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• Materials required
• 48-72 hrs old slant culture of
bacillus sp., malachite green
& Saffranin ,glass slide ,
inoculation loop , etc.
• The glass slide was cleaned well and a smear of given
organisms were made on the slide and dried and heat
• Malachite green was applied and the slide was placed
on the hot water bath allowing the smear preparation
to retain for 2-3 minutes.
• Care was taken to see that the stain does not get
• The slide was cooled and then washed with running
• Saffranin was applied and kept for 30 sec.
• Again the slide was washed in water and examined
under oil immersion.
• The glass slide was cleaned.
• A smear of the given culture was made on the glass slide using
• The smear was air dried or heat fixed.
• Gram crystal violet was flooded over the smear and left for 60 sec.
• Then it was washed with water .
• Gram iodine was applied as a mordant for 30 sec.
• The slide was again washed with water.
• Decolorizing agent was added drop wise till the primary stain stops
draining from the smear and washed with water.
• Saffranin was added as a counter strain for 60 sec.
• Again the slide was washed with water and then dried with blotted
sheet and examined under the microscope.
• Methyl red
• Dissolve 0.1g of methyl red in 300ml of 95% ethyl
alcohol .dilute to 500ml with distilled water.
• 5ml of MR-VP broth is dispersed into the test tubes
and sterilized at 151b pressure.
• The tubes were inoculated with loop inoculation .
• Inoculated tubes were incubated for 24-38 hrs at 37c.
• One un inoculated tube will serve as a control .
• After incubation methyl red solution was added
and observed the results.
• 5ml of simmon citrate agar was dispersed in test
tube and sterilized.
• After sterilization ,slant was prepared along with
• Inoculate the given culture by means of slant &
stab inoculation .
• One un-inoculated tube will serve as a control.
• All the tubes are incubated at 37c for 24 hrs.
• After incubation observe the results.
• Prepare trypticase soy agar plates.
• inoculate the given culture by means of simple
• Incubate all the plates at 37c for 24 hrs .
• After incubation 3% hydrogen peroxide were
added to the plates.
• Observed the results.