STAINING

1. STAINING OF
MICROORGANISMS
PART I
Dr. Dinesh Kr Jain, MD., Assistant professor,
Department of Microbiology,SMS Medical college, Jaipur

2. WHAT IS A STAIN
• A stain is a substance that adheres to a cell, giving the cell color so
they are much more visible.
• Different stains have different affinities for different organisms, or for
different parts of organisms.
• They are used to differentiate different types of organisms or to view
specific parts of organisms.
2

3. INTRODUCTION
• Stains were introduced in the mid-19th century which are responsible for
the major advances that have occurred in clinical microbiology &
diagnostic microscopy during the past 100 years.
• Stains consist of aqueous or organic preparations of dyes or groups of
dyes that impart a variety of colors to microorganisms
• Dyes may be used as
Direct stains of biologic materials
As indicators of pH shifts in culture media
As oxidation–reduction indicators to demonstrate the presence or lack of
anaerobic conditions
To demonstrate physiologic functions of microorganisms using so-called
supravital techniques.

4. STRUCTURE OF DYES
• Most of the biologically useful dyes are derived from coal tar.
• The fundamental chemical structure of most dyes is the benzene ring.
Dyes are generally composed of two or more benzene rings connected
by well-defined chemical bonds that are associated with color
production (chromophores).
• Dyes differ from one another in the number and arrangement of these
rings and in the substitution of hydrogen atoms with other molecules.
• Most stains used in microbiology are derived from aniline and are
called aniline dyes.

5. MECHANISM OF COLOR DEVELOPMENT
• Not totally understood,
It is theorized that certain chemical radicals have the property of absorbing
light of different wavelengths, acting as chemical prisms
The depth of color of a dye is proportional to the number of chromophore
radicals in the compound.
All biologic dyes have a high affinity for hydrogen. When all the molecular
sites that can bind hydrogen are filled, the dye is in its reduced state and is
generally colorless. In the colorless state, the dye is called a leuko
compound. Looking at this concept from the opposite view, a dye retains its
color only as long as its affinities for hydrogen are not completely satisfied.
Because oxygen generally has a higher affinity for hydrogen than many
dyes, color is retained in the presence of air

6. TYPES OF STAINING TECHNIQUES
 Simple Stains
 Differential Stains
Gram’s Staining & it’s Modifications
Ziehl-Neelsen Staining & it’s Modifications
 Special Stains
 Staining of Flagella
 Staining of Capsule
 Staining of Endospore

7. TYPES OF STAINING TECHNIQUES-
Continue
• Fluorescent Staining
• Staining of Parasites
• Staining of fungi
• Staining of Viruses
• Tissue staining
• Romanowsky stain
• Nuclear Stain
• Intercellular lipids
• Polysaccharide

8. SIMPLE STAINS
• Gives same color to all bacteria & cells.
• Show presence of organisms & also nature of cellular contents in exudates.
• Simplest method & Staining is easy to perform.
• The actual staining process may involve immersing the sample (before or
after fixation and mounting) in dye solution, followed by rinsing and
observation.
• The stain can be poured drop by drop on the slide.
• Stains are referred to as acidic or basic. These designations do not
necessarily indicate their pH reactions in solution, but rather, whether a
significant part of the molecule is anionic or cationic.

9. ACIDIC STAINS
 They are negatively charged & stain Basic molecules and
structures, such as cytoplasmic structures.
 Examples are:
• Eosin
• Acid Fuchsin

10. BASIC STAINS
 These are cationic (positively charged) basic radicals which stain
negatively charged molecules and Structures, such as nucleic acids and
proteins.
Basic stains, will bind electrostatically to negatively charged molecules
such as many polysaccharides, proteins and nucleic acids. These stains
will readily give up a hydroxide ion or accept a hydrogen ion, which
leaves the stain positively charged. Since the surface of most bacterial
cells is negatively charged, these positively charged stains adhere readily
to the cell surface.
Examples:
• Loeffler's methylene blue
• Polychrome methylene blue

11. DIFFERENTIAL STAINS
•Imparts Different Colors To Different Bacteria.
I. Gram’s Stain & Its Modifications
II. Ziehl-Neelsen Method & Its Modifications

12. GRAM’S STAINING
• It was introduced by Danish
physician Hans Christian Gram in
1884 to differentiate Streptococcus
pneumoniae from Klebsiella
pneumoniae in Lung tissue.
• It was originally described in a
publication titled “The differential
staining of Schizomycetes in tissue
sections & in dried preparations” in
Fortschitte der Medicin; 1884 , Vol
2, pages 185-189.

