stains use in microbiology

1. STAINS USED IN
MICROBIOLOGY
- BY DR.MEHAK MANRO

2. INTRODUCTION
• As bacteria consists of clear protoplasmic
matter, differing slightly in refractive index
from the medium in which they are growing, it
is difficult with the ordinary microscope to see
them in unstained condition.
• Staining , therefore is of primary importance
for the recognition of bacteria.

3. STAINING METHODS
TYPES:
• Simple staining: Imparts same colour to all the
bacteria.
e.g. Loeffler’s methylene blue.
• Differential staining: Imparts different colours
to different bacterial structures.
e.g Gram stain, Zeihl Neelson stain

4. Simple staining
• Loeffler’s methylene blue:- shows characteristic
morphology of polymorphs, lymphocytes and
other cells more clearly than the strong stains.
• Polychrome methylene blue: formed by slow
oxidation( ripening) of Loeffler’s methylene blue
which forms a violet compound that gives stain
its polychrome properties. (addition of 1%
potassium carbonate to the stain quickens
ripening) e.g. McFadyean’s reaction.
• Dilute carbol fuchsin: made by diluting Ziehl-
Neelson’s stain with 20 times its volume of water.

5. Negative staining
• This provides a uniformly coloured
background against which unstained bacteria
stand out in contrast .
• Useful for demonstration of capsule.
• E.g
1. India ink
2. Nigrosin

6. GRAM STAINING
•Most widely used differential
staining in microbiology.
•Differentiates bacteria into 2 groups:
1. Gram positive.
2. Gram negative.

8. Original formulation of DR. Gram
• Primary stain- Aniline gentian violet
• Mordant- Lugol’s iodine
• Decolouriser- Absolute alcohol
• Secondary stain- Bismarck Brown

9. Cell Walls Of Gram positive and Gram
Negative Bacteria.

11. Principle of Gram staining
• The protoplasm of Gram positive bacteria is more
acidic than gram negative bacteria.
• This accounts for their retaining of basic primary dye
more strongly than Gram negative bacteria.
• The peptidoglycan layer of Gram positive bacteria is
more thick than that of Gram negative bacteria and
thus retain the dye iodine complex more strongly than
Gram negative bacteria.
• The high lipid content of Gram negative bacteria makes
them permeable to secondary dye after
decolourisation.
• The presence of magnesium ribonucleate in Gram
positive cell membrane helps in holding the dye-iodine
complex more firmly whereas Gram negative bacteria
do not possess this substance.

12. Components of Gram stain
• Primary stain: crystal violet ( methyl violet,
gentian violet)
• Mordant: Gram’s iodine (lugol’s iodine)
• Decolouriser: acetone( alcohol,
alcohol:acetone mixture)
• Counter stain: safranin(dilute carbol fuchsin,
neutral red, sandiford stain.

14. Intrepretation
• Gram positive bacteria stain dark purple.
• Gram negative bacteria, tissue cells, debris in
inflammation and leucocytes stain light red.

15. Various modifications of Gram stain
1. Kopeloff and Beerman’s modification
Primary stain- methyl violet
Decolouriser- acetone/acetone-alcohol mixture.
2. Jensen’s modification
Primary stain- methyl violet
Decolouriser- absolute alcohol
Counterstain- neutral red.

16. 3. Weigert’s modification
Primary stain- carbol gentian violet.
Decolouriser- aniline-xylol.
4. Preston and Morrel’s modification
Primary stain- crystal violet
Decolouriser- iodine-acetone.

17. 5. Hucker’s method:
1. Crystal violet(1minute)
2. Gram’s iodine(1minute)
3. Acetone(5sec)
4. Safranin(1minute)
6.Quick’s Gram method 7. Gram method
(single slides) for multiple slides
1. Crystal violet(5sec) 1.methyl violet(30sec)
2. Iodine(5sec) 2.lugol’siodine(30sec)
3. Acetone(2sec) 3.iodineacetone(30sec
4. Basic fuchsin(5sec) 4. basic fuchsin(30sec

18. ZEIHL-NEELSON STAINING
-Acid fast staining
-Differential staining

19. Principle of Ziehl-Neelsen stain
• Acid fastness has been ascribed to the high lipid
content and variety fatty acids and higher
alcohols found in tubercle bacilli.
• A lipid peculiar to the acid fast bacilli, a high
molecular weight hydroxy acid wax containing
carboxyl groups(mycolic acid) is acid fast in the
free state.
• Either heat or detergent( Tergitol) is required to
allow the stain to penetrate the capsule.
• Once stained, acid fast bacteria resist
decolourisation whereas other bacteria are
decolourised with acid alcohol.

