S.Y B.PHARM SEMESTER III
PHARMACEUTICAL MICROBIOLOGY PRACTICAL
EXPERIMENT NO. 04
GRAM STAINING TECHNIQUE
AIM: To study bacterial morphology by Gram staining
REFERENCE: -
1.
Experimental Microbiology
book (As Per PCI Syllabus) by Savita Mandan, Umesh Laddha, Sanjay Surana,
Published by Career Publication, First edition, Page No 73 - 75.
2.
Pharmaceutical Microbiology
(As Per PCI Regulation) by Prof Chandrakant Kokare, Published by Nirali
Prakashan, First Edition, Page No. 6.3 – 6.6
3.
Practical pharmaceutical microbiology
book by Prof Rageeb, K.D Baviskar, N.G Patil, Published by S. Vikas And Company
(Medical Publisher), Edition 2018, Page No 45–48.
REQUIREMENTS:
CULTURES: Broth cultures of Escherichia coli (A) or Bacillus
cereus (B) or Staphylococcus aureus (C).
STAIN: Crystal violet and Safranin.
REAGENTS: Ethyl alcohol (95%), Grams iodine.
APPARATUS: Staining tray, glass slides, Bunsen burner,
inoculating loop, glass slide.
EQUIPMENT’S: Microscope.
PRINCIPLE:
GRAM STAINING TEST:-
It is differential staining
technique. It helps to distinguish the bacteria into two groups Gram positive
and Gram negative. It is based on difference in structure of cell wall of
bacteria, four different reagents are used in Gram staining technique.
1) Primary stain (Crystal
violet): It stain both cell wall of Gram positive and negative bacteria, after
staining both type of microbial cell will retain violet colour.
2) Mordant (Grams iodine
solution): It forms insoluble complex with stain and fix colour to bacterial
cell. It forms crystal violet iodine-magnesium ribonucleate complex in Gram
positive bacteria. Such complex is not formed in Gram negative bacteria as
magnesium ribonucleate is absent.
3) Decolorizing agent (ethyl
alcohol 95%): Lipids are in very negligible amount in cell wall of Gram-positive
bacteria or almost absent. Therefore, the shrinkage of cell wall takes place
due to dehydration and decrease the permeability for CV-I-Mg ribonucleate
complex. In case of Gram-negative bacterial cell wall lipids are present in
abundant amount. Therefore, when Gram negative bacterial cell is treated with
alcohol it extracts lipids from its cell wall and cell wall becomes porous. Staining
reagents are washed out through this porous cell wall of Gram-negative bacteria
and it becomes colourless.
4) Counterstain (Safranin): Gram
negative cells are decolorized so it will absorb the counterstain and appear as
pink coloured cell. Gram positive cell will retain the stain of primary stain
and appear as violet colours.
GRAM STAINING PROCEDURE:
GRAM STAINING PROTOCOL:- (GRAM STAINING PROCESS)
1. Take
three different glass slides and label it as A, B and C.
2. Prepare
bacterial smears of each microorganism Escherichia coli on slide A, Bacillus
cereus on Slide B and Staphylococcus aureus on slide C.
3. Fix smear
by heat.
4. Place a
slide on the staining tray and flood the smear with crystal violet.
5. Allow the
staining solution to react with bacterial cell for 30 to 60 seconds.
6. Wash the
smear with water and few drops of Grams iodine solution, keep it for one minute.
7. Wash the
slides carefully with alcohol 95 % till washing solution becomes violet
coloured.
8. Again,
wash slides with water and add few drops of counterstain allow reacting for few
seconds.
9. Wash the
slides and dry it.
10.
Add one to drops of cedar wood oil and
observe under oil immersion lens.
OBSERVATION AND RESULT:
GRAM STAINING RESULT
A. Smear prepared on slide A
shows pink coloured rod shaped bacteria i.e. Gram-negative bacilli.
B. Smear prepared on slide B
shows violet coloured rod shaped bacteria i.e. Gram-positive bacilli.
C. Smear prepared on slide C
shows deep violet coloured spherical shaped bacteria i.e., Gram positive
staphylococci.
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