13. GRAM’S STAINING
• The original Gram’s Staining was slightly different from what we use
today.
• He used Primary stain – Gentian violet
Mordant - Lugol`s iodine
Decolourizer - Absolute alcohol
Counterstain - Bismarck brown
• This is most frequently used differential stain for diagnostic identification
of bacteria into two major groups :
1. GRAM-POSITIVE
2. GRAM-NEGATIVE

14. PRINCIPLE
• Exact mechanism of Gram staining
is not known. It may, however be
attributed to:
1. Typically, Gram positive
bacteria have thicker cell wall
(approximately 40 layers of
peptidoglycan 60%-90%)
compared to gram negative
bacteria , which have only 2-3
layer of peptidoglycan (10%-
20%)

16. PRINCIPLE- continue
2. Gram reaction is dependent on permeability of the bacterial cell wall
and cytoplasmic membrane, to the dye-iodine complex . In Gram
positive bacteria , the crystal violet dye-iodine complex combines to
form a larger molecule which precipitates within the cell.
Alcohol /Acetone mixture which act as decolorizing agent, cause
dehydration of multi-layered peptidoglycan of the cell wall. This causes
decreasing of the space between the molecules causing the cell wall to
trap the crystal violet iodine complex within the cell.

17. PRINCIPLE- continue
• In the case of Gram negative bacteria ,the alcohol dissolves the outer
lipopolysaccharide layer of the cell wall and also damage the
cytoplasmic membrane.
• As a result, the dye-iodine complex is not retained within the cell and
permeates out of it during decolorization.
• Hence the Gram positive bacteria do not get decolorized and retain
primary dye appearing violet/purple. In case of Gram negative
bacteria when a counterstain is added, they take up the color of the
counterstain and appear red/pink.

18. PRINCIPLE- continue
3. Slightly lower cytoplasm pH (2.0) in Gram positive helps the basic
dye to bind more efficiently than the Gram negative bacteria who
have slightly elevated pH(3.0).
4. A compound of magnesium ribonucleate and basic protein
concentrated at the cell membrane helps Gram positive bacteria retain
the primary stain. Gram negative bacteria do not possess this
substance.
• At the end of Gram staining ,Gram positive bacteria appear violet
or purple whereas Gram negative bacteria appear red/pink.

19. Bacteria
All bacteria will be stained Bluish or Bluish purple
Stain will be fixed due to formation of a complex of crystal violet and
Gram`s iodine
Primary stained retained Gram
positive bacteria
Primary stain do not retained and
take counterstain Gram negative
bacteria

20. REAGENTS
• PRIMARY STAIN- a basic pararosaniline (triphenyl methane) violet
dye,
Examples: crystal violet (2%),
methyl violet (0.5%),
gentian violet (1%) ( crystal violet + methyl violet)
• MORDANT - Gram`s iodine / lugol`s iodine
Gram`s iodine- Iodine crystal - 1gm
Potassium iodide - 2gm
Distilled water - 100ml.

