Gram staining

Gram stain kits is used to stain

microorganisms from cultures and specimens

by the differential gram method.

The Gram stain is used to differentiate

bacteria into two groups based on the cell color

after staining:

1. Gram +ve bacteria which are bluish purple.

2. Gram ₋ve bacteria which are pinkish red.

Requirements for Gram staining:

1. Glass slides.

2. Bunsen flame.

3. Gram stain kit.

4. Microscope.

5. Wire loop.

Reagents in Gram stain kit:

1. Gram’s crystal violet

solution.

2. Gram’s iodine solution.

3. Gram’s decolorizing

solution.

4. Gram’s safranine solution.

Procedure for making a bacterial smear

Gram Staining method:

Flood the fixed smear with Gram's crystal violet Solution.

Let stand for 2 minutes.

Pour off the stain and gently wash with tape water from a

faucet or a plastic water bottle.

Flood with Gram's iodine Solution. Allow it to remain for 2

minutes (function as mordant which increases interaction

between the cell and dye so that cell is stained more strongly.

Pour off the iodine solution and gently wash with tape water.

Shake off the excess water from the surface.

Decolorize with Gram's Decolorizer Solution (95% Ethanol)

for 3 seconds until the blue dye no longer flows from the

smear. Further delay will cause excess decolorization in the

gram-positive cells.

Gently wash the smear with

tape water.

Counterstain with Gram's

safranin solution for 2

minutes.

Wash off the safranin solution

with water.

The slide is to be shaken to

remove most of the water

and air-dried.

Examine the finished slide

under a microscope .

Ziehl Neelsen staining

The Ziehl-Neelsen (Zn) technique is used to stain

Mycobacterium species including M. tuberculosis, and

M. leprae.

Mycobacteria, unlike most other bacteria, do not stain

well by the Gram technique.

The stain binds to the mycolic acid in the

mycobacterial cell wall. After staining, an acid

decolorizing solution is applied.

This removes the red dye from the background cells,

tissue fibers, and any organisms in the smear except

mycobacteria which retain (hold fast to) the dye and

are therefore referred to as acid fast bacilli “AFB”.

Hot and cold Zn techniquesIn the ‘hot’ Zn technique the phenolic-carbol

fuchsin stain is heated to enable the dye to

penetrate the waxy mycobacterial cell wall.

This heating releases phenol vapours which are

TOXIC.

Techniques which do not heat the stain are

referred to as ‘cold’ techniques.

Requirements Glass slide Bacterial specimen Hot Plate Methanol for smear fixation Staining reagents i.e.a) Primary stain Carbol fuchsin (Red

color)b) Decolorizer (95% Ethanol + 3% HCl)

andc) Counter stain Methylene blue Microscope

Steps in Staining:1. Smear preparation and Fixation Prepare thin smear of given sample

on a glass slide. Allow the slide to air dry.

Fix with methanol: Cover the slide with methanol for 10-

15 minutes until the alcohol completely evaporates.

In this way Mycobacterium becomes completely inactive and attached firmly to the slide.

2. Staining(i) Application of Carbol Fuchsin:Two methods are used i.e.

a) Hot Method Cover the smear with Carbol

Fuchsin and heat the slide by placing it on Hot plate for 3-5 minutes.

Do not allow the stain to boil and dry but it should only evaporate.

By this way stain easily goes into the cell.

b) Cold Method

The smear is flooded with Carbol Fuchsin

along with a wetting agent (Turgitol).

Turgitol reduces surface tension

between the cell wall of mycobacteria

and the stain.

So that stain easily goes into the cell.Following application of primary stain, all cells will appear red.

(ii) Rinse with tap water.

(iii)Add the acid-alcohol decolorizing (95% ethanol and 3% HCl) slowly drop wise until the dye no longer runs off from the smear (20-30 seconds).

(iv) Wash well with tap water.

(v)Counterstain with methylene blue for 2 minute.

(vi) Wash with water, air dry and observe using oil immersion (at 100X) lens.

Kinyoun- Gabett staining kit

Requirements

1.Kinyoun- Gabett staining kit.

2.Glass slides

3. Thermal block (for the hot method).

4.Methanol.

5.Microscope.

Kinyoun- Gabett staining Kit:

1) Kinyoun solution:1. Basic fuchsin.2. Phenol.3. Ethyl alcohol.4. Demineralize

d water.

2) Gabbett solution:1. Methylene

blue.2. Sulfuric acid.3. Ethyl alcohol.4. Demineralize

d water.

Kinyoun- Gabett staining method

Smear preparation and fixation:

1.Smear the sample to be examined in a thin

layer on a clean slide.

2.Allow to dry.

3.Fix with methanol: cover the slide with

alcohol for 10- 15 minutes until the alcohol

completely evaporates.

A. Cold method

1. Place the slide in a staining dish filled with

Kinyoun solution for 3 hours.

2. Rinse with water.

3. Cover the smear with Gabbett solution for 5

minutes.

4. Rinse with water.

7. Wipe the back of the slide clean, and place

it in a draining rack for the smear to air-dry.

B. Hot method

1. Place the slide on a thermal block.

2. Cover the smear with filter paper which is slightly

smaller than the slide.

3. Float the paper with Kinyoun solution.

4. Heat until steam is given off.

5. Leave in contact for 10 minutes. Regularly flood the

filter paper to prevent drying.

6. Rinse with water.

7. Cover with Gabbett solution.

8. Rinse with water.

9. Air dry.

Results

AFB . . . . . . . . . . . . . . . . . . . . Red, straight

or slightly curved rods, occurring singly or

in small groups

Reporting of sputum smears

1. More than 10 AFB/field . . . . . . . . . . . report +++

2. 1–10 AFB/field . . . . . . . . Report ++

3. 10–100 AFB/100 fields . . report +

4. 1–9 AFB/100 fields . . . . . report the exact

number

5. When no AFB are seen after examining 100

fields:

Report the smear as ‘No AFB seen’. Do not report

Negative’ because organisms may be present

but not seen. Up to three specimens (one

collected as an early morning specimen) may

need to be examined to detect M. tuberculosis in

sputum.