Veterinary Clinical Biochemistry and Laboratory Diagnosis-II

STAINING REACTION

Gram Staining

Introduction

• The Gram stain was introduced by Hans Christian Gram, a Danish Physician in1884 .This is most frequently used differential stain that divides bacteria into two major groups as gram –positive and Gram – negative.

Principle

• Bacterial cells in smear stained with crystal violet are purple in colour. The addition of mordent Gram’s iodine, is a stabilizer that causes the dye to form large crystals that are trapped by peptidoglygan layer in cell wall. The peptidoglygan layer in Gram positive cell wall in thicker (20-80 nm) than Gram negative cells (8-10 nm). Apart from this Gram positive bacteria contain magnesium ribonucleoprotein in their cell wall which forms complex with crystal violet and Gram’s iodine are resistant to decolorize and do not take the counter stain. The Gram negative bacteria do not contain magnesium ribonucleoprotein , they do not form the complex ,the violet stain is decolorized with decolourizer as decolouriser dissolves the lipid in the outer membrane and removes dye from the cells and take the counter stain. Mordants are substances combines with the primary stain which stains the cell structures and make it more insoluble complex .Gram’s iodine is used .

Decolorizing Agents

• Acetone ,absolute alcohol(95%),acetone alcohol, and iodine acetone are some of commonly used decolorizing agents. Among this acetone is fastest and most specific decolorizing agent.

Method

• Prepare thin smear, allow to dry and fix with heat.
• Cover the slide with crystal violet solution and allow act to act for 1 minute.
• Wash in water.
• Flood the smear with Gram’s iodine solution for 1 minute.
• Wash in water.
• Decolorize with absolute alcohol or acetone for few seconds, until the smear to lose colour.
• Wash with water.
• Counter stain with dilute carbol fuschin solution for 30 seconds.
• Wash in water, blot, dry and examine under oil immersion.

Result

• Gram-positive organisms Violet
• Gram-negative organisms Pink or Red

Gram positive cocci	Gram negative coccobacilli

Based on staining reaction

• Microscopic study under staining reaction reveals the shape and characteristic grouping and arrangement of cells, their size and presence of intracellular inclusions eg. spores, metachromatic granules. In addition to morphology, Gram stained preparation divides bacteria into two categories as Gram positive and Gram negative. For morphologic studies it is preferable to examine young cultures growth on non selective nutrient media.
• Gram stained reaction, when observed in conjunction with the types (cocci and bacilli) and arrangements of bacterial cells can be used to make presumptive identification

Gramp positive ccocci in clusters	Staphylococci sp;
Gram positive cocci in chains	Streptococci sp,
Gram positive, lancet shaped diplococci	Streptococci pneumoniae
Large gram positive bacilli	Bacillus or Clostridium species;
Small gram positive Bacilli	Listeria species
Small gram positive Bacilli with “Chinese letter"arrangements	Coryneforms (diphtheriaoids )
Gram negative, kidney shaped diplococci	Neisseria species.
Gram negative rods , Curved	Vibrio species
Gram negative rods corkscrew forms	Camphylobacter species.

• Acid fast staining procedure used to demonstrate Mycobacterium species.
• Modified acid fast staining used to demonstrate Brucella sp.