aeover

Principle
differential stain. It was developed by Christian gram in 1884
The grams stain is a groups. Itis very
2 major
identifying and classifying the bacteria in to useful stain te
Gram +ve: Which retain the stain (violet) hence appears deep violet or purple colour.
1.

2. Gram- ve: Which retain the counter stain saftranine and appear pink colo.
in their cell walls than gram +ve
Gram -ve bacteria have greater lipid content bacteria
in alcohol and acetone treatment. By this treatment lipids will be extracted.
porosity to gram -ve cells. Cell walls retain
This resutsLipidins aresclube,
the stain and appear pink. The cell
bacteria become dehydrated when treated with alcohol or: acetone and permeability walls ofingrcrease
is, arm ie
crystal violet iodine complexX cannot be extracted. Hence, its cells remain decreased. Theretre
violet in colour.
Requirements
1. Bacterial culture or pure culture
Inoculation loop
3. Crystal violet
4. Grams iodine

95% ethyl alcohol or acetone

6. Saffranine
7. Distilled water

8. Spirit larnp,glass slides, droppers etc

Procedure

Preparation of a smear
1. Place a loop full of given culture with an inoculation loop on a
clean grease free slide and spread the
culture with inoculation loop into athin smear.
2. If the culture is from the solid media,
place a small drop of water on the grease free slide and
spread the culture with inoculation loop in the water thorougny
drop into a thin smear.
Fixation of smear
1. Allow the smear on the slide to dry in
the air or by heat fixing by passing the side with
the flame 2 to 3 times. smear side up ov
2. Mark the srnear
area on the bottom side of the
slide with a marker pen.

16
 smear of the given culture
Staining of the
Flood the smear with the crystal violet solution by adding drop by drop through the dropping bottle and
keep it for 30 seconds.
2
Washoff the excess stain from the smear by pouring water from the wash botle.
Flood the smear With Grams iodine and keep it for 30 seconds.
4. Wash off the excess Grams iodine from the smear by adding tap water.
6 Add alcohol/ acetone onto the smear by keeping the slide in slanting position and allow the decolourizer
to react with smear for about 5 to 10seconds only.
A Immediately pour water onto the smear for removing eXcess alcohol/ acetone from the smear.
7 Add counter stain with saffranindrop onto the smear and allow it to react fro 30 seconds.
8. Wash off the excess stain from the smear by tap water
Dry the smear by gently blotting the slide between filter paper folds.
 a microscope:
Observation of the stained smear under
loW power objective usingcoarse and fine
1. Focus the smear under
power objective from focus and shift
the
high power
adjustment onthte micrOscope.
2. Move away the low objective iinto positon anditk
on the microscope.
the smear using fine acjustment knob
from focus and add a drop of
3. Move away the high power objective immersion oil on the
4
focus.
Shift the100x oil immersion objective into position and focus the smear using adjustrnent
sTnear under
microscope.
Observe the shape of cells, arrangement oficells, Gramreaction and
6
endospores, if any
Draw a representative diagram of the cuiture observed in a microscopic field.
Precautions
The slides should be well washed. Young cultures should only be used.
should be avoided.
Excess heating during firai:
Observations &Conclusions

1. Morphological features:

2. Organism Type:

3 Colour:

Inference