it includes the staining methods,slide preparation methods and important points for GPAT exam

1. Staining Techniques
By
B.Lakshmi satya(PhD)
Assoc. Professor
VIPER

2. STAIN/DYE
• Dye
_ an organic compound obtained naturally or
synthetically
– make internal and external structures of cell more
visible by increasing contrast with background
– chemical composition is
• chromophore groups
– chemical groups with conjugated double bonds
– Imparts color to benzene
• Auxochrome
_conveys the property of ionization
_enables it to form salts
_ helps dye bind with a cell
• Benzene ring
_ colourless organic solvent

3. • Benzene ring and chromophore together are called as
chromogen
• Chromogen is a colored compound but not a stain
• Along with auxochrome it is called as stain

4. Types of stains
• Depending on the molecular structure
- Triphenyl methane dyes
- Oxazine dyes
- Thiazine dyes
• Depending on the electric charge present on the
chromophore.
- acidic dyes
- basic dyes

5. Types of stains
• Basic dye: cationic chromogen
-upon ionization chromogen portion exhibits +ve
charge
-therefore they have strong affinity towards –vely
charged constituents of the cell like nucleic acids
-the chloride or sulphate salts of coloured bases
-eg: methylene blue, crystal violet, safranin, basic
fuchsin, Eosin Y, malachite green.

6. Types of stains
• Acidicic dye: anionic chromogen
-upon ionization chromogen portion exhibits -ve charge
-therefore they have strong affinity towards +vely charged constituents
of the cell like proteins
- Na,K,Ca and ammonium salts of coloured bases
-eg: picric acid, congo red, nigrosine/indian ink, sodium eosinate, Rose
Bengal stain
Basic dyes are mostly used due to the presence of _ve charge on
bacterial surface.
Ph may alter staining effectiveness. Basic dyes effective at higher Ph
and acidic dyes effective at lower Ph.

7. Theory of staining
• Chemical theory: Ionization
sometimes through covalent binding
there is no evidence of formation of a new compound
through chemical reactions
eg: DNA, schiffs reagent, Feulgen
• Physical theory : absorption, adsorption,
Osmosis, Solubility
dye can be extracted by washing with acid or alcohol.

8. Staining techniques
• Classified into
– Simple stain: single stain is used
for study of morphology
– Differential stain: two or more stains.
for differentiation into groups.
Eg:gram staining, acid fast staining
– Structural or special stains: two or more stains
for study of internal and external structures
Eg:flagella stain, capsule stain, spore stain, nuclear
Staining differentiates organisms due to differences in
chemical composition of organism.

9. Preparation of Specimens
(increases visibility of specimen)
• Wet mount or hanging drop method
• Fixation
– heat fixing
• Causes coagulation of proteins
• preserves overall morphology but not internal structures
– chemical fixing
• Preserves internal structures
• Penetrates into cell components makes them immobile,
insoluble, inactive.
• protects fine cellular substructure and morphology
• for more delicate organisms
• Eg: glacial acetic acid, HCHO, glutaraldehyde, acetone and
ethanol.

10. Heat fixation

11. Wet mount method

12. hanging drop method

13. Wet mount/hanging drop method are used to
- study morphology of spiral bacteria
Eg:syphilis organism in dark field microscope
- Motility
- Cytological changes during cell division to determine rate of division
- To study cell inclusion bodies eg:vacuoles and lipids.
• These methods are used in slide preparation for dark field and phase
contrast microscopy.
• When same is used for bright field the light intensity should be adjusted
properly. Partially close to the substage condenser diaphram

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Inc. Permission required for reproduction
or display.
14
Simple staining
– a single staining agent is used
– basic dyes are frequently used
• dyes with positive charges
• e.g., crystal violet

15. Steps in simple staining
• 24 hr culture
• Cell suspension preparation
• Smear preparation
• Air drying
• Heat fixation
• Application if reagent

16. Microscopic images of few organisms

17. Differential Staining
• To differentiate organism into groups based on
the differences in their cellular components
• More than 1 dye is used
– e.g., Gram stain
– e.g., acid-fast stain
Copyright © The McGraw-Hill Companies,
Inc. Permission required for reproduction
or display.
17

