Aim: To study Grams reaction of a given bacterial sample.

Principle:

Gram staining was introduced by Danish bacteriologist Christian Gram in 1880. The Gram stain is a differential
stain, commonly used in the microbiology laboratory that differentiates bacteria on the basis of the chemistry
and their cell wall composition. Since cytoplasm of bacterial cell is colourless, we therefore need a staining
method to differentiate bacteria. Most bacteria can be divided into two groups based on the composition of
their cell wall:

1. Gram-positive bacteria: The cell walls of the have a thick peptidoglycan layer beyond the plasma
membrane. Characteristic polymers called teichoic and lipoteichoic acids stick out above the
peptidoglycan and it is because of their negative charge that the cell wall is overall negative. Gram
positive cell walls stain blue/purple with the Gram stain.
2. Gram-negative bacteria: The cell walls are more complex. They have a thin peptidoglycan layer and an
outer membrane beyond the plasma membrane. The space between the plasma membrane and the
outer membrane is called the periplasmic space. The outer leaflet of the outer membrane is composed
largely of a molecule called lipopolysaccharide (LPS). This contributes to the overall negative charge of
the cell. Lipoproteins join the outer membrane and the thin peptidoglycan layer. Gram-negative cells will
stain pink with the Gram stain.
Differential staining requires the use of at least four chemical reagents that are applied sequentially to a heat-
fixed smear: The primary stain, the mordant , the decolourising agent and the counter-stain.
The overall procedure involves four steps:
 Heat fixed smear on the slide is first treated with a basic dye, crystal violet. At this stage all the bacteria
take up the primary stain and appear violet.
 In the second step, after washing, the smear is treated with 2% Potassium Iodine solution which acts as
mordant and is known as Gram’s Iodine. This intensifies the colour of the stain inside the cell and all
the cell will appear purple-black at this point.
 In the third step, the smear is treated with 70% alcohol solution to decolourise the stained cells. During
this process, some bacteria will lose the purple colour while some will retain it.
 In the fourth step, after washing, smear is treated with a counter-stain, safranin. At this stage all the
bacteria which had lost the primary stain will take up the counter stain and appear pink/red. The rest will
remain purple.
Thus after this process the bacteria will appear either red or violet. The purple coulored ones are called gram-
positive and the red coloured ones are called gram-negative.
Here the alcohol is a lipid solvent and it extracts some of the lipid content of the outer liposaccharide layer of
gram-negative bacteria leaving minute pores through which the Gram’s Iodine and primary stain leaves out. In
gram positive bacteria the Gram’s Iodine and the primary stain complex binds well to the cell wall more firmly
and is difficult to remove by the alcohol decolouriser.

Requirements:
 24 hours old bacterial broth
 Gram’s Iodine
 Basic Dyes (Crystal Violet, Safranin)
 70% alcohol solution
 Compound microscope
 Cedarwood oil as oil immersion
 Inoculating loop
 Glass slide
 Rectified Spirit
 Spirit lamp
Procedure:
1. A 24 hours old bacterial liquid culture was prepared and taken in a test tube.
 2. The glass slides were cleaned thoroughly with water followed by wiping with rectified spirit and it was
made sure that the slide was clean and grease free.
3. The inoculating loop was sterilized by heating it in spirit lamp fire till it became red hot and was allowed
to cool down.
4. After the inoculating loop was sterilized, it was put inside the bacterial culture and a film of the broth
was taken out in the inoculating loop.
5. This film was spread out evenly on the glass slide and a smear was prepared.
6. The smear was allowed to air dry and then it was heat fixed.
7. Crystal violet (primary stain) was put on the smear and was left undisturbed for 1 minute.
8. After 1 minute the slide was washed under running water very gently.
9. Added Gram’s Iodine (Mordant) to the washed slide and left undisturbed for 1 minute.
10. Again the slide was washed using running water very gently.
11. The smear was then decolourised by washing it immediately with 70% alcohol solution drop wise until
the crystal violet failed to get washed away from the smear.
12. The smear was then counter stained with safranin and left undisturbed for one minute.
13. After one minute the slide was washed with tap water gently.
14. The slide was then allowed to air dry and after completely drying, the slide was observed under
compound microscope at 1000X total magnification using 10X objective lens and 100X oil immersion
lens. The observations were noted.

Observations:
 The colour of the bacteria was observed as pink.
 The shape of the bacteria was observed as cocobacillus.
 The arrangement was observed as singlet/chain.
Results:

Precautions:
 It was made sure that while heat fixing the smear, excessive heat was not provided as it might destroy
the bacterial culture.
 Care was taken while decolourising the smear using 70% alcohol, as over decolourisation would
remove the primary stain in gram positive bacteria and also cause them to appear gram negative and
on the other hand under decolourisation would make it difficult to counter stain the gram negative
bacteria.