Loeffler’s Flagella Mordant Tannic acid 20% aqueous 100 mL Ferrous sulfate crystals 20 g Loeffler’s Flagella Stain 10% alcoholic solution of Basic fuchsin 10 mL Distilled water 40 mL Microbiology and Bacteriology 825

 

7. Rinse in water again.

8. Stain with one of the following counterstains: Safranin,

Neutral red, or 1:10 Carbol fuchsin.

9. Rinse in water and allow it to dry by standing it

vertically, or by blotting it with filter paper.

Results

Because the gram-positive organisms retain the crystal

violet after decolorization, they appear dark blue in color.

The gram-negative organisms are decolorized and take up

the counterstain and therefore, appear pink in color.

Reagents

1. Crystal violet—0.5% solution in distilled water.

2. Iodine-(Lugol’s)—10 g iodine, 20 g potassium iodide

in 1000 mL of distilled water. Dissolve the potassium

iodide in 250 mL water and then add 10 g of iodine.

When dissolved make up to 1000 mL with distilled

water (This solution is three times stronger than Gram’s

iodine and is preferable).

3. Acetone.

4. Counterstain.

a. 1 g Neutral red

2 mL 1% Acetic acid

Distilled water to make 1000 mL

b. Safranin

1.7 g safranin

50 mL alcohol

Distilled water to make 500 mL

c. Dilute carbolfuchsin

1:10 dilution of strong carbol fuchsin.

Ziehl-Neelsen Stain

This stain is another method of categorizing certain

bacteria, depending on their ability to resist decolorization

by acid and alcohol. A very strong stain is used, basic

fuchsin in a phenol solution and heat is applied in order

that the stain can penetrate the waxy covering certain

bacteria.

Method

a. Make a smear of the material and allow to dry at room

temperature.

b. Flood the whole slide with strong carbol fuchsin and

heat gently underneath the slide until steam is seen

rising from the slide (Do not overheat, avoid boiling

of the stain).

c. Rinse in water and flood the slide with 25% sulfuric

acid. Leave this until the smear is pale pink in color.

d. Rinse in water and pour on alcohol for a few minutes.

e. Counterstain with malachite green, methylene blue or

picric acid.

f. Dry by standing the slide vertically—do not blot dry as

the tubercle organisms may get attached to the paper

and later may get transferred to another slide.

Results

The tubercle bacillus resists decolorizing by acid and

alcohol (i.e. it is both acid and alcohol fast) it will remain

bright red while all other organisms and material will take

on the color of the counterstain.

Troubleshooting (AFB-Staining)

Problem: False positive results

Possible causes Solutions

1. Sputum collected without washing the Patient should wash their mouth thoroughly while procuring sputum to minimize

mouth or in an unclean container specimen contamination with food particles, mouthwash or oral drugs.

 Patient should be asked to collect sputum in a clean container free from waxes,

inorganic materials and artefacts

 Artefacts may be mistaken for acid-fast bacilli

2. Oil immersion lens is not cleaned during Oil immersion lens should be cleaned after every observation to avoid contaminaobservation of slides ting other slides

Microbiology and Bacteriology 823

3. Contaminated water with acid-fast bacteria used Use clean, non-contaminated water for washing of slides during staining

for washing of slides during staining procedure

4. Carbol fuchsin held on the slide for long Allow the stain to stand for exactly 5 minutes with the application of heat. While

time with improper heating heating, ensure that the stain is not boiled. Heat only till steam starts rising

 from the slide. Leave the slide to cool for 2 minutes before decolorizing

5. Less decolorization done for thick smear The number of times for decolorization is to be increased for thick smears.

preparation Decolorization is to be carried out till the pink color disappears and the smear

appears colorless

Problem: False negative results

Possible causes Solutions

1. Sputum collected inadequately, i.e. only Thick yellowish green mucoid sputum collected from an early morning deep

the saliva productive cough should be used as a specimen

2. Failure to select suitable sputum portion Select a suitable portion, i.e. thick yellowish green mucoid portion of the sputum

for smear preparation preparation

3. Longer time duration given for counter- Allow the counterstain B to stain for 15–20 seconds before washing.

stain, i.e. more than 30 seconds

4. Inadequate examination of the smear Smear should be examined thoroughly from one edge to the other covering

100 fields or more

Modified Ziehl-Neelsen’s Stain

Used for leprosy where the bacteria are less acid fast. The

method is as mentioned above except that 5% Sulfuric acid

is used instead of 25%.

Reagents

Carbol fuchsin:

Basic fuchsin 10 g

Alcohol—100 mL

5% aqueous phenol—1000 mL.

Decolorizing agents:

25% sulfuric acid, or

5% sulfuric acid (for M. leprae) or

Acid-alcohol 3% HCI in alcohol.

Counterstains:

Loeffler’s methylene blue or

Malachite green—0.05% aqueous solution or

Methylene blue—0.1% aqueous solution or

Picric acid-saturated aqueous solution.

Special Stains

Used to stain flagella, capsules, spores and granules.

