Gram Staining

Introduction

Gram staining, also called Gram's Method, is named after Hans Christian Gram who developed the method in 1884, which allows for one to distinguish between gram-postive and gram-negative bacteria using a combination of different chemical stains.  In this test, bacteria that retain the crystal violet dye do so because of a thick layer of peptidoglycan. In contrast, negative bacteria do not retain the violet dye and appear as red/pink. 

Background

Gram staining can be used to differentiate bacterial species into two large groups based on their physical properties. While gram staining is not the main method for diagnosing or identifying bacteria, it can be used to establish whether there are significant numbers of bacteria or if there are multiple types of bacteria. Gram stains are sometimes used in the medical field to quickly determine bacterial infection while waiting for culture results. 

Gram Postive Vs gram Negative bacteria

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Materials

  • Sample to be evaluated
  • Crystal Violet Solution
  • Gram's Iodine Solution
  • Decolorizing Solution
  • Counter Stain
  • Bibulous paper (optional)
  • Microscope with oil immersion objective
  • Deionized Water (DI)

Note: A gram stain kit may be used instead of individual staining reagents. 

Reagents and Solutions

Counter Stain

  • 2.5 g of Safranin O per 100 mL of 95% ethanol. (store up to 1 year at room temperature)

Crystal Violet Solution 

  • Crystal Violet Stock (Solution A) 
    • Dissolve 20 g of crystal violet (85% dye) in 100 mL of 95% ethanol. (Store up to 1 year at room temperature)
  • Oxalate stock (Solution B)
    • Dissolve 1 g of ammonium oxalate in 100 mL of water.  (Store up to 1 year at room temperature)
  • Working Solution (Crystal Violet Solution)
    • Dilute Solution A (1:10) with DI water and mix with 4 volumes work of Solution B.  (Store up to 6 months in a glass bottle at room temperature.)

Decolorizing Solution

  • Mix equal volumes of 95% ethanol and acetone.  (Store up to 1 year in a glass bottle at room temperature.)

Gram's Iodine Solution

  • Dissolve 1 g of  iodine crystals and 2 g of potassium iodide in 5 mL of water.
  • Add 240 mL of water and 60 mL of 5% (w/v) sodium bicarbonate solution and mix. (Store up to 6 months in an amber glass or foil covered bottle at room temperature.)

Method

Preparation of a Smear

  1. Hold an inoculating loop in flame until red hot, and allow to cool for ~30 s. Using the sterilized loop, prepare a thin film of sample to be stained on a glass slide. The culture can come from a thick suspension of a liquid culture, or a pure colony diluted in water.  (Be sure to use a small fresh culture as heavy or old cultures can stain erratically.)
  2. Air dry the smear then heat-fix by passing the slide over a flame two or three times (do not leave the slide over the flame, as this will overheat the sample and possibly shatter the slide.)

Stain Smear

  1. Flood the heat-fixed smear with crystal violet solution. Let stand for approximately 30 seconds. 
  2. Wash the stain off gently with DI water using a pipette or very slow stream. 
  3. Cover the smear with gram's iodine solution for 1 minute, then wash off the stain again with DI water.

Decolorize and Counter-Stain

  1. Decolorize the stain by tilting the slide slightly and slowly dropping decolorizing stain above the smear allowing it to run down through smear.  Decolorize for approximately 10 second or until purple color ceases to flow away from smear. (It is important that the smear is neither over or under decolorized.  Excessive decolorization may remove enough dye to give false gram-negatives while insufficient decolorization may provide false gram-positives.)
  2. Wash slide with DI water for approximately 5 seconds.
  3. Cover the slide with counterstain for approximately 30 seconds.
  4. Wash with DI water and shake of excess water. Allow to air dry, or blot with tissue.

Note: A well-prepared smear should be barely visible to the unaided eye

Examine Slide

  1.  Examine the bacteria under or without oil immersion (900x to 1000x) to distinguish gram positive and gram-negative bacteria. 

References

Coico, R. (2006), Gram Staining. Current Protocols in Microbiology, 00: A.3C.1-A.3C.2. doi:10.1002/9780471729259.mca03cs00

Gladwin, Mark, Bill Trattler. 2003. Clinical Microbiology made Ridiculously Simple. Miami: MedMaster.

Gram Staining : Principle, Procedure, Interpretation and Animation. LaboratoryInfo.com, 19 Jan. 2016, laboratoryinfo.com/gram-staining-principle-procedure-interpretation-and-animation/.