Because microbial cytoplasm is usually transparent, it is necessary to stain microorganisms before they can be viewed with the light microscope. In some cases, staining is unnecessary, for example when microorganisms are very large or when motility is to be studied, and a drop of the microorganisms can be placed directly on the slide and observed.

1. VEENA P KUMAR
1 MSC.MICROBIOLOGY
SCHOOL OF BIOSCIENCE,
MGU
BACTERIAL STAINING
TECHNIQUES
VEENA P KUMAR
MSC.MICROBIOLOG
Veena P Kumar
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2. INTRODUCTION
 As bacteria consist of clear protoplasmic matter,
differing but slightly in refractive index from the
medium in which they are growing, it is difficult
with the ordinary microscope, except when
special methods of illumination are used, to set
them in the unstained condition.
 Staining, therefore, is of primary importance on
the recognition of bacteria.
 Staining may be simple staining and differential
staining.
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3. DYES
 Why we need to stain bacteria?
 Bacteria are transparent and colorless, so
they would be invisible to naked eye if
observed under a microscope thus
bacteria should be stained with certain
dyes in order to visualize bacterial cell or
their internal structures using the light
microscope.
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4.  DYE (stain) :
Colored organic compound in the form of
salt, composed of positive and negative ion, one
of these ions is responsible for color called
chromogen.
 TYPES OF DYES:
1. BASIC DYES
2. ACIDIC DYES
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5. DYES(CONTD.)
 BASIC DYES:
 In which chromogen is the positive ion
(cation).
 Basic dye has the form: dye+Cr-
 E.g.; crystal violet, methylene blue and
safranin.
 ACIDIC DYES:
 In which the chromogen is negative
ion(anion).
 Acidic dye has the form :Na+dye-
 E.g.; nigrosin and India ink.
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7. SIMPLE STAINING
 These show not only the presence of
organism but also the cellular contents of
exudates.
 A single stain is used.
 Examples are Loffler's methylene blue,
polychrome methylene blue, dilute Carbol
fuschin.
 Simple staining is of positive staining and
negative staining.
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8. POSITIVE SIMPLE STAINING
 1. Add one loopful of the sample (mixture of
microorganisms) onto a glass slide.
 2. Allow it to air-dry.
 3. Heat-fix the specimen on the glass slide,
unless the specimen is heat-fixed, the bacterial
smear will wash away during the staining
procedure.
 4. Flood slide with crystal violet and wait 1 min.
or safranin and wait 3-4min.
 5. Wash the smear with tap water to remove
the excess of stain.
 6. Blot dry, then add cederwood oil (immersion
oil) and examine under a microscope.
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9. NEGATIVE SIMPLE STAINING
 1. Place a small drop of nigrosin at the end of
the slide.
 2. Place a loopful of sample ( mixture of
microorganisms) and mix with drop of
nigrosin.
 3. Using the edge of another slide, spread
the drop out across the slide.
 4. Allow to air dray.
 5. Place one drop of immersion oil and
examine under a microscope.
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11. RESULT OF SIMPLE STAINING PROCEDURE
 Simple positive staining: all bacteria are
colored.
 Simple negative staining: background is
dark, bacteria are without any color .
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12. DIFFERENTIAL STAINING
 This type of staining is to differentiate two
organisms.
 Mainly used differential staining methods are
1. GRAM’S STAINING.
2. ACID-FAST STAINING.
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13. GRAM’S STAINING
 Gram staining is developed in 1884 by the
Danish physician Christian Gram , is the
most widely used method in bacteriology.
 It is first and usually the only method
employed for the diagnostic identification of
bacteria in clinical specimen.
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14. HANS CHRISTIAN GRAM
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15. PRINCIPLE
 This procedure separates bacteria into two
groups: Gram positive bacteria and Gram
negative bacteria.
 Crystal violet is first applied, followed by the
mordant iodine, which fixes the stain.
 Then the slide is washed with alcohol, and
the Gram positive bacteria retain the crystal
violet iodine stain; however, the Gram
negative bacteria lose the stain.
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16.  The Gram negative bacteria subsequently
stain with the safranin dye, the counterstain,
used next. These bacteria appear red under
the oil immersion lens, while Gram positive
bacteria appear blue or purple, reflecting the
crystal violet retained during the washing
step.
 Gram-positive cells have a thick
peptidoglycan cell wall that is able to retain
the crystal violet-iodine complex that occurs
during staining, while Gram-negative cells
have only a thin layer of peptidoglycan.
 Thus Gram-positive cells do not decolorize
with ethanol, and Gram-negative cells do
decolorize. This allows the Gram-negative
cells to accept the counter stain safranin.
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17. PROCEDURE
 Step 1- Crystal violet
(primary stain) for 1min.
water rinse.
 Step 2- Iodine(mordent )
for 1min. Water rinse
 Step3- Alcohol
(decolorizer) for 10-30
seconds.
 Step4-saffranin
(counterstain) for 30-
60sec.water rinse. Blot dry.
 Cell stains purple.
 Cells remain purple.