18. Gram Staining
• Invented by Christian gram in 1884
• Used to differentiate bacteria.
• Yeast cells stain as gram negative bacteria.
• Few protozoa respond to this.
• Useful in identification of bacteria
• Not all bacteria can be definitely classified by this
technique. This gives rise to Gram- variable and
Gram indeterminate groups as well

21. Gram Staining
• Gram staining reagents
– Crystal violet: primary stain
– Iodine: mordant
– Alcohol or acetone-alcohol:
decolourizer
– Safranin: counterstain
Gram positive: purple
Gram negative: pink-red
Staphylococcus aureus
Escherichia coli

22. Steps in Gram staining
• 24 hr culture
• Cell suspension
• Smear preparation
• Air drying
• Heat fixation
• Application if reagents in below sequence

23. Steps in gram staining

24. Principal involved
• Structural differences in the cell wall
Bacteria is a prokaryotic cell contains cell wall and no nucleus
Theory Gram positive Gram negative
Peptidoglycan More layers and cross links Less layers
Lipid Low(1-4%) High(11-20%)

26. Figure 4.13b, c

27. • Braun’s lipo proteins: binds outer membrane and
peptidoglycan very firmly.
• Outer membrane contains lipopolysaccharides (LPS)
• LPS -protects cell wall from antibody attack
avoids host defenses
protects entry of bile salts, Antibiotics & toxic
substances that can kill it
contributes -ve charge on bacteria surface
• LPS contains 3 parts
lipid A-major constituent & toxic
So LPS acts as endotoxin and shows some symptoms that arise in Gr-ve bacterial infections
core poly saccharide
O side chain-Constitutes major antigen

28. Differences Between Gram Positive and Negative Cells
Gram-positive cell walls
• Thick cell wall(20-80nm)
• 90% peptidoglycan(40-90%
dry cell weight)
• Teichoic acids (provide –ve
charge)
• 1 layer
• Not many polysaccharides
• Less periplasmic space
Gram-negative cell walls
• Thin & complex cell wall (2-
7nm peptidoglycan & 7-8nm
outer membrane)
• 5-10% peptidoglycan(5-10%
dry cell weight)
• No teichoic acids
• 3 layers
• Outer membrane has braun’s
lipids, polysaccharides
• More periplasmic space

29. Differences Between Gram Positive and Negative Cells
Gram-positive cell walls
• Mesosomes (localized
infoldings-more in bacilli)
are present
Gram-negative cell walls
• Mesosomes are absent

30. Precautions
Need to be careful in the following areas
• 24 hr culture
• Heat fixation/methanol
• Clean slide
• Thin smear
• decolourization(not too long period or too short)
• culture age, media, incubation atmosphere,
staining method

31. Gram staining Experiment
• https://www.youtube.com/watch?v=AZS2wb7
pMo4

32. Acid fast staining
• It was first discovered by Earlich in 1881 and
modified by Zeihl & Neelsen.
• It is a differential stain used mainly to detect
mycobacteria.
• ACID FAST means bacteria which protect the
primary dye to be washed of from the action
of acid alcohol decolourizer.

33. • Z N stain is a modification of Ehrlich’s original method
for differential staining of acid fast bacilli by use of
aniline gentian violet followed by strong nitric acid.
• The ordinary aniline dye solution do not readily
penetrate the acid fast bacilli.
• So by use of powerful staining solution that contain
phenol and application of heat , the dye can be made
to penetrate the bacillus.
• Phenol will solubilise the cell wall and heat will increase
the stain penetration.
• Once stained the tubercle bacilli will withstand the
action of powerful decolorizing agents for considerable
period of time , retains the primary stain when every
thing else has been decolorized.

34. Acid fast Staining
(for identification of Mycobacterium sps)
– Corbol fuchsin: primary
stain
– Heating/surfactant:
mordant
– Alcohol or acid: decolourizer
– Safranin: counterstain
– Acid fast: red
– Non acid fast: Blue

35. Carbol
fuchsin
Acid
alcohol
Methylene
blue
Reddish-pink
Blue
Acid Fast
Nonacid Fast Kinyoun Acid-Fast Staining Procedure
1. A sample of cells is mixed with a drop of
water on a clean slide to make a smear.
After air drying, the slide is heat fixed.
2. Slide is flooded with carbol fuchsin (primary
stain basic fuchsin + mordant carbolic acid)
and allowed to sit for 15 minutes.
Slide is rinsed until water coming off the slide
is clear.
3. Slide is decolorized with acid alcohol (3% HCl
and 95% alcohol) 20 seconds, then rinsed.
4. Slide is flooded with methylene blue (counter
stain) for 60 seconds and then rinsed.