21. DECOLOURIZER
removes the primary stain from gram negative bacteria making it colour-less
Decolorizer Duration
Acetone 2-3 seconds Fastest, short period of exposure is difficult to
control when many slides have to be stained
simultaneously
Absolute alcohol 1 minute Consist of 100% ethanol
Acetone-alcohol (1:1) 10 seconds 95% ethanol
Iodine-acetone ( preston &
morrell modification)
2-30 seconds Addition of small concentration of iodine(0.35%) to
acetone slows its rate of decolourization (>30sec)
without reducing its specificity
Limitation: irritating aerosols may be produced
Weak Iodine(0.035%)-acetone 10 seconds Avoid aerosol irritation & gives good Gram
differentiation

22. COUNTERSTAIN
Gram negative bacteria takes the counterstain and appears
pink/red
COUNTER STAIN Composition Duration
Dilute Carbol
Fuchsin
1 volume of Ziehl-Neelsen carbol fuchsin with
12-20 volumes of distilled water
10-30 seconds
Strongest counterstain
Basic Fuchsin 0.5 gm Basic fuchsin in 1 liter Distilled water 10-30 seconds
For general use
Neutral Red 1 gm Neutral red
2 ml 1% acetic acid in 1 liter Distilled water
2-4 minutes
For Gonococci & other
intracellular gram negative
bacteria
Safranin 0.5 gm Safranin in 1 liter distilled water 30-45 seconds

23. MODIFICATIONS OF GRAMS
Modifications Primary stain Decolorizing agent Counter stain
Kopeloff & Beerman’s Methyl violet Acetone Basic fuchsin
Burke’s Methyl violet Acetone Safranin
Jensen’s Methyl violet 95-100% Alcohol Neutral red (0.1%)
Preston & Morrell’s Crystal violet Iodine acetone Dilute carbol fuchsin
Wiegert's Carbol gentian violet Aniline –xylol Carmalum solution
Hucker`s Crystal violet Acetone-alcohol
(1:1)
Safranin

24. METHOD OF MAKING SMEAR
• Take a grease-free clean slide and make an oval shaped mark at the
center by using a glass marker about 2cm in diameter.
• Slide Dimensions : 75×25mm or 3×1 inches
• Thickness: 1mm
• For purulent specimen- use a sterile wire loop, make a thin preparation.
Do not centrifuge a purulent fluid, e.g. CSF containing pus cells.
• For non-purulent fluid specimen- centrifuge the fluid and make a smear
from a drop of well mixed sediment.

25. • Plate Culture- emulsify a colony in sterile distilled water and make a thin
preparation on a slide.
• Broth culture- transfer a loop full to a slide and make a thin preparation.
• Sputum- use a piece of clean stick to transfer and spread purulent and
caseous material on a slide.
• Swabs-roll the swab on a slide.
• Faeces –use a piece of clean stick to transfer small amount of material,
spread to make a thin preparation.
• Air dry the smear followed by Fixation

26. Gram Staining Procedure

27. Gram-positive Bacteria Observed
Under Oil Immersion Appear Purple
Gram-negative Bacteria Observed
Under Oil Immersion Appear Pink.

28. ATCC Control For Gram Staining
Gram positive
Staphlococcus aureus
ATCC-25923
Gram negative
Escherichia coli
ATCC-25922

29. • Crystal violet/ Methyl Violet for 5
seconds
• Iodine solution on tilted slide for 5
seconds
• Acetone for 2 seconds
• Basic Fuchsin counterstain for 5
seconds
Quick
Gram
Method
for single
Slides

30. GRAM METHOD FOR MULTIPLE SLIDES
• Space of 1-2 cm between adjacent slides to prevent a cross flow of reagents.
• It’s not possible to tilt each slide after rinsing with water, thus enough
reagent is added to displace residual water
• Attach a length of flexible rubber or plastic tubing to a high level water-tap
with moderate stream of water.
• Start applying from same side of slide ( preferably left )
• Slowly acting decolorizer such as iodine-acetone is used.
• Methyl
violet
• 30 sec
Primary
Stain
• 1% lugol’s
Iodine
• 30 sec
Morda
nt
Acetone
30 sec
Decolor
izer
Basic
Fuchsin
30 sec
Counter
stain

31. Interpretation of Result
• Examine the slide for microorganisms characteristic morphologies and
arrangements including gram-positive versus gram-negative,
• Mention cocci, bacilli, spirochetes, curved-rods, large or small in
singlets, pairs, clusters, chains, or diplococci.
• Indicate pleomorphic, coccobacillary or diphtheroids if applicable.
• If bacterial spores are present, indicate cellular location such as
terminal or subterminal and shape such as oval or round.
(Note-Spores do not stain with Gram stain reagents but will appear as
clear areas within the cells.)