 Gram positive cells
remain purple. Gram
negative cells became
colorless.
 Gram positive cells
remain purple. Gram
negative cells appear
red.
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18. Veena P Kumar
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19. OBSERVATION
Gram positive cocci in chains Gram negative bacilli
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20. ACID-FAST STAINING
 This is also known as ziehl-neelsen staining.
 This method is a modification of
Ehrlich’s(1882)original method for the
differential staining of tubercle bacilli and
other acid fast bacilli.
 Stain used consists of basic fuschin with
phenol added.
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21. PRINCIPLE
 This technique differentiates species of
Mycobacterium from other bacteria. Because
the cell wall is resistant to water-based
stains, acid-fast organisms require a special
staining technique.
 Heat or a lipid solvent is used to carry the
first stain, carbolfuchsin, into the cells.
 Then the cells are washed with a diluted
acid alcohol solution.
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22. PROCEDURE CNTD.
Mycobacterium species resist the effect of
the acid alcohol and retain the carbolfuchsin
stain (they appear bright red under the
microscope). Other bacteria lose the stain
and take on the subsequent methylene blue
stain (they appear blue under the
microscope).
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23. PROCEDURE
1. Place a drop of NaCl onto the glass slide.
2. Using a sterilized and cooled inoculation loop,
obtain a very small sample of a bacterial
colony.
3. Gently mix the bacteria into the NaCl drop.
4. Let the bacterial sample air dry.
5. Using slide holder, pass the dried slide
through the flame of Bunsen burner 3 or 4
times, smear side facing up.
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24. 6.Flood slides with Kinyoun carbolfuchsin for 5 minutes.
7.Rinse gently with water until the water flows off clear.
8. Flood slides with acid-alcohol (3% HCl in ethanol) for
3~5 seconds.
9. Rinse gently with water until the water flows off clear.
10.Flood slides with methylene blue for 1 minutes.
11.Rinse gently with water until the water flows off clear.
12.Allow slides to air dry before viewing.
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26. OBSERVATION
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27. ZN METHODS FOR WEAKLY ACID FAST
ORGNISMS
1. Leprosy bacilli are acid-fast, but usually to
lesser degree than the tubercle bacillus.
They are stained in films or sections in the
same way as the tubercle bacillus, except
that 5% sulphuric acid is used for
decolorization in the place of 20% sulphuric
acid or acid alcohol.
2. Sections of tissues containing ‘clubs’ formed
by actinomycetes, mycobacteria and
nocardia can be stained by ZN stain and
decolorized with 1% sulphuric acid to
demonstrate the acid fastness of the clubs.
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28. 3. Brucella differential stain. Brucella abortus in
infected tissue or exudate may be
distinguished by the latter by its weakly acid
fast reaction. Stain with dilute (1-in-10) carbol
fuschin, without heating, for 15 seconds.
Decolorize with 0.5% acetic acid solution for 15
seconds, wash thoroughly with tap water and
counter stain with Loeffler’s methylene blue for
1min.
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29. SPECIAL STAINS
1. Volutin-Granule stain
2. Endospore stain
3. Capsule stain
4. Flagella stain
5. Giemsa stain
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30. VOLUTIN-GRANULE STAINING
 Volutin granules are a type of cytoplasmic
inclusion bodies found in many bacteria as
well as in some fungi, algae, protozoa.
 These granules are composed of mainly of
phosphate, RNA and proteins.
 These granules are found most prominent in
old cultures before starvation occurs.
 The method of volutin granule staining is
known as ALBERT-LAYBOURN METHOD.
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31. Veena P Kumar
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32. PRINCIPLE
 Albert’s stain contains cationic dyes like toludine
blue and malachite green.
 Due to the highly acidic nature of granules, they
can be selectively stained by acidified basic
dyes.
 The toludine blue preferentially stain volutin
granules while malachite green stain the
cytoplasm.
 Later due to application of Albert’s iodine, the
dye molecules are fixed by precipitation.
 Well developed granule of volutin (phosphate)
may be seen in unstained wet preparations as
round refractile bodies within the bacterial
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33. PROCEDURE
A thin uniform smear of culture was made. It was air dried and heat
fixed.
Lower the slide with Albert’s stain A and allowed to react for 3-5min.
The slide was then washed under running tap water.
Flood the slide with Albert’s iodine and allowed to react about 1min.
Slide was then washed and blot dried.
The slide was then observed under oil immersion objective of a
microscope.
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34. OBSERVATION
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35. ENDOSPORE STAINING
 The morphology of bacterial endospore is best
observed in unstained wet films under the
phase contrast microscope, where they appear
as large, refractile, oval or spherical bodies with
in a bacteria mother cells or else from the
bacteria.
 If spore-bearing organisms are stained with
ordinary dyes , or by gram’s stain , the body of
the bacillus is deeply colored, whereas the
spore unstained and appears as a clear area in
the organism.
 This is the way in which spores are most
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36. PRINCIPLE
 The spores are thick walled structures and
very resistant to physical and chemical
agents.
