March 9, 2021 [LAB REPORT]

“GRAM STAINING.”
LAB REPORT:

 Submitted by: HAFIZ M.ANAS SARFRAZ.

"IN THE NAME OF ALLAH, THE MOST GRACIOUS,

LEARNING OUTCOME:
After this lab report we can understand the following outcomes:
 Differential staining.
 Gram staining.
 Differentiation between gram + and gram negative bacteria.
 Use of oil emersion lens 100x of microscope.

STAINING
The staining is the procedure which is done to give colours to the bacteria so we can
easily visualize them easily.

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So there are different type of staining which leads to different procedures but here we
only perform gram staining.

EQUIPMENTS:
The major requirements of our work is given bellow:
 Bacterial culture.
 Inoculating loop.
 Spirit lamp.
 Glass slide.
 Normal saline.
 Tap water.
 70 % Ethanol.
 Crystal violet.
 Gram iodine.
 Dropper.
 Sefaranin.
 Cider wood oil.
 Microscope.

GRAM STAINING

The procedure for GRAM STAINING is given below:

PROCEDURE:
1. First make a smear by placing a drop of normal saline on glass slide and now pick
the bacterial colony by inoculating loop.

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 2. After the sterilization the loop use the loop to place the picked bacterial sample and
spread to the glass slide and make a smear.

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 3. Now dry the smear by passing on flame 1 to 3 times slightly in a way not to
damage the glass slide.

4. .Put the primary stain on the glass slide which is crystal violet and it is positive in
nature and wait for 1 min.

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 5. Now apply washing with tap water.

6. Then we apply the MORDENT to make the CVS complex by putting the drops of
GRAM IODINE which is negatively in nature and wait for 1 min.

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 7. The apply washing again with tap water.

8. Now apply the DECLORIZER which is 70 % ethanol and wait for about 15_20
sec. The decolorizer effects the gram negative bacteria only so it has an outer
membrane made up of LIPID POLYSACHARIDE mostly so by adding ethanol it
diffuse out and also the CVS complex diffuse with it and gram positive bacteria
does not effected with the ethanol because it outer membrane does not have LPS
in higher amount. GRAM + retain primary colour.

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 9. Apply the washing procedure again.

10.Now apply the secondary stain which is SEFRANIN to stain the GRAM
NEGATIVE bacteria so it got pink colour and GRAM POSSITIVE bacteria retain
the primary colour which is purple in nature.

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 11.Apply the washing procedure again.

12.After air dry put a drop of CEADER WOOD OIL on the glass slide and place it
under microscope on 100x oil emersion lens and observe the results.

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 OBSERVATIONS
Microscopic visuals shows that the bacillus bacteria (rod shape) and they retain purple
colour so GRAM POSSITIVE + in nature.