32. No. of Neutrophils Per 10X Low – Power Field Grade
<10 0
10-25 +1
>25 +2
mucus +1
No. of Epithelial Cells Per 10 x Low – Power Field
10-25 -1
>25 -2
• Bartlett’s Grading System
For evaluation of sputum samples

33. Murray and Washington’s Grading System
Epithelial Cells per Low- Power Field Leukocytes per Low-Power Field
Group 1 25 <10
Group 2 25 10-25
Group 3 25 25
Group 4 10-25 25
Group 5 <10 25
NOTE: Only Group 5 Specimens are clinically relevant

34. Gram-positive Cocci in chains
Intracellular Gram-negative
diplococci within pale-staining
segmented neutrophils.

35. Mixed gram-positive and gram-
negative morphologies
Traditional Gram’s stain from a
legionellosis demonstrating
polymorphonuclear leukocytes and
macrophages, but “no organisms
seen” since legionellae stain poorly
with the safranin counterstain

36. USES OF GRAM STAIN
• Differentiation of bacteria into Gram-positive and Gram-negative. This differentiation
is helpful in determining the use of subsequent culture media & biochemical tests.
• To Start Empirical Treatment: Gram stain gives a preliminary clue about the bacteria
present so that the empirical treatment with broad spectrum antibiotics can be started early
before the culture report is available.
• Presumptive Identification For Fastidious Organisms, such as Haemophilus which
takes time to grow in culture
• Preliminary Clue To Put Anaerobic Culture Anaerobic organisms, such as Clostridium,
which do not grow in routine culture.
• Staining certain fungi such as Candida and Cryptococcus (appear gram-positive).
Presence of spore (unstained area) & its position can be determined .
• Presence of inflammatory cells (phagocytes) are key indicator of an infectious process.
• It can identify non bacterial forms such as Yeast, Trichomonas, Strongyloides larvae,
Pneumocystis carinii cysts & Toxoplasma gondii trophozoite.

37. LIMITATIONS
1. Over-decolorization may result in the identification of false gram-negative results.
2. Under decolorization may result in the identification of false gram-positive results.
3. Smears that are too thick or viscous may retain too much primary stain, making
identification of proper Gram stain reactions difficult. Gram-negative organisms may not
decolorize properly.
4. Cultures older than 16-18 hours will contain living and dead cells. Cells that are dead
will be deteriorating and will not retain the stain properly.
5. Stain may form precipitate with aging. Filtering through gauze will remove excess
crystals.
6. Gram stains from patients on antimicrobial therapy may have altered Gram stain
reactivity due to the successful treatment.
7. Occasionally, pneumococci identified in the lower respiratory tract on a direct smear will
not grow in culture. Some strains are obligate anaerobes.

38. PITFALLS IN INTERPRETATION OF
GRAM STAINING

39. MEDICALLY IMPORTANT BACTERIA THAT
CANNOT BE SEEN IN GRAM STAIN
MICROORGANISMS REASON ALTERNATIVE
Mycobacteria Including
M. tuberculosis
High Lipid In Cell Wall, So
Dye Cannot Penetrate
Acid Fast Stain
Treponema pallidum Too Thin To See Dark Field Microscopy
Mycoplasma pneumoniae No Cell Wall, Very Small Giemsa
Legionella pneumophila Poor Uptake Of Red
Counterstain
Prolong Time Of Counterstain
Chlamydia Intracellular, Very Small Giemsa
Rickettsiae Intracellular, Very Small Giemsa

40. ZIEHL-NEELSEN STAINING
PRINCIPLE
• Acid-fast mycobacteria contain mycolic acid in their outer
membrane, making the cells waxy and resistant to staining with
aqueous based stains such as the Gram stain.
• The primary stain, carbol fuchsin is applied to the cells, and heat
and phenol are used to allow the stain to penetrate into the waxy
surface of acid-fast microorganisms.
• The excess stain is removed with treatment by acid alcohol (ethanol
and hydrochloric acid).
• A secondary stain, methylene blue, is then applied to the cells.
• NOTE: Colour Blind workers are advised to use picric acid
solution (7g/l in water) which yields a yellow background.