 The spores have a capacity to survive for
long periods even in unfavourable
environmental conditions.
 The heat resistant of spore is due to the high
content of calcium- dipicolinic acid.
 Spore are differentially stained using special
procedures that help dye to penetrate the
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37.  An aqueous primary stain, malachite green is
applied and steamed to enhance the
penetration of the impermeable spore coat.
 Once stained the endospore does not readily
decolorize even with the application of
decolorizer and they appear , but the cytoplasm
of the cell takes the color of saffranine and
appears red
 A modified Ziehl-Neelsen stain in which weak,
0.25% sulphuric acid is used as decolorizer,
yield a red spore in blue-stained bacteria. Lipid
granules also stained red, appearing like small
spherical spores.
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38. PROCEDURE
Films are dried and fixed with minimal flaming.
1. Place the slide over a beaker of boiling water ,
resting it on the rim with bacterial film uppermost.
2. When, within several seconds, large droplets have
condensed on the under side of the slide, flood it
with 5% aqueous solution of malachite green and
leave to act for 1min. While the water continues to
boil.
3. Wash in cold water.
4. Treat with 0.5% of saffranine or 0.05% basic fuschin
for 30 sec.
5. Wash and dry.
This method colors the spores green and the vegetative
bacilli red. Lipid granules are unstained.
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39. OBSERVATION
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40. CAPSULE STAINING
 Chemically, the capsular material is a
polysaccharide, a glycoprotein or a polypeptide.
 Capsule staining is more difficult than other
types of differential staining procedures because
the capsular materials are water soluble and
may be dislodged and removed with vigorous
washing.
 Bacterial smears should not be heated.
 The capsule is non-ionic, so that the dyes
commonly used will not bind to it.
 Two dyes, one acidic and one basic, are used to
stain the background and the cell wall,
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41. PRINCIPLE
 Negative staining methods contrast a
translucent, darker colored, background with
stained cells but an unstained capsule. The
background is formed with India ink or nigrosin
or congo red.
 A positive capsule stain requires a mordant that
precipitates the capsule. By counterstaining with
dyes like crystal violet, methylene blue or
carbolfuchsin, bacterial cell wall takes up the
dye. Capsules appear colorless with stained
cells against dark background.
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42. PROCEDURE
 1. Using sterile technique, add a loopful of bacterial
culture to tube with 1 ml NaCl.
 2. Add one drop of carbol fuchsin into the tube and
mix gently.
 3. Heat the mixture under flame 1min.
 4. Place a drop of mixture onto the glass slide.
 5. Place a drop of nigrosin onto the same glass slide
next to drop of mixture of bacteria and the dye.
 6. Use the other slide to drag the nigrosin-cell mixture
into a thin film along the first slide.
 7. Allow to air dry for 5-7 minutes (do not heat fix).
 8. Examine the smear microscopically (100X) for the
presence of encapsulated cells as indicated by clear
zones surrounding the cells.
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43. OBSERVATION
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44. FLAGELLAR STAINING
 Flagellar staining provides taxonomically
valuable information about the presence and
distribution pattern of flagella on prokaryotic
cells.
 Bacterial flagella are fine thread like organelles
of locomotion that are so slender(about 10 to 30
nm in diameter) they can only seen directly
using electron microscope.
 To observe bacterial flagella under light
microscope ,their thickness is increased by
coating them with mordents such as tannic acid
and potassium alum, and then stain with
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45. FLAGELLA
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46. PROCEDURE
1. Grow bacteria for 16-24hrs on a non
inhibitory medium, e.g. tryptic soy agar or
blood agar.
2. Touch a loopful of water on to the edge of a
colony and let motile bacteria swim into it.
3. Then transfer a loopful into a loopful of
water on a slide to get a faintly turbid
suspension and cover it with a cover slip.
4. The bacterial suspension is thus prepared
with a minimum of agitation , which would
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47. PROCEDURE CNTD.
5. After 5-10min, when many bacteria have
attached to the surface of the slide and cover-
slip, apply 2 drops of Ryu’s stain to the edge of
the cover slip and leave the stain to diffuse into
the film.
6. Examine with the microscope after standing
5-15 min. at ambient temperature.
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48. OBSERVATION
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49. NUCLEAR STAINING BY GIEMSA’S METHOD
 PRINCIPLE
 Giemsa’s stain is prepared by mixture of two
stains that are methylene blue ( basic dye) and
eosin( acidic dye) so the resulting stain has
properties of both dye.
 The chromatide of a cell is highly acidic in
nature so when it reacts with Giemsa’s stain it
gives a reddish purple color to DNA.
 The RNA of a cell is removed by acid hydrolysis
step in this step the smear is treated with 1 N
HCL solution in a water bath of about 60°c.
 Where as DNA material has triple bonds in
between some base pairs so DNA
molecule doesn’t get hydrolyzed and hence
only DNA molecule get stain and cytoplasm
remains colourless.
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50. Veena P Kumar
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51. OBSERVATION
 The cytoplasm appears colourless where as
nuclear material appears purple in colour.
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