Gram staining

MBBS, DMLT, BMLT and allied health sciences

1. Gram Staining Arun Kumar Parthasarathy Ph.d Dept. of Microbiology D.Y Patil Medical College, Kolhapur

2. Stain A stain , or dye, is a molecule that can bind to a cellular structure and give it color. They have chromophore groups , with conjugated double bonds that give the dye its color. They can bind with cells by ionic, covalent, or hydrophobic bonding. Staining techniques make the microorganisms stand out against their backgrounds. They are also used to help, examine the structural and chemical differences in cellular structures, and look at the parts of the cell

3. Classification of dye (Stain) Examples:- Methylene blue, Crystal violet, Safranin , and Malachite green. Examples:- Eosin and Picric acid, Example:- Leishman Stain

4. Gram Staining The Gram stain was developed by Hans Christian Gram in 1884 and modified by Hucker in 1921. The Gram stain separates bacteria into two groups: (1) Gram-positive microorganisms that retain the primary dye (Crystal violet) and (2) Gram-negative microorganisms that take the color of the counter stain (usually Safranin O).

5. Materials Clean grease free slide Microscope with oil immersion objective Inoculation loop Bacterial culture /suspension Bunsen burner Gram stain Reagents a. Crystal Violet (Primary stain) b. Grams Iodine (Mordant or Fixative ) c. Grams decolourizer (95% ethanol or Acetone) d. Saffranin (Counter stain )

6. Smear Preparation Take clean glass slide Add one drop of normal saline or distilled water with help of inoculating loop Open bacterial culture plate pick one or 2 colonies with help of inoculating loop and mix with water Heat fixation (10-15 secs )

7. Gram staining Protocol Keep slide in staining rack Add Crystal violet dye – wait for one minute After one minute wash slide with slow running tap water After washing, add grams iodine – wait for one minute After one minute- wash slide with slow running tap water Add grams decolourizer (95% alcohol)- wait for 20-25 secs Wash slide with slow running tap water Add saffranin (counter stain) wait for 30 seconds Wash with slow running tap water Allow to air dry Observe under oil immersion (100X) objective

8. Interpretation under microscope Gram positive cocci- Purple color Gram Negative bacilli- Pink color Gram positive budding yeast , Pus cells (Pink color), Epithelial cells (Blue color)

9. Theories of Gram stain PH Theory:- Cytoplasm of gram positive bacteria is more acidic hence, can retain basic dye (Crystal violet) for longer time. Iodine acts as fixative (it combines with primary stain to form CV-I complex which gets retained inside the cell 2. Magnesium Ribonucleate Theory:- Magnesium ribonucleate and basic proteins are concentrated at the cell membrane of gram positive bacteria. Mg ribonucleate retain the basic dye (crystal violet) hence, gram positive bacteria appear violet/purple in color

10. 3. Cell wall Theory:- Most important theory Gram Positive cell wall - Thick peptidoglycan layer (50-100 layers) with tight cross linkages It acts as permeability barrier and prevent the loss of Crystal violet dye Alcohol or acetone shrink the pores of the thick peptidoglycan Hence, large dye-iodine complex are not able to penetrate the tightened peptidoglycan layer . Gram Positive bacteria – Crystal violet (Purple) in color Gram Negative cell wall:- Thin peptidoglycan layer and not tightly cross linked Contain lipopolysaccharide layer, which gets disrupted easily by the alcohol or acetone Forming larger pores that allows the dye-iodine complexes to escape from cytoplasm Take only counter stain (Saffranin) Gram negative bacteria- Saffranin (Pink) in color

11. Examples Gram Positive Cocci Staphylococcus (Cocci in clusters) Streptococcus (Cocci in chains) Enterococcus (Cocci in Pairs) Gram Positive Bacilli Bacillus spp Corynebacterium spp Clostridium spp Gram Negative Cocci Neisseriae spp Veillonella spp Gram Negative Bacilli E.Coli Klebsiella spp Proteus spp Enterobacter spp Pseudomonas spp

12. Uses of gram staining To differentiate bacteria into gram positive and gram negative Identification of bacteria To start empirical treatment

13. Modification of Gram stain Kopeloff and Beerman’s modification- primary stain and counter stain are methyl blue and basic fuschin – anaerobic bacterial identification Jensen’s modification- use absolute alcohol as decolourizer and neutral red as counter stain- useful for meningococci and gonococci (Gram negative cocci) .