Gram stain is one of the most common staining technique used for examining specimens suspected to contain bacteriologic agents. The direct microscopic examination of specimens and cultures can provide a rapid presumptive diagnosis meanwhile gram staining is a differential stain that provide morphological information regarding the shape of cell,the type of cell arrangement that is single chained, clustered of gram negative and gram positive bacteria
Principle of Gram stain
There are certain bacteria when stained with dyes such as Methyl Violet, Crystal Violet or Gentian Violet (which is a mixture of two preceding dyes) and then treated with Iodine acting as a mordant fixing the stain so that subsequent treatment with a decolourizing agent like alcohol or acetone does not remove the primary stain colour.
Other organisms turn to be decolourized by this process. Thus, if a mixture of various organisms are stained and subjected to decolourizing agents, it is found that some retain the the primary stain and these are termed Gram-Positive whereas others are completely decolourized and are termed as Gram-Negative.
In order to render the decolourized organism visible, and to distinguish them from those retaining the colour, a counterstain is then applied. This counterstain is usually red so that the gram-negative organism which appear reddish pink in colour may easily be differentiated from gram-positive organism, which retain the original violet stain.
This difference in the ability of gram positive bacteria to pick up the primary stain is due to the presence of a thick peptidoglycan on their cell wall while those gram negative bacteria possess a thin peptidoglycan able to pick the primary stain but is decolorized when flooded with alcohol decolorizer leaving the peptidoglycan unstained reason why it picks up the counter stain Carbol fuchsin
Types of Specimen for Gram stain
Smear prepared from any of the following can be use for gram staining
- throat swab
- nasal swab
- ear discharge
- pleural fluid
- urethral discharge
- centrifuged deposit of urine
- bacterial culture
- vaginal discharge etc.
Materials for gram staining
- Crystal violet (0.5%)
- Safranin (0.5%)
- Iodine crystals
- Potassium iodide
- Acetone (100%) or Ethanol (95%)
- Ethyl alcohol
- Distilled water
How to prepare Reagents or Stains
You can buy the already prepared stain but is also important to know how to prepare your reagents to reduce unnecessary expenditures
Preparation of 0.5% Crystal violet
Mix the following together
|Crystal violet||0.5 gm|
|Distilled water||100 ml|
Preparation of 1% Gram’s Iodine
|Potassium Iodide||2.0 gm|
|Distilled water||100 m|
- Start by grinding potassium iodide and iodine
- Dissolve the grind powder in not more than 20 ml of distilled water.
- Make up the volume to 100 ml with distilled water
Preparation of 0.5% Safranin
|Distilled water||100 ml|
- Dissolve 0.5 gm safranin in 10 ml alcohol
- make up the volume to 100 ml with distilled water
Gram stain procedure
- Make a smear of the suspected specimen on a clean glass slide
- Heat-Fix the smear by passing the slide over flame 2-3 times quickly
- Cover the slide with crystal violet solution and allow to act for about 30 seconds
- Pour off stain (crystal violet solution) and holding the slide at an angle downwards pour on the iodine solution on the slide so that it washes away the crystal violet
- Cover the slide with fresh iodine solution then allow it to act for 1 minute.
- Decolorize the slide with 100% acetone or ethanol. First tip off the iodine and hold the slide at a steep slope. Then pour acetone over the slide from its upper end, so as to cover its whole surface until colour ceases to come out of the preparation.
- Decolorization is very rapid and is usually complete in 2-3 seconds. After this period of contact, wash thoroughly with water under a running tap
- Now cover the slide with the counterstain (safranin) for 30 seconds
- Wash with water and blot dry
- Examine the smear under 100X objectives oil immersion microscopy
Result and Interpretation of Gram staining
|Violet (Purple) stained bacteria||Gram-Positive|
|Reddish pink bacteria||Gram- Negative|
Gram staining Quality Control
In order to obviate errors from over decolorizing with alcohol, a control smear of a known gram positive organism such as. a pure culture of Staphylococcus aureus may be prepared on one end of slide and another smear of gram negative organism like Escherichia coli be prepared on the other end of the slide.
These smears should be gram stained as described. The smear prepared from Staphylococcus aureus should show violet coloured bacteria whereas that of Escherichia coli should show pink coloured bacteria