This document discusses various staining techniques used to visualize cells and internal structures. It describes the basic components and principles of dyes and stains, including chromophores, auxochromes and benzene rings. It outlines different types of stains categorized by molecular structure and electric charge. Basic techniques like simple staining, gram staining and acid-fast staining are explained in detail. The document compares properties of gram-positive and gram-negative cell walls. It provides examples of structures that are acid-fast and the importance of Ziehl-Neelsen staining for detecting Mycobacterium tuberculosis bacilli.
this slide will help you to understand the behavior of different types of bacteria in different culture media. its is made with an exmaple experiment which can provide better understadng. selective, differential and enriched media is given with detailed description in the example.
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this slide will help you to understand the behavior of different types of bacteria in different culture media. its is made with an exmaple experiment which can provide better understadng. selective, differential and enriched media is given with detailed description in the example.
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identification of bacteria- lecture 7.pptxOsmanAli92
he culture media are classified in many different ways: Based on the physical state Liquid media Solid media Semisolid media Based on the presence or absence of oxygen Anaerobic media Aerobic media Based on nutritional factors Simple media Synthetic media Complex
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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.
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
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
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%)
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
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
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
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
49. Spore stain (Schaeffer-Fulton)
– double staining technique
– bacterial endospore is one color and vegetative cell
is a different color
Bacillus subtilis