How to Gram Stain - wikiHow

How to Gram Stain

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.]]Gram staining is a quick procedure used to look for the presence of bacteria in tissue samples and to characterise bacteria as Gram-positive or Gram-negative, based on the chemical and physical properties of their cell walls.[1] The Gram stain should almost always be done as the first step in diagnosis of a bacteria infection. The 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.[2]

Steps

1. Get a tissue sample for the 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 (centrifuge the fluid and obtain the sediment for the 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 the likelihood of finding the bacteria, centrifuge the middle ear fluid sample to allow it to settle into three layers: supernatant (top), buffy coat (middle), and red cells (bottom); get the buffy coat layer for the Gram stain). Essentially any human tissue can be obtained for the 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 two to three 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 it minimises 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 shade.[3][4] Basic fuchsin, which stains anaerobic bacteria more intensely, may be substituted for safranin, but it is less commonly used.[5]
11. Gently rinse off excess safranin with tap water.
12. Drain slide and allow it to air dry. The Gram stain is done.
13. Examine the slide under the microscope. 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 classified 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.

Video

Tips

• Wear gloves for protection.
• 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 flammable. 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 wikiHows

• How to Treat a Bacterial Infection
• How to Know the Difference Between Bacteria and Viruses
• How to Grow Bacteria in a Petri Dish
• How to Put a Slide Together for a Microscope
• How to Use a Microscope to Observe Microorganisms

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.

1. ↑ Bergey, David H.; John G. Holt; Noel R. Krieg; Peter H.A. Sneath (1994). Bergey's Manual of Determinative Bacteriology (9th ed.). Lippincott Williams & Wilkins.
2. ↑ Gram, HC (1884). "Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten" (in German). Fortschritte der Medizin 2: 185–9.
3. ↑ Beveridge TJ, Davies JA (November 1983). "Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain". Journal of bacteriology 156 (2): 846–58.
4. ↑ Davies JA, Anderson GK, Beveridge TJ, Clark HC (November 1983). "Chemical mechanism of the Gram stain and synthesis of a new electron-opaque marker for electron microscopy which replaces the iodine mordant of the stain". Journal of bacteriology 156 (2): 837–45.
5. ↑ http://www.med-chem.com/procedures/GRAMSTAIN.pdf

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