GRAM STAINING

Gram staining is a type of Differential staining technique, widely employed in the microbiology. It's the most important and basic bacterial identification procedure followed in almost all labs. Gram staining was originally discovered by Christian Gram in 1884.

 

 GRAM STAIN REAGENTS :

Primary stain - crystal violet or methyl blue or gentian violet.

Mordant - Gram's iodine or Lugol's iodine.

Decolourizer - Acetone or alcohol.

Counter stain - Safranin or carbol fuschin or neutral red.

 

PROCEDURE :

•  Take a clean grease free glass slide and label it.
• Add a drop of saline in the marked area.
• Emulsify a loop full of colony taken from a culture plate.
• Allow it to dry and heat fix the smear.
• Flood the smear with the primary stain - Crystal Violet for 1 minute.
• Rinse the slide in running tap water.
• Add Gram's Iodine - a mordant to the slide for 1 minute.
• Wash the slide with running tap water.
• Add the decolourizer - Acetone for 5 seconds and wash the slide.
• Finally add the counter stain - Safranin for 30 seconds.
• Wash the slide, allow it to dry and observe it under oil immersion(100X).

 

PRINCIPLE : 

         Bacteria that are Gram stained will fall into two groups - Gram Positive and Gram Negative. 

Gram Positive bacteria's appears Purple/violet colour whereas,

Gram Negative bacteria's are sappears as Pink/red colour.

This is due to the differences in the structure and composition of the cell wall of gram positive and gram negative organisms.

Gram positive organisms have thick peptidoglycan layer and low lipid content on their cell wall. The crystal violet forms a complex with the mordant - crystal violet-Iodine complex (CV-I) which gets trapped inside the thick peptidoglycan layer. When treated with a decolourizer like acetone or alcohol, due to low lipid content,, cells become dehydrated, the pore size and the permeability reduces. Hence the CV-I complex cannot be extracted.Thus, they retain their primary stain i.e crystal violet and appear as Purple/violet colour colonies.

                                           

The Gram negative organism has different cell wall structure compared with gram positive organism. It has higher Lipid content i.e Lipopolysaccharides and relatively very less peptidoglycan layer. When stained with crystal violet, initially it takes up the primary stain and forms as CV-I complex. But eventually CV-I complex is washed off of the cell when treated with decolourizer (acetone/alcohol). Due to high lipid content, the acetone extracts the lipid layer which results in increased porosity and permeability for the CV-I complex to escape the cell.  Thus the cell is decolorized and subsequently takes up the counter stain safranin. Hence, it appears Pink/red in colour. 

                                 

                                              

EXAMPLES :

Gram Positive bacteria - Staphylococcus, Streptococcus, Enterococcus, etc.,

Gram Negative bacteria - E.coli, Klebsiella, Salmonella, Shigella, etc.,

LIMITATIONS :

• Old cultures may produce false results.
• Decolourizing is the crucial step, if decolourized for long time, gram positive may appear as gram negative organism. Insufficient decolourizing may cause gram negative organism to appear as gram positive.