Scribd
Upload
151 views3 pages

Negative Staining: Course: MCB 103 Lab Section: 1 Name: Noor-E-Khadiza Shama ID: 1921168 Date: 18/02/20

1. The document describes an experiment using negative staining to observe the morphology of Bacillus cereus bacteria without heat fixing altering their shape or size. 2. Negative staining uses acidic dyes like nigrosin that stain the background dark while leaving the negatively charged bacteria unstained and visible. 3. The procedure involves making a smear of B. cereus mixed with nigrosin on a slide, allowing it to air dry, and examining under the microscope at high powers to view the rod-shaped bacteria against the dark background without distortion.

Uploaded by

Shama
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
151 views3 pages

Negative Staining: Course: MCB 103 Lab Section: 1 Name: Noor-E-Khadiza Shama ID: 1921168 Date: 18/02/20

1. The document describes an experiment using negative staining to observe the morphology of Bacillus cereus bacteria without heat fixing altering their shape or size. 2. Negative staining uses acidic dyes like nigrosin that stain the background dark while leaving the negatively charged bacteria unstained and visible. 3. The procedure involves making a smear of B. cereus mixed with nigrosin on a slide, allowing it to air dry, and examining under the microscope at high powers to view the rod-shaped bacteria against the dark background without distortion.

Uploaded by

Shama
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd

Course: MCB 103 Lab

Section: 1
Name: Noor-E-Khadiza Shama
ID: 1921168
Date: 18/02/20

Experiment 5

NEGATIVE STAINING

PURPOSE
1. Heat fixing alters the morphology of bacteria. As negative staining does not use heat
fixing, so morphology of bacteria is not altered and their shape and size is clearly seen.
2. It is also used for identification of certain bacteria like spirochetes or bacterial structures
like capsule and spores which is difficult to stain.

PRINCIPLE
The negative staining uses dyes like Nigrosin or India ink which are acidic dyes. Due to its acidic
nature, it gives up a proton and becomes negatively charged. Because the bacterial cell wall is
also negatively charged, it will repel the dye. The glass of the slide will stain but the bacterial
cell will be left unstained giving a clear appearance over a dark background, more like negative
film of a photo and hence the name “Negative Staining”

MATERIALS & EQUIPMENT


1. Bunsen burner
2. Inoculating loop
3. 2 glass slides
4. Nigrosin dye
5. Microscope
6. Culture of bacillus cereus (old capsules are formed around old bacteria)

PROCEDURE
1. A small drop of Nigrosin dye is placed at one corner of a clean slide
2. An inoculating loop is sterilized by holdingh in a Bunsen burner flame and a loopful of
inoculum from Bacillus cereus is taken and placed in the Nigrosin dye over the slide.
3. The inoculum is mixed gently with the dye using the loop.
4. A second slide is taken and held at 45o angle in the front of the dye and bacterial
mixture.
5. The edge of the second slide is pulled across to form a thin smear
6. The smear is air dried by keeping near a flame
7. Finally the slide is examined under microscope at 40X power and oil immersion at 100X
power objective lens

RESULT & OSERVATION


The slide was observed under a microscope and the following image was seen.
DISCUSSION
Nigrosin only attached to the glass slide which gave a black background. The bacteria being
negative repel the negative dye and appeared clear against the dark background. The structure
of the bacteria is seen to be rod shaped so we can say that the shape is not distorted as the
process doesn’t include heat fixing.

PRECAUTION
1. The smears should not be too thick, as in thick smear, there will be far too many bacterial cells
to see individually
2. While taking the bacterial colony from the agar, the inoculating loop should not be too hot as it
might kill the bacteria
3. The colony should be picked gently so that the agar does not break
4. After sterilizing the inoculating loop, do not touch the loop somewhere else as it might cause
contamination.

You might also like