41. METHOD OF MAKING SMEAR
1. Vortex concentrated sediment, un-concentrated sputum, other purulent
material, or stool. Place 2 to 3 drops on the slide from the end of the
broom-stick parallel to the slide and slowly spread the liquid uniformly to
make a thin smear.
2. For cerebrospinal fluid (CSF) sediment, vortex thoroughly and apply to
the slide in heaped drops. A heaped drop is allowed to air dry, and a
second application of sediment is placed on the same spot and allowed to
dry. A minimum of three layers, applied to the same 1-cm diameter circle,
should facilitate detection of small numbers of bacilli. (Note: Some
laboratories have stopped performing acid-fast stains on CSF because
positive stains are extremely rare.)
3. Fix the smear at 80° C for 15 minutes or for 2 hours at 65° to 70° C on an
electric hot plate. (Note: Survival of mycobacteria at this temperature has
been reported; handle all specimens with proper precautions.)

42. Ziehl-Neelsen staining
Procedure
4. Rinse with Tap Water & Decolorize
with 25% sulphuric acid
1. Pour 1 % Carbol Fuchsin
5. Let it stand for 10-15 seconds &
rinse gently with tap water
6. Pour 0.1% methylene blue &
leave for 1 minute
3. Carbol fuchsin 3-5 mins2. Gently heat until vapour rises

43. Acid fast bacilli will appear stained pink,
straight curved rods with blue background due
to methylene blue

44. Reporting
• Read at least for 5 minutes & 100 high power field before reporting a
negative result.

45. REVISED RNTCP GUIDELINES
AFB observation Grade
More than 10 AFB per field in atleast 20 fields 3+
1-10 AFB per field in atleast 50 fields 2+
10-99 AFB per 100 fields 1+
1-9 AFB per 100 fields Scanty
(Actual no.)
No AFB seen in atleast 100 field Negative

46. Acid-fast organisms/structures Sulfuric acid (%) needed
for Decolorlzation
Mycobacterium tubercuJosis 25%
Mycobacterium leprae 5%
Nocardia 1%
Acid·fast parasites such as Cryptosporidium,
Cyclospora, Isosopra, Microsporidia, Taenia
saginata (segments and eggs), hooklets of hydatid
cyst and eggs of Schistosoma mansoni
1%
Bacterial spore 0.25-0.5%
Sperm head 0.5-1%
Legionella micdadei 0.5-1%

47. Kinyoun modification (Cold Method)
Identification of acid-fast Mycobacterium spp. and parasites such as
Cryptosporidium and Isopora spp.
PRINCIPLE
• Acid-fast mycobacteria contain mycolic acid in their outer membrane,
making the cells waxy and resistant to staining with aqueous based stains
such as the Gram stain.
• The primary stain, carbol fuchsin, is applied to the cells and phenol is used
to allow the stain to penetrate into the waxy surface of acid-fast
microorganisms.
• The excess stain is removed with treatment by 1% sulfuric acid.
• A secondary stain, methylene blue, is then applied to the cells.

48. RESULTS
• Acid-fast organisms, Mycobacterium spp., will appear pink.
• Nonacid-fast organisms will appear blue.
• In addition, background material should stain blue.
LIMITATIONS
• May be less sensitive than the Ziehl-Neelsen method.
• Smears that are too thick may not properly stain.

49. • Smear prepared from material
obtained from a necrotic
tuberculoma of the lung stained
with the kinyoun acid-fast stain.
Note the relatively short, thin,
beaded, slightly curved, red-
staining acid-fast bacilli

50. SPECIAL STAINS
I. STAINING OF FLAGELLA
II. STAINING OF CAPSULE
III. STAINING OF ENDOSPORE

51. FLAGELLAR STAINS
• To visualize the presence and arrangement of flagella for the
presumptive identification of motile bacterial species.
• Most motile bacteria possess flagella, the shape, number, and
position of which are important in the identification,
particularly when biochemical reactions are weak or equivocal.

