Course: MCB 103 Lab

Section: 1
Name: Noor-E-Khadiza Shama
ID: 1921168
Date: 04/02/20

Experiment 4

GRAM STAINING
PURPOSE
1. To find out whether a bacterium is gram positive or gram negative
2. To distinguish between gram positive and gram negative bacteria based on their
morphological characteristics.
3. To see the arrangement of each type of bacteria

PRINCIPLE
Gram staining is the most common differential staining used to distinguish between gram
positive and gram negative bacteria. Gram staining uses four components, a primary stain
(crystal violet),a mordant (iodine) that fix the stain, a decolorizing agent(alcohol) to remove the
primary stain and a counter stain (safranin).

Gram positive bacteria has thick peptidoglycane made cell wall and also has teichoic acid in its
cell wall but it lacks an outer membrane. After adding crystal violet followed by iodine, it form
crystal violet iodine complex inside the bacteria which is retained by the thick peptidoglycane
cell wall, giving the bacteria a dark violet color. So, during alcohol wash, the stain does not
come out of the cell wall and the cell does not absorb safranin.
Gram negative bacteria has a thin peptidoglycane made cell wall and has a lipopolysaccharide
made outer membrane. After adding crystal violet and iodine, when alcohol is added for
decolorizing, the crystal violet iodine complex comes out of the bacterial cell and the bacteria is
decolorized. During counter staining when safranin is added, the cell absorbs the color and
becomes pink.

MATERIALS & EQUIPMENT

1. Inoculating loop
2. Saline
3. Bunsen burner
4. Microscope slide
5. Solid culture of E.coli and Staphylococcus aureus (24 hour old)
6. Staining tray
7. Stop watch
8. Light Microscope
9. Pipette
10.Crystal violet (primary stain)
11.Safranin (counter stain)
12.Iodine (mordant)
13.95% Ethanol (decolorizing agent)

PROCEDURE
1. 2 clean glass slides are taken
2. The entire length of an inoculating loop is heated in the Bunsen burner flame to
sterilize it.
3. A tube containing saline is taken with one hand and the tube cap is removed
with the fingers of the other hand holding the loop.
4. The mouth of the tube is held in the flame.
5. Then using the inoculating loop, some saline solution is spread over both the
slides.
6. The loop portion of the inoculating loop is then held in the flame and then kept
in the air for some time to cool down
7. After the loop cools down, it is gently scuff over the agar plate containing E.coli
to pick a colony.
8. The colony is put into saline over the slide and using the loop, a thin smear is
prepared.
9. Using the same process, a smear for Staphylococcus aureus is also prepared.
10.Then before keeping the loop , it is again sterilized by holding in the Bunsen
burner flame.
11. The slides are kept near the Bunsen burner for the smears to dry, after drying it
will look cloudy.
12. Then to head-fix the smears , the slides are cautiously passed through the flame
two or three times so that they don’t wash off during washing procedure.
13.After passing the slides each time, the slides are pressed against the palm of the
hand to cool it down.
14.Then both the slides are kept over a staining tray
15.At first, crystal violet which is the primary stain is added to both the slides using
a pipette and kept for 1 min and then rinsed with water
16.Then iodine solution which is the mordant is added to the slides and kept for 1
min and then rinsed with water
17.Then the decolorizing agent which is 95% ethanol is added and kept for 20 sec
and then rinsed with water
18.Finally, the counter stain which Safranin is added to the slides and kept for 1 min
and then rinsed with water
19.The back and sides of the slides are dried by blotting with a tissue paper and kept
for drying near the flame
20.Once the slides are dry ,they are observed under the microscope at 40X power
and oil immersion under 100X power objective lens
RESULT & OBSERVATION
Both the slides were observed under microscope and the following results were seen.

DISCUSSION
The slide having round shape Staphylococcus aureus sample was dark violet in color which
shows that it is a gram positive bacteria and it has thick peptidoglycane made cell wall. We can
also see that the cells of Staphylococcus aureus remain attached to one another like a chain.
The slide having rod shaped E.coli sample was red in color which shows that it is a gram
negative bacteria and it has thin peptidoglycane made cell wall. The cells of E.coli remain
scattered and separated.

LIMITATIONS
1. It can only be used to distinguish bacteria based on their cell wall composition
2. Acid fast bacteria cannot be distinguished by gram staining
3. It cannot be used to stain endospores or cellular structures like flagella

PRECAUTION
1. The smears should not be too thick, because in a thick smear, the dye will not penetrate and there
will be far too many bacterial cells to see individual shapes.
2. The slide should not be put over the Bunsen burner flame for too long as the slide might crack.
3. While taking the bacterial colony from the agar, the inoculating loop should not be too hot as it
might kill the bacteria.
4. The colony should be picked gently so that the agar does not break
5. After sterilizing the inoculating loop, do not touch the loop somewhere else as it might cause
contamination.
6. The smear should be gently rinsed with water as washing it harshly will wash away the bacteria