36. Mycobacteria structure
• Contain large amount of
fatty waxes (mycolic acid)
within their cell wall 
resist staining by ordinary
methods
• Require a special stain for
diagnostic  Acid Fast
stain.
http://www.med.yale.edu/labmed/casestudies/images/cs4_mycolic_acid.jpg

37. Specimen having Mycobacterium sps

38. STRUCTURES THAT ARE ACID FAST
• All Mycobacteria - M. tuberculosis, M. leprae and
atypical Mycobacterium
• Actinomyces – Nocardia ,Rhodococus
• Head of sperm
• Bacterial spores
• Cysts of some coccidian parasites:
Cryptosporidium parvum, Isospora belli, Cyclospora
cayetanensis
• A few other parasites:
Taenia saginata eggs, Hydatid cysts, especially their
hooklets stain irregularly with ZNstain

39. Importance Of Z N Stain For M. Tb
Bacilli
• Acid fast staining reaction of mycobacteria along
with their characteristic size and shape is a
valuable aid in the early detection of infection
and in the monitoring of therapy for
mycobacterium disease,.
• The presence of acid fast bacilli in the sputum,
combine with a history of cough, weight loss and
chest radiographic evidence of pulmonary
infiltrate, is the presumptive evidence of active
tuberculosis.

40. Modifications in the zeihl-neelsen
method
• For weakly acid fast organisms
• 5% H2SO4 for M. leprae (cigar bundle appearance)
• 1% H2SO4 for Actinomyces in tissue
• 0.5% H2SO4 for cultures of Nocardia
• 0.25-0.5% H2SO4 for spores and for oocysts of
Cryptosporidium and Isospora
• 0.5% acetic acid ---- Brucella (dilute carbol fuchsin, no
heating)
• H2SO4 does not decolourize as strongly as the HCl. This
makes it useful for staining organism that are weakly
acid fast
• Secondary stain is brilliant green(M.Leprae) or
methylene blue

41. ZN stain methods
• Cold ZN stain:
Kinyoun’s Method, Gabett’s Method
• ZN stain for spores:
Muller’s method, Dorner’s method, Schaffer fulton
stain, Muller chermock tergitol method
• For tissue sections:
Ellis and Zabrowarny stain, Fite faroco stain, Wade
fite stain
• Modified bleach ZN method
• Cooper’s modifications

42. Kinyoun’s Method
• Same as zeihl-nelson method
• No heating of slides as mordant
• The carbol fuchsin of Kinyoun has a greater
conc. of phenol and basic fuchsin so heating is
not required.
• Secondary stain is methylene blue.

43. Gabett’s Method
• It is a two step method
• No heating as mordant
• Decolourization and counter staining are done
in one step.

44. summary
• Various method of modification of Z N stain are
helpful by their modification to see less acid fast
structure , acid fast bacilli in tissue section and
also spores. It also causes less damage to this
structure.
• It also increases the sensitivity of stain.
• 20% H2SO4 is used for M.tuberculosis
• M. tuberculosis is both acid fast and alcohol fast,
while saprophytic mycobacteria are only acid fast.
• At least 10000 bacilli/ml should be present for
this method.

45. • Which alcohol is better?
• Several alcohols have been studied, and it has been
reported that the more complex the alcohol, the slower
the decolorization action. As the carbon chain lengthens,
decolorization is slower.
• Conn found in practice, however, no known advantage
can be gained by substituting the higher alcohols for
ethyl alcohol.

46. Acid fast staining Experiment
https://www.youtube.com/watch?time_continu
e=23&v=YzTgHU-aCqo&feature=emb_logo

47. Special Stains
• Capsule stain
– Klebsiella pneumonia

48. Flagella Staining
mordant applied to increase thickness of flagella

49. Spore stain (Schaeffer-Fulton)
– double staining technique
– bacterial endospore is one color and vegetative cell
is a different color
Bacillus subtilis