20. Acid fast stain basic requirements
1. Primary stain- carbol fuchsin
2. Decolouriser- 20% sulphuric acid / 3% HCL in
95%alcohol.
3. Counter stain- methylene blue / malachite
green/ picric acid
• Acid fast structures stain – red
• Non acid fast structures stain - blue

22. Modifications of ZN staining method:
• KINYOUN MODIFICATION (cold method):
Surface active detergent TERGITOL is used,
phenol-9% is used.
• 20%H2SO4 – for Mycobacterium tuberculosis.
• 5%H2SO4 – for Mycobacterium leprae.
• 1%H2SO4 – for Nocardia spp., clubs of
Actinomycetes, coccidian parasites.
• 0.25%H2SO4 – for spores.

23. Brucella Differential stain:
Brucella abortus – weakly acid fast organism:
• Primary stain: dilute (1 in 10) carbol fuchsin for
15 minutes.
• Decolourizer: 0.5% acetic acid for 15 seconds.
• Secondary stain: loeffler’s methylene blue for 1
minute.

24. ALBERT’S STAIN
• Used to stain granules of volutin(
polymetaphosphate) of diphtheria bacillus.
• Best seen in young cultures( 18-24 hours)
• Principle: with basic dyes such as toluidine
blue, volutin granules stain metachromatically,
a reddish purple colour.

25. ALBERT- LAYBOURN METHOD:
Staining solution:
• Toluidine blue - 1.5gm
• Malachite green - 2gm
• Glacial acetic acid - 10ml
• Alcohol( 95% ethanol)- 20ml
• Distilled water - 1L
Albert’s iodine:
• Iodine - 6gm
• Potassium iodide - 9gm
• Distilled water - 900ml

27. Staining of spores
Spores are highly resistant and metabolically
inactive forms.
1. Best observed in unstained wet films under
phase contrast microscope.
2. Modified Zeihl Neelson stain( 0.25%
sulphuric acid as decolouriser).
3. Malachite green stain

29. DEMONSTRATION OF CAPSULES:
1. Negative staining or Relief staining- Best
method – wet film India Ink method.
2.Phase contrast microscope.
3. Loeffler’s polychrome methylene blue: slow
oxidation(ripening) of Loeffler’s methylene
blue forms a violet compound that gives the
stain its polychromatic properties.
e.g.Mc Fadyean’s reaction

31. Staining of spirochaetes:
• Too thin to be demonstrated by ordinary stains.
• Best observed- unstained wet films under dark
ground microscope
• Silver impregnation method:
artificially thickens bacteria with deposit of silver
and renders them visible.
• Fontana’s method:
Spirochaetes are stained brownish -black on a
brownish -yellow background.

33. Staining of flagella:
• Because of their extreme thickness-
Best demonstrated under – electron microscope.
• Films made with phototungstic acid for negative
staining are used.
• Ryu’s stain:
1. Crystal violet
2. Mordant solution: phenol, tannic acid
,aluminium potassium sulphate.

35. THE ROMANOWSKY STAINS:
• The original Romanowsky stain was made by
dissolving in methyl alcohol, the compound
formed by interaction of watery solution of eosin
and zinc free methylene blue.
• Various modifications :
1. Leishman’s
2. Wright’s
3. Jenner’s
4. Giemsa’s

36. PRINCIPLE:
Oxidized methylene blue + Eosin Y
(basic dyes) (acidic dyes)
Bind acidic nuclei bind to basic cytoplasm
blue to purple colour red colouration.
The peculiar property of Romanowsky stains-
impart reddish-purple colour to the chromatin of
malaria and other parasites.

39. References:
• MACKIE $ McCARTNEY- 14th edition
• Ananthnarayan $ Paniker- 10th edition
• Koneman’s Textbook of Diagnostic
Microbiology- 6th edition