charged bacterial cell particles and stain these cells purple.
  • Many labs use Hucker violet crystals, which contain ammonium oxalate to prevent a precipitate from forming.

 Using a pipette, place a few drops of crystal violet (a dye sometimes called gentian violet). Wait 30 to 60 seconds. Crystal violet (CV) in an aqueous solution dissociates into CV+ and chloride (Cl-) ions. These ions penetrate the cell wall and membrane of both Gram-positive and Gram-negative cells. The CV+ ions interact with negatively charged bacterial cell particles and stain these cells purple.
  • Many labs use Hucker violet crystals, which contain ammonium oxalate to prevent a precipitate from forming.

 Tap the slide and rinse it with a wash bottle filled with distilled water or under the tap, taking care to run the water from top to bottom over the slide, but avoiding directing the stream of water onto the specimen. Do not rinse excessively, as this may wash away the staining of Gram-positive bacteria


 Use a pipette to pour in the iodine solution, let it stand for at least one minute, and rinse carefully using the same method as before ée negatively charged iodine combines with CV+ to form a large molecule within the inner and outer layers of the cell. This traps the crystal violet color within the cell.
  • Iodine is caustic. Avoid inhalation, ingestion, or skin contact.


 A 1:1 mixture of acetone and ethanol is typically used for this critical step, which requires precise timing. Hold the slide at an angle and pour the destainer until the stream remains clear, with no purple staining. This should normally take ten seconds or less, but may take much less depending on the concentration of the alcohol-acetone mixture. Stop immediately; otherwise, the destainer may remove all the purple stain from both Gram-positive and Gram-negative cells, and staining should be restarted from the beginning. Immediately rinse off any remaining destainer from the slide, following the technique described above.
  • Pure acetone (95% or higher) can be used instead of the alcohol and acetone solution. The more acetone in the solution, the faster the bleaching will occur, and the more precise the timing will need to be.
  • If you are having difficulty timing this step, try adding the food coloring drop by drop.


 A re-stainer, most often safranin or fuchsin, is used to enhance the contrast between Gram-negative and Gram-positive bacteria by staining the de-stained (Gram-negative) bacteria pink or red  . Leave the re-stainer on the slide for at least 45 seconds and then rinse it off .
  • Fuchsin dyes many Gram-negative bacteria (e.g., Haemophilus bacteria and Legionella) a particularly strong color. Therefore, it is particularly recommended for beginners.

 You can let it air dry or with absorbent paper provided for this purpose  . The Gram stain is now complete.

Analyze the coloring


 Place the slide under the microscope light. Bacteria vary greatly in size, so the required magnification can range from x400 to x1,000 igher magnifications, an oil immersion objective is recommended. Place a drop of oil on the coverslip, avoiding any movement during application to prevent bubbles. Rotate the turret until the objective is in position, in contact with the oil.
  • Immersion in a drop of oil can only be achieved with a lens designed for this purpose and not with an ordinary lens.

 Examine the slide under a light microscope. Gram-positive bacteria appear purple because the crystal violet is trapped within their thick cell walls, whereas Gram-negative bacteria appear pink or red because the crystal violet has passed through their thin cell walls and the pink re-stain has penetrated them.
  • If the sample is too thick, you may get false positive results. Perform a stain if all the bacteria are Gram-positive to ensure the result is correct.
  • If the staining has lasted too long, you may get false positive results. Restain a new sample if all bacteria appear Gram-negative to ensure the accuracy of your results.

 If you are unsure about the correct identification of a bacterium, consult a reference imaging database, categorized by shape and Gram stain reaction. Such databases are available online, for example, at this site: National Microbial Pathogen Database . To facilitate identification, examples of common or diagnostically significant bacteria are listed below by shape and Gram stain reaction.


 Bacteria are classified based on their appearance under a microscope, usually as coccus (round or shelled) or rod-shaped (cylindrical). Here are some Gram-positive bacteria (stained purple) classified by their shape:
  • Gram-positive cocci are generally either staphylococci (when in clusters) or streptococci (when in chains),
  • Gram-positive rod-shaped bacteria include bacilli, clostridia, coryneforms, and listeria .

 Gram-negative bacteria (stained pink) are generally classified into three groups. Cocci are spherical bacteria, rods are long, thin bacteria, and coccid (oval) rods are an intermediate category.
  • Gram-negative cocci are most often Neisserias.
  • Gram-negative rod-shaped bacteria include E. coli , Enterobacter , Klebsiella , Citrobacter , Serratia , Proteus , Salmonella , Shigella , Pseudomonas , and many others. Vibrio cholerae can appear as classic or curved rods  .
  • Gram-negative oval bacteria (or "coccobacilli") include Bordetella , Brucella , Haemophilus , and Pasteurella .


 Some bacteria show little staining due to the fragility of their cell walls. Occasionally, a mixture of purple and pink can be seen within the same cells or among different cells from the same sample. This may indicate that the sample is too old (more than 24 hours old), but some bacterial species are difficult to characterize, regardless of their age. This may require more specific tests to narrow down the number of hypotheses, such as Ziehl-Neelsen staining, observation of growth in culture, TSI agar tests, and genetic testing.
  • Actinomyces , Arthobacter , Corynebacterium , Mycobacterium , and Propionibacterium are considered Gram-positive bacteria, but often appear inconclusively stained.
  • Small and thin bacteria such as treponemes , Chlamydia and Rickettsia are difficult to stain correctly.

 Waste disposal procedures may vary depending on the laboratory and the equipment and products used. Typically, the liquid in the staining tray is disposed of in closed bottles designated for hazardous or contaminant waste. Slides are rinsed with 10% bleach and disposed of in a sharps container.

Advice

  • The relevance of the staining result depends largely on the quality of the sample analyzed: it is fundamental to teach patients how to provide usable samples (for example, the difference between sputum and mucus from a chesty cough).
  • Ethanol acts more slowly than acetone as a bleach.
  • Follow normal safety precautions for laboratory work.
  • Practice with a sample taken from the inside of the cheek, which usually has both Gram-positive and Gram-negative bacteria. If you only see one type of bacteria, you are likely using too much or too little bleach 
  • You can use a wooden clothespin to handle microscope slides
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