52. PRINCIPLE
• The Leifson staining technique (or modification) is most commonly used
in clinical laboratories and is not difficult to perform, providing exact
details that are followed in each step of the procedure.
• Bacterial flagella can be stained by alcoholic solutions of rosaniline dyes
that form a precipitate as the alcohol evaporates in the staining
procedure. Basic fuchsin (pararosaniline acetate) serves as the primary
stain with tannic acid added to the solution as a mordant. A counterstain,
such as methylene blue, may be used to better visualize the bacteria in
instances in which the primary stain is weak or does not react at all with
the bacterial cell wall.

53. METHOD
1. LEIFSON METHOD
Most commonly used Method
• Reagents
1. Primary Stain- Basic fuchsin (para
rosaniline acetate) 1.2% in 95%
alcohol
2. Mordant-tannic acid 3% in water
3. Sodium chloride, 1.5% in water
4. Final Stain-1:1:1
Counterstain-methylene blue for
better visualization
• Stain for 5–15 minutes by alcoholic
solutions of rosaniline dyes ,
allowing a precipitate to form as the
alcohol evaporates
• Observe the stained slide under the
oil-immersion (100×) objective of
the microscope -staining red to blue-
black flagella should be observed

54. 2. RYU METHOD
Easy to perform and gives good results
• Solution I-
a. 5% phenol, 10.0 ml
b. Powdered tannic acid, 2.0 g
c. Saturated aluminum potassium
sulfate 12-hydrate (crystals)
• Solution II- Saturated solution
of crystal violet in alcohol
• Final Stain: Solution I: II in
10:1 ratio
Method
• Flood the air-dried smears with
the staining solution for 1–5
minutes.
• Wash the staining solution off in
tap water. After the smears have
dried, examine them under the
oil-immersion objective of the
microscope. Cell bodies &
flagella stain violet.

55. 3. Wet-Mount Technique
Heimbrook and colleagues have described use of the wet-mount
technique of Mayfield and Innis and the stain of Ryu as a rapid, simple
way of staining flagella.
• Bacteria grown on a non inhibitory medium for 16 to 24 hours is
touched by wire and then touching a drop of water on a slide.
• A coverslip is placed over the drop of a faintly turbid suspension, and
the slide is examined for motile cells.
• After 5 to 10 minutes, or when about half of the cells are attached to
the glass slide or coverslip, two drops of the Ryu stain are applied to
the edge of the coverslip and allowed to flow under the coverslip by
capillary action.
• The cells are examined for the presence of flagella after 5-15 minutes
at room temperature.

56. INTERPRETATION
• Look for morphology of flagella, the shape, number, and position
• Polar
• Monotrichous—single flagellum at one or both poles
• Multitrichous—two or more flagella at one or both poles
• Subpolar—flagella near pole with base of flagella at right angle to long axis
• Lateral—flagella projecting from the middle of bacterial cell
• Peritrichous—flagella haphazardly arranged all around bacterial cell
• Positive control
peritrichous, Escherichia coli;
polar, monotrichous, Pseudomonas
aeruginosa;
multitrichous, Burkholderia cepacia
• Negative Control
Acinetobacter baumannii

57. Alcaligenes spp.,
peritrichous flagella
Pseudomonas aeruginosa,
polar flagella

58. STAINING OF CAPSULE
PRINCIPLE
Bacterial capsules are non ionic so neither acidic or basic stains will
adhere to their surfaces.
Therefore the best way to visualize them is to stain the background
using acidic stain and to stain the cell itself using a basic stain.
Example : India ink
which leaves the capsule as a clear halo surrounding a purple cell in a
black field.

