NOVEMBER 2021

GRAM STAINING ON COLLECTED WATER SAMPLE

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Objective of experiment
This test differentiates the bacteria into Gram-Positive and Gram-Negative Bacteria, which helps in the
classification and differentiation of microorganisms.

Introduction:
The two major groups of bacteria can be divided into gram-positive and gram-negative. The Gram stain
technique is based on the differential structure of the cellular membranes and cell walls of the two groups.

Gram-positive organisms contain a highly cross-linked layer of peptidoglycan that retains the primary dye,
crystal violet (CV), following the application of the mordant, iodine (I). The iodine and crystal violet form a
complex within the peptidoglycan. When decolorizer is applied to the cells, the CV-I complex remains
within the cell, making it appear dark purple to blue.

The gram-negative organisms do not contain a thick cross-linked layer of peptidoglycan. The peptidoglycan
is loosely distributed between the inner cell and the outer cell membranes. Following the application of the
crystal violet and iodine, the CV-I complexes are not trapped within the peptidoglycan. Application of the
acid-alcohol decolorizer dehydrates the outer cellular membrane, leaving holes in the membrane and
effectively washing or removing the CV-I complex from the cells. The cells appear colorless. To make the
colorless cells visible, a secondary stain, safranin, is applied, leaving the gram-negative cells pink.
 PROCEDURE FOR GRAM STAINING:

EQUIPMENT:

 Electric microscope
 Microscope slide
 Looper
 Gas burner
 Distilled water

REAGENTS

 Crystal Violet, the primary stain

 Iodine, the mordant
 A decolorizer i.e alcohol (95%)
 Safranin, the counterstain

Procedure:

1. Prepare and sterilize the microscope slide.

2. Sterilize the looper by heating on the burner
3. Smear a loopful of sample broth on the slide.
4. Heat fix the sample on the slide by passing it through the flame about 3 times
5. Flood the smear with crystal violet, the primary stain, for 30 seconds.
6. Gently rinse off the excess stain with distilled water.
7. Flood the smear with Gram’s iodine, the mordant, for 1 minute.
8. Gently rinse off the excess iodine with distilled water.
9. Run the acid-alcohol decolorizer over the smear until the solution appears clear.
10. Gently rinse with distilled water for 30 seconds.
11. Flood the smear with safranin, the secondary or counterstain, for 30 seconds.
12. Gently rinse off the excess stain with distilled water.
13. Blot dry with bibulous paper.
14. Place the slide on the microscope stand and view under 100X objective lens

OBSERVATION
A purple color was observed on the specimen
DISSCUSSION

The purple color is an indication of gram-positive bacteria. This is because Gram positive result
in Blue/Purple Color and Gram-Negative result in Red/Pink Color

Some examples of Gram-Positive: Streptococcus, Staphylococcus, Corynebacterium, Listeria,

Bacillus, Clostridium, etc

CONCLUSION/ RECOMMENDATIONS:

Therefore, from results of this study, the groundwater in the study area can be regarded to
contain a gram positive bacteria. Hence the water should be treated using appropriate methods
before it is used for domestic purposes or as drinking water.