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.
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.
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
Distilled water to make 1000 mL
Distilled water to make 500 mL
1:10 dilution of strong carbol fuchsin.
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
a. Make a smear of the material and allow to dry at room
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
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
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.
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
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
Microbiology and Bacteriology 823
for washing of slides during staining procedure
from the slide. Leave the slide to cool for 2 minutes before decolorizing
preparation Decolorization is to be carried out till the pink color disappears and the smear
Problem: False negative results
the saliva productive cough should be used as a specimen
for smear preparation preparation
stain, i.e. more than 30 seconds
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
5% sulfuric acid (for M. leprae) or
Acid-alcohol 3% HCI in alcohol.
Malachite green—0.05% aqueous solution or
Methylene blue—0.1% aqueous solution or
Picric acid-saturated aqueous solution.
Used to stain flagella, capsules, spores and granules.
Stains for Diphtheria Bacillus
Distilled water to make 100 mL.
Spread the stain on the film for 1 minute and wash in tap water.
Dark blue granules in pale blue bacillus.
Solution I Toluidine blue 0.15 g
Dissolve the dyes in alcohol and add to the water and
acetic acid. Let stand for one day and filter.
Apply solution I for 3 to 5 minutes, wash in tap water, blot
and dry. Apply solution II for one minute, wash, blot and
The granules stain bluish black, the cytoplasm green and
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.
The bacilli show deep blue granules, the remainder of the
organism assumes a pink color.
a. Saturated alcoholic solution of basic fuchsin or gentian
violet 1 part to distilled water 19 parts
b. 20% aqueous copper sulfate solution.
Place a few drops of solution (a) on slide. Heat to steaming
and leave on slide 30 seconds.
Capsule appears as faint blue halo around dark purple cell.
be used for demonstrating cryptococci.
1. Ziehl-Neelsen carbol fuchsin
2. Sulfuric acid 0.5% or methylated spirit
1. Stain with carbol fuchsin for 5–10 minutes, heating
3. Decolorize with 0.5% sulfuric acid or methylated spirit.
If the acid is stronger than 1%, spores of many bacilli
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,
The spores are stained bright red and the bacilli blue.
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.
1. Treat the film 3 times, 30 seconds each time, with the
2. Wash off the fixative with absolute alcohol to act for
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
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
7. Wash well in distilled water and dry.
The spirochetes are stained brownish black on a brownish
Cotton blue/methylene blue 0.05 g
Dissolve the phenol crystals in the liquids by gently
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.
Tannic acid 20% aqueous 100 mL
Microbiology and Bacteriology 825
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
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