59. Negative staining by India ink and Nigrosin stain
• Bacteria are mixed on a slide with an acidic dye such as congo red or the
black stain, India ink, Nigrosin. The mixture is smeared across the face
of the slide and allowed to air dry.
• Because the stain carries a negative charge, it is repelled by the bacteria,
which also have a negative charge. The stain gathers around the cell.
Since a chemical reaction has not taken place, and because heat fixing
has been avoided, the cells appear less shriveled or distorted. They often
appear larger than stained cells and more natural.
• India ink – aqueous solution of fine carbon particles (unable to penetrate
the cells).

60. Negative staining by India ink and Nigrosin stain:
• Capsule appears as a clear
refractile halo around the
bacteria where as both the
bacteria and the background
appear black.
• India Ink preparation,
illustrating the irregular-
sized,encapsulated, spherical
yeast cells of Cryptococcus
neoformans

61. M'FAYDEAN CAPSULE STAIN
• .
• It is used for demonstration of
capsule of Bacillus anthracis by
using polychrome methylene
blue stain.
• M fadyean's reaction-
amorphous purple capsule
surrounding blue bacilli
(Polychrome Methylene Blue
Stain)

62. Quellung reaction
This microscopic “precipitin test” can be used to identify pneumococci or to
determine the capsular serotype of individual pneumococcal isolates by
adding antisera mixed with Methylene Blue.
• Reaction of the anti-capsular antibodies
with the carbohydrate material of the
capsule causes a micro precipitin reaction
on the surface of the organism and a
change in the refractive index of the
capsule itself.
• A small amount of methylene blue is
added to the preparation to allow
visualization of the cells and to provide
contrast so that subtle refractile changes in
the capsule can be more easily discerned.
Microscopically, the capsule appears
to “swell.”

63. STAINING OF ENDOSPORE
• The spores are highly resistant to normal staining procedures due to their
tough protein coats.
• The most commonly used method is Schaeffer-Fulton method.
• Principle: The primary stain in endospore staining procedures (malachite
green) is driven into the cells with heat since malachite green is water
soluble and doesn’t adhere well to the cell & since vegetative cells have
been disrupted by heat, malachite green rinses easily from the vegetative
cells allowing them to readily take up the counterstain.

64. Place dried & heat
fixed slide over beaker
of boiling water with
bacterial film uppermost
Within seconds,
condensation of droplets
on underside of slide; pour
5% aqueous solution of
malachite green & leave
for 1 mins with water
continue to boil.
Wash in cold water & treat
it with 0.5% safranin or
0.05% basic fuchsin for 30
seconds.Wash & dry
Observe it under
microscope

65. Schaeffer-Fulton Stain

66. Method Primary Stain Decoloriser Counterstain Interpretation
Grams Stain Crystal Violet Acetone Safranin Spore-colorless
Bacteria-Violet
Modified Ziehl-
Neelsen Staim
Carbol Fuchsin 0.25-0.5%
Sulphuric acid
Loeffler’s
Methylene Blue
Spore-red
Bacteria-Blue
Dorner Stain Carbol Fuchsin Acid Alcohol Nigrosin Spore-red
Bacteria-colorless
Variation in
Dorner Stain
Carbol Fuchsin Nigrosin Spore-red
Bacteria-colorless
Schaeffer-Fulton
Stain
Malachite Green Water Safranin Spore-green
Bacteria-red
Bartholomew &
Mittwer Method
Malachite Green Water Safranin Spore-green
Bacteria-red
Abbot’s Method Methylene Blue Acid Alcohol Aniline-fuchsin Spore-Blue
Bacteria-red
Moeller Stain Carbol Fuchsin Acidified
Ethanol
Methylene Blue Spore-red
Bacteria-Blue
Modified Moeller
Stain
Kinyoun’s Carbol-
fuchsin
Kinyoun’s
carbol-fuchsin
Loeffler’s
Methylene Blue
Spore-red
Bacteria-blue

67. REFRENCES
• Bailey & Scott’s Diagnostic Microbiology
• Koneman’s color atlas and textbook 7th Edition
• Mackie & McCartney Practical Medical Microbiology 14th Edition

68. THANK YOU
13th September, 1853
166th Birth-Anniversary of Christian Gram