Differential staining

Like comment share

1. GONO BISHWABIDYALAY

Nolam,Mirzanagar,savar,Dhaka-1344




DEPARTMENT OF MICROBIOLOGY

Course : Basic techniques in microbiology

Topic : Differntial Staining

Shahriar Shamim

2. Gram stain

The gram stain was developed in 1884 by the Danish Bacteriologist Hans Christian Gram. Gram
stain is a differential stain which divides most bacteria into two groups – Gram positive and
Gram negative bacteria.

Principle

Some bacteria resist decolorization and stained dark blue or violet by gram staining
due to thick peptidoglycan layer. On the other hand some bacteria do not resist decolorization
and stained red or pink by gram statining due to thin peptidoglycan layer.

Equipment

Glass slide, Bunsen burner , Permanent glass marker, Sterilized loop, Immersion oil,
Microscope,Sample (pus) .

Reagents


1. Crystal violet stain. (purple)
2. Safranin stain. (red)
3. Gram's Iodine.
4. 95% Acetone Alcohol.

3. Procedure

1. Take a clean greze free glass slide
2. By using a sterile loop, take a loopful sample (pus) and make a smear
3. Air dry and heat fixed the slide by passing 2-3 times over a flame
4. Apply Crystal Violet ( Primary dye - It is used to stain the all cells blue ) For 1 minutes and
wash with running tap water
5. Apply Grams Iodine (Used as Mordant interacts with CV+ and forms large complexes of
Crystal Violet and Iodine CV-I within the inner and outer layers of the cell ) For 1 minutes and
wash running tap water
6. Apply 95% Acetone Alcohol (Decolorization Agent - It removes the blue color of some
species but not others) For 10-15 sec and wash with running tap water
7. Apply Safranin ( Secondary dye – It is used to stain red those cells that have been
previously decolorized) For 45 sec and wash with running tap water
8. Observe the slide under microscope with oil immersion ( Cederwood oil – It is used to
increase the intensity of light)

4. Results of the Gram staining:

Gram-positive bacteria appear blue or purple color

Gram-negative cells will appear pink to red color

Examples of Gram-positive and Gram-negative bacteria:

Gram-positive bacteria: Staphylococcus aureus, Streptococcus pyogenes, Corynebacterium
diphteriae

Gram-negative bacteria: Neisseria meningitidis, Escherichia coli, Pseudomonas aeruginosa









Acid Fast Stain or Ziehl-Neelsen Method

Acid fast bacteria – The organism which contain hydrophobic waxy like lipid (mycolic)
material on their cell wall these organisms are called AFB.

Acid Fast stain is a differential stain. This stain is used to diffentiate acid fast organisms from
non-acid fast organisms.

Principle

The cell walls of acid fast organism ( genus mycobacterium and Nocardia) contain a waxy like
lipid materials are called mycolic acid and it is impermeable to primary dye carbol fuchsin but
resist decolorization and stained red. Others organism are decolorized and stained with blue.

Equipment

Glass slide, Bunsen burner , Permanent glass marker, Sterilized loop, Immersion oil,
Microscope, Sample(sputum).

5. Reagents

1. Carbolfuchsin stain
2. Methylene blue stain.
3.Acid-alcohol.

6. Procedure

1. Take a clean greze free glass slide
2. By using a sterile loop, take a loopful sample (Sputum) and make a smear
3. Air dry and heat fixed the slide by passing 2-3 times over a flame
4. Apply heated with Carbol fuchsin ( Primary stain – It is used to stain all cells red) For 5
minutes and wash with running tap water
5. Apply Acid Alcohol (Decolorization Agent - It removes red color from some species but not
others) For 1 minutes and wash with running tap water
6. Apply Methylene Blue ( Secondary dye – It used to stain blue those cells that have been
previously decolorized ) for 2 min with wash tap water
7. Observe the slide under microscope with oil immersion ( Cederwood oil – It is used to
increase the intensity of light)

Results of the Acid Fast Stain or Ziehl-Neelsen Method:

Acid Fast Bacteria appear bright red color

Non-Acid Fast Bacteria appear blue color

Examples of acid fast and non-acid fast bacteria:

Acid fast bacteria: Mycobacterium tuberculosis, Mycobacterium avium

Non-acid fast bacteria: Escherichia coli, Staphylococcus aureus,