Course: MCB 103 Lab

Section: 1
Name: Noor-E-Khadiza Shama
ID: 1921168
Date: 28/01/20

Experiment 3

SIMPLE STAINING
PURPOSE
Simple staining process is used to determine the shape, size and arrangement of a microscopic
cell like bacterium. Staining helps to see cells clearly as unstained microscopic cells are nearly
transparent when observed under a light microscope

PRINCIPLE
Basic stains like methylene blue and crystal violet give up a negative ion (OH -) or accept a
hydrogen ion (H+) and becomes positively charged. Since the surface of most bacteria are
negatively charged, the positively charged stain readily adhere to its surface and the shape of
the cell can be seen clearly under the microscope

MATERIALS & EQUIPMENT

1. Inoculating loop
2. Saline
3. Bunsen burner
4. Microscope slide
 5. Solid culture of Staphylococcus aureus
6. Basic dye (Crystal violet)
7. Staining tray
8. Stop watch
9. Light Microscope
10.Pipette

PROCEDURE
1. A clean microscope slide is 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, a drop of saline is picked and spread over the slide.
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 to pick a bacterial colony.
8. The colony is put into saline over the slide and using the loop, a thin smear is prepared
9. The loop is again sterilized to the full length by holding in the Bunsen burner flame
10. The slide is kept near the Bunsen burner for the smear to dry, after drying it will look
cloudy.
11. Then to head-fix the smear, the slide is cautiously passed through the flame two or three
times so that they don’t wash off during washing procedure.
12.After passing the slide each time, the slide is pressed against the palm of the hand to cool
it down.
13.Then the smear is stained by adding plenty of stain (crystal violet) and allowing it to
remain covered with stain for 30 seconds
14.Then the slide is kept over a staining tray and rinsed off using distilled water.
15.The back and sides of the slide is dried by blotting with a tissue paper
16.The slide is again kept for drying near the flame.
17.After drying, the smear is placed on the stage of the microscope and focused under 4X
power objective lens
 18.It is also observed under 10X and 40X power lenses. And using oil immersion it is observed
under 100X power objective lens

RESULT & OBSERVATION

After simple staining, the Staphylococcus aureus was observed under microscope and it look
like the following image. The cells of Staphylococcus aureus remain attached to one another like
a chain.

DISCUSSION
The Staphylococcus aureus looked round as it is a cocci. The smear was thin so structure of the
cells could be seen clearly. The cells are clearly visible as they are stained properly.

LIMITATIONS
1. Cannot distinguish gram positive and gram negative cells
2. Endospores cannot be stained using simple staining
3. Structures like flagella cannot be stained using simple staining

PRECAUTION
1. The smear 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 and arrangements
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