Staining description to differentiate bacteria

1. Microscopic examination of
bacterial cells through staining
techniques
By,
Harshitha K N

4. Plant Pathogenic Gram + ve bacteria Plant Pathogenic Gram – ve bacteria
Clavibacter/ Corynebacterium tritici Ralstonia solanacearum
Streptomyces scabies Xanthomonas oryzae pv. oryzae
Erwinia amylovora
Pectobacterium carotovorum
Agrobacterium tumefaciens
Pseudomonas
Yellow ear rot
Potato scab

6. Stain
• Stains and dyes are used to highlight the specimen at the
microscopic level to study it at higher magnification for
histopathological studies and diagnostic purposes.
• Simple staining uses only one staining reagent and is used to
determine shape, dimensions, and arrangement of microbial cells.
• Differential staining uses more than one staining reagent (dye) to
differentiate cellular structures
• In some techniques the stains are applied separately, in others they
are mixed and applied in one solution

7. Simple staining
• Procedure for simple staining
• Methylene blue is probably the most widely used stain for
bacteriological techniques.
• Dissolve the dye (methylene blue 0.3 gm) in alcohol (30 ml) and
then dilute with the distilled water (100 ml) and filter the solution
through paper
• 24-28 hrs old bacterial culture, inoculation needle clean glass slides,
cover slips and methylene blue stain.

8. 1. Take a drop of culture aseptically by means of a loop and spread it
uniformly and drop the smear and heat fix by drawing the slide through
burner heat.
2. Flood the smear with the stain and wash the stain by holding under the
slow running tap water.
3. Drain the water and gently blot dry the slide between the folds of
blotter/tissue
4. Mount the slide and observe the bacterial cells under oil immersion
objective
5. Record the observations- shape, size and arrangement of bacterial cell

9. Hans Christian Gram (1884)

14. Crystal violet
• This basic dye is positively charged and, therefore, adheres to the cell
membranes of both gram negative and positive cells.
Iodine/ Mordant
• Iodine fixes the crystal violet into the cell wall of the bacteria by
working as a mordant.
• A mordant forms a complex that adheres tightly to the cell wall.
95% Ethanol
• Ethanol acts as a solvent which causes the crystal violet-iodine
complex to dissolve away and rinse out of the cell wall if the cells
are gram negative.

15. Safranin/ Counter stain
• The safranin directly stains the bacteria that has been decolorized
gram-negative bacteria pink color for easier identification