Stains for Diphtheria Bacillus

Ponder’s Stain

Toluidine blue 0.02 g

Glacial acetic acid 1 mL

Absolute alcohol 2 mL

Distilled water to make 100 mL.

Method

Spread the stain on the film for 1 minute and wash in tap water.

Result

Dark blue granules in pale blue bacillus.

Albert’s Stain

Solution I Toluidine blue 0.15 g

Malachite green 0.2 g

Glacial acetic acid 1 mL

95% alcohol 2 mL

Distilled water 100 mL

Dissolve the dyes in alcohol and add to the water and

acetic acid. Let stand for one day and filter.

Solution II Iodine 2 g

Potassium iodide 3 g

Distilled water 300 mL

Method

Apply solution I for 3 to 5 minutes, wash in tap water, blot

and dry. Apply solution II for one minute, wash, blot and

dry.

Result

The granules stain bluish black, the cytoplasm green and

other organism light green.

Modified Neisser’s Method

Neisser’s methylene blue

Methylene blue 1 g

Ethyl alcohol (95%) 50 mL

Glacial acetic acid 50 mL

Distilled water 1000 mL.

Method

a. Stain with Neisser’s methylene blue for 3 minutes.

b. Wash off with iodine solution used in Gram’s method

and leave some solution on the slide for 1 minute.

824 Concise Book of Medical Laboratory Technology: Methods and Interpretations

c. Wash in water and counterstain with neutral red

solution used in Gram’s method for 3 minutes.

d. Wash in water and dry.

Result

The bacilli show deep blue granules, the remainder of the

organism assumes a pink color.

Staining of Capsules

Hiss’s Method

a. Saturated alcoholic solution of basic fuchsin or gentian

violet 1 part to distilled water 19 parts

b. 20% aqueous copper sulfate solution.

Method

Place a few drops of solution (a) on slide. Heat to steaming

and leave on slide 30 seconds.

Wash off with solution (b).

Result

Capsule appears as faint blue halo around dark purple cell.

India Ink Method

The capsule is seen as a clear halo around the microorganism against the black background. This method may

be used for demonstrating cryptococci.

Staining of Spores

Modified Ziehl-Neelsen Method

1. Ziehl-Neelsen carbol fuchsin

2. Sulfuric acid 0.5% or methylated spirit

3. Loeffler’s methylene blue.

Method

1. Stain with carbol fuchsin for 5–10 minutes, heating

until steam rises.

2. Wash in tap water.

3. Decolorize with 0.5% sulfuric acid or methylated spirit.

If the acid is stronger than 1%, spores of many bacilli

are decolorized.

4. Wash in tap water. Now the smear is examined and if

both bacilli and spores are red, it is decolorized again.

If the spores alone are stained, it is counterstained. Let

the counterstain to act for 2 minutes. Wash in water,

blot and dry.

Result

The spores are stained bright red and the bacilli blue.

Staining of Spirochetes

Fontana’s Method

a. Fixative Acetic acid 1 mL

 Formalin 2 mL

 Distilled water 100 mL

b. Mordant Phenol 1 g

 Tannic acid 5 g

 Distilled water 100 g

c. Ammoniated silver nitrate

 Add 10% ammonia to 0.5% solution of silver nitrate in

distilled water until the precipitate formed just dissolves.

Now add more silver nitrate solution drop by drop until

the precipitate returns and does not redissolve.

Method

1. Treat the film 3 times, 30 seconds each time, with the

fixative.

2. Wash off the fixative with absolute alcohol to act for

3 minutes.

3. Drain off the excess of alcohol and carefully burn off

the remainder until the film is dry.

4. Pour on the mordant, heating till steam rises and allow

to act for 30 seconds.

5. Wash well in distilled water and again dry the slide.

6. Treat with ammoniated silver nitrate, heating till steam

rises, for half minute, when the film becomes brown

in color.

7. Wash well in distilled water and dry.

Result

The spirochetes are stained brownish black on a brownish

yellow background.

Staining of Fungi

Lactophenol Cotton Blue

Phenol crystals 20 g

Lactic acid 20 mL

Glycerol 40 mL

Cotton blue/methylene blue 0.05 g

Distilled water 20 mL

Dissolve the phenol crystals in the liquids by gently

heating and then add the dye.

Take a portion from the fungal growth and place it on

a drop of lactophenol cotton blue on a slide. Then place a

cover slip over the drop and press gently. Blot to remove

excess stain. Seal with varnish or nail polish.

Staining of Flagella

Loeffler’s Method

Loeffler’s Flagella Mordant

Tannic acid 20% aqueous 100 mL

Ferrous sulfate crystals 20 g

Loeffler’s Flagella Stain

10% alcoholic solution of

Basic fuchsin 10 mL

Distilled water 40 mL

Microbiology and Bacteriology 825

Method

Flood the smear with the mordant for 5 minutes. Wash

with distilled water. Add heated Loeffler’s flagella stain

and allow to act for 3 minutes. Wash with distilled water

and dry (The slides should be very clean).

Result

Organisms stain red and flagella pink.