Gram stain

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A Gram positive organism
A Gram negative organism
The area used for Gram Stains can become very messy very quickly!

In 1884 the Danish bacteriologist Hans Christian Gram developed a staining method to differentiate between Streptococcus pneumoniae and Klebsiella pneumoniae. This basic staining technique can be used to group bacteria into two major types depending on the make-up of their cell walls (Gram-negative and Gram-positive bacteria). Gram positive organisms are violet while Gram negative ones are red/magenta.

Contents

Principles

So-called Gram-positive organisms have a peptidoglycan cell wall. When stained using a basic para-rosaniline dye (such as crystal violet or gentian violet) followed by a mordant (such as iodine), this peptidoglycan cell wall takes up the dye. Subsequent application of a decolourisation agent (such as ethanol or acetone) doesn't lead to removal of the dye. This is because the peptidoglycan cell wall has a low lipid content leading to poor permeability to the organic decolourising solvent. Also, it is thought that the cell wall structure allows the dye-iodine complex to become trapped within it.

Gram-negative bacteria on the other hand have a lipid outer membrane, separated from a thin peptidoglycan cell wall by the periplasmic space. The organic decolouriser easily permeates the lipid-rich outer membrane which dissolves, taking the stain with it.

Subsequent counter-staining with a red dye is carried out in order to make any decolourised organisms visible.

Methods

Gram's method

Gram's original method utilised aniline-gentian violet as the first stain, with Lugol's iodine as the mordant. This was then decolourised with absolute ethanol and counter-stained with Bismarck brown. This method has been surpassed by subsequent improvements to the stain, counter-stain and decolouriser.

Jensen's modification

This is the method most commonly encountered in UK laboratories.

Components

Primary stain: Methyl violet/Crystal violet

  • Methyl violet 6B/Crystal violet - 5g
  • Distilled water - 1 litre

Mordant: Iodine solution

  • Iodine - 10g
  • Potassium iodide (KI) - 20g
  • Distilled water - 1 litre

Dissolve the potassium iodide in 250ml water, then add the iodine. When fully dissolved, add the remaining water to 1 litre. Leave to stand overnight before use.

Decolouriser

NB: Acetone decolourises much faster than ethanol.

Counter-stain: Neutral red/Safranin

  • Neutral red/safranin - 1g
  • Distilled water - 100ml

or

  • Neutral red/safranin - 1g
  • Acetic acid 1% solution - 2ml
  • Distilled water - 1 litre

Procedure

  1. Fix specimen with 95% methanol or using heat fixation.
  2. Stain with primary stain for 20 seconds.
  3. Wash off and flood with iodine solution for 1 minute.
  4. Wash with decolouriser for a few seconds. The trick is to keep washing with decolouriser until no more crystal violet is seen being dissolved. This is very difficult in practice and over-decolourisation is often a problem.
  5. Wash immediately with water.
  6. Add counterstain and leave for 30 seconds.
  7. Wash again and allow to dry, using hot-plate if necessary.

In reality, the length of time for each stain and counterstain is not fully agreed upon.

Pitfalls

  • Over-decolourisation can lead to Gram-positive organisms appearing Gram-negative, as judicious use of the decolouriser has led to the primary stain being washed away. Some advocate the use of a streak of a control organism such as Staphylococcus aureus alongside the test organism on the slide - if this appears Gram-negative you can be sure you have over-decolourised your stain and may need to start again. Some say avoiding over-decolourisation is an art.
  • Fibrin will stain Gram-positive and may sometimes look similar to small Gram-positive cocci or bacilli.
  • Likewise, stain deposit left behind when a slide is not washed sufficiently can give misleading results.
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