Gram Stain

Gram staining is a quick procedure to look for the presence of bacteria in tissue samples and to characterise bacteria as Gram-positive or Gram-negative, based on chemical and physical properties of their cell walls. The Gram stain should almost always be done as the first step in diagnosis of a bacteria infection.

Gram stain is named after the Danish scientist Hans Christian Gram (1853 – 1938), who developed the technique in 1882 and published it in 1884 as a technique to discriminate between two types of bacteria with similar clinical symptoms: Streptococcus pneumoniae (also known as the pneumococcus) and Klebsiella pneumoniae bacteria.

Steps

 * 1) Get tissue sample for Gram stain. For example, if a pneumonia is suspected, get a sputum sample. If an urinary tract infection is suspected, get a urine sample. If an intestinal infection is suspected, get a stool sample. If a brain infection is suspected, get a cerebrospinal fluid (CSF) sample (need to centrifuge the fluid and obtain the sediment for gram stain). If a skin infection is suspected, get a skin biopsy sample. If an ear infection is suspected, get a middle ear fluid sample (to increase yield, or likelihood of finding the bacteria, centrifuge the middle ear fluid sample to allow it to settle into three layers: supernatant (top), buffy coat (middle), red cells (bottom); get the buffy coat layer for Gram stain). Essentially any human tissue can be obtained for Gram stain.
 * 2) Add 1-2 drops of the tissue sample onto a glass slide. Spread it evenly on the slide to form a thin smear, which can be done by sliding the edge of another glass slide across the glass slide containing the tissue sample. Allow it to air dry.
 * 3) Heat fix the smear, by quickly passing it 2-3 times through a flame, or heat it on top of an electric slide warmer. Do not overheat, to avoid distortion. Alternatively, the smear may be fixed by methanol instead, by adding 1-2 drops of methanol onto the dried smear, draining off excess methanol, and allowing it to air dry. Methanol has the advantage that it does not lyse red cells and minimise damage to host cells, giving a cleaner background.
 * 4) Flood the smear with crystal violet. Wait thirty seconds. Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl–) ions. These ions penetrate through the cell wall and cell membrane of both gram-positive and gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells to stain the cells purple.
 * 5) Gently rinse off the crystal violet with tap water. Do not rinse excessively, which might remove the stain from Gram positive bacteria.
 * 6) Flood the smear with iodine. Leave for at least three seconds. Iodine, in the form of negatively charged ions, interacts with CV+ to form large complexes of crystal violet and iodine (CV–I complexes) within the inner and outer layers of the cell. Iodine acts as a trapping agent to retain the purple crystal violet colour in the cell.
 * 7) Gently rinse off the iodine with tap water.
 * 8) Decolourise by adding alcohol or acetone to the smear while holding the slide at an angle to allow the declouriser to drain. Stop when runoff becomes clear, within seconds. The decolourisation step is critical and must be timed correctly. If left on too long, the decolourising agent will remove the crystal violet stain from both gram-positive and negative cells. A decolouriser, such as alcohol or acetone, interacts with the lipids of the bacterial cell membrane. A gram-negative bacteria will lose its outer membrane, allowing the CV–I complexes to wash out from the gram-negative bacteria along with the outer membrane. In contrast, a gram-positive cell will become dehydrated from an ethanol treatment, trapping the large CV–I complexes within the multilayered peptidoglycan cell wall of the gram-positive bacteria. After decolourisation, the gram-positive cell remains purple and the gram-negative cell loses the purple stain.
 * 9) Gently rinse off excess decolouriser with tap water.
 * 10) Flood the smear with safranin counterstain. Wait thirty seconds. Counterstain is applied last to stain the decolourised gram-negative bacteria a pink or red. Basic fuchsin, which stains anaerobic bacteria more intensely, may be substituted for safranin, but it is less commonly used.
 * 11) Gently rinse off excess safranin with tap water.
 * 12) Drain slide and allow it to air dry. Gram stain is done.
 * 13) Examine the slide under the microscope.

Tips

 * Wear gloves for protection.
 * Gram-positive bacteria appear purple as stained by crystal violet, which is trapped within their thick cell walls. Gram-negative bacteria appear pink as stained by the safranin counter-stain, as their thin cell walls allow the crystals violet to wash out during decolourisation. Bacteria are further classifed by their shape under the microscope, most commonly as cocci (spherical) or rods (cylindrical). The most common bacterial species in each of the four groups thus classified are as follows:
 * Gram positive cocci are either Staphylococci (meaning cocci in clusters) or Streptococci (meaning cocci in chains).
 * Gram positive rods include Bacillus, Clostridium, Corynebacterium, and Listeria.
 * Gram negative cocci are Neisseria spp.
 * Gram negative rods are subclassified as follows:
 * Gram negative "coccoid" rods (or coccobacilli) include Bordetella, Brucella, Haemophilus, Pasteurella.
 * Other Gram negative rods (not coccobacilli) include E. coli, Enterobacter, Klebsiella, Citrobacter, Serratia, Proteus, Salmonella, Shigella, Pseudomonas, and many others.
 * As declouriser, ethanol acts slower than acetone.
 * To ensure good staining results, use clean glass slides, make thin smears, do not overheat, and avoid excessive rinsing.

Warnings

 * Acetone and ethanol are inflammable. Use with caution.
 * Iodine is corrosive. Avoid inhalation, ingestion, or skin contact. Wear gloves, and work in a well-ventilated area.

Things You'll Need

 * Tissue sample
 * Centrifuge (helpful for certain specimens)
 * Glass slides
 * Pipette
 * A source of flame, or a slide warmer, or methanol
 * Water
 * Crystal violet
 * Iodine
 * Declourising agent, such as alcohol or acetone
 * Safranin

Related Tips and Steps

 * How to Treat a Bacterial Infection
 * How to Know the Difference Between Bacteria and Viruses
 * How to Grow Bacteria in a Petri Dish

Sources and Citations

 * Isenberg, H.D. (ed). 1992. Clinical Microbiology Procedures Handbook . American Society for Microbiology, Washington, D.C.
 * Isenberg, H.D. (ed). 1995. Essential Procedures for clinical Microbiology. American Society for Microbiology, Washington, D.C.