STAINING TECHNIQUES

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INTRODUCTIONAs 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 for the recognition of bacteria.

Staining may be simple staining and differential staining.

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DyesBasic dyes: Methylene blue, Basic fuchsin, Crystal violet,

Safranine, Malachite green have positively charged groups (usually from penta-valent nitrogen) and are generally sold as chloride salts. Basic dyes bind to negatively charged molecules such as nucleic acids, many proteins and surfaces of bacterial and archeal cells.

Acidic dyes: Eosin, Rose Bengal and Acid fuchsin possess groups such as carboxyls (-COOH) and phenolic hydroxyls (-OH). Acidic dyes, in their ionized form, have a negative charge and bind to positively charged cell structures.

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SIMPLE STAINING• These show not only the presence of

organisms but also the nature of the cellular content in exudates.

• A single stain is used.• Examples are Loeffler’s methylene

blue, polychrome methylene blue, dilute carbol fuchsin.

• Simple staining is of positive staining and negative staining.

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Positive staining

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Negative staining• Negative staining procedure helps to

study the cell shape, cell breakage, refractable inclusion bodies and spores besides poly- hydroxy butyrate granules.

• It is useful for those bacteria which are difficult to stain.

• Very slender bacteria like spirochaetes that are not detectable by simple staining methods can be viewed by negative staining.

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Principle Negative staining requires the use of acidic stains

such as Indian ink and nigrosin. The acidic stain with its negatively charged chromogen will not penetrate the cell because of the negative charges on the surface of the bacteria. So unstained cells are diffferentiable against the dark background. Since heat fixation is not required, the cells are not subjected to the destaining effect of chemicals and heat, their natural size, shape and arrangement can be seen by this method. It is possible to observe bacteria that are difficult to stain.

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Procedure • A loopful of undiluted Indian ink was placed on one

end of clean slide.• A loopful of inoculum was transferred into the drop

of stain.• By using a second dirt-free slide with smooth edge

over the suspension, it was spread uniformly along the edge.

• The suspension was spread to the end of the slide so as to form a uniform smear.

• The slide was then air dried and observed under oil immersion objective.

• Colorless bacteria are seen against a dark background.

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Observation

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DIFFERENTIAL STAINING • This type of staining is to

differentiate two organisms.• Mainly used differential staining is

GRAM’S STAINING

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HANS CHRISTIAN GRAM

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HANS CHRISTIAN GRAMINTRODUCED GRAM’S STAINING

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GRAM’S STAINING• Gram Stain 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 specimens.

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Principle • Violet dye and the iodine combine to form an

insoluble, dark purple compound in the bacterial protoplasm and cell wall.

• This compound is dissociable in the decolorizer, which dissolves and removes its two components from the cell.

• But the removal is much slower from Gram-positive than from the Gram-negative bacteria, so that by correct timing the former stay dark purple whilst the latter become colorless.

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Principle • The difference between the two

types of bacteria is that the Gram positive have thicker and denser peptidoglycan layers in their cell walls, which makes them less permeable to the stain than those of the Gram negative bacteria.

• The iodine has a critical role in enhancing this difference.

• It seems to bind temporarily to the peptidoglycan and make it even less permeable to the dye.

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PROCEDURE• Step 1- Crystal violet

(primary stain) for 1 minute. Water rinse.

• Step 2- Iodine (mordant) for 1 minute. Water rinse.

• Step 3 – Alcohol (decolorizer) for 10-30 seconds. Water rinse.

• Step 4 - Safranin (counterstain) for 30-60 seconds. Water rinse. Blot dry.

• Cells stain purple.

• Cells remain purple.

• Gram-positive cells remain purple. Gram negative cells become colorless.

• Gram positive cells remain purple. Gram-negative cells appear red.

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Procedure

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Observation Gram positive cocci in chains Gram negative bacilli

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PAUL ERHLICH

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PAUL ERHLICHFIRST IDENTIFIED Mycobacterium tuberculosis

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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 with aniline-gentian violet followed by strong nitric acid.

• Stain used consists of basic fuchsin, with phenol added.

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PrincipleAcid fast bacteria retain primary stain

(carbol fuchsin) even after washing with a strong acid. It appears red while non-acid fast bacteria are decolorized on washing with acid and takes the color of the counter stain (methylene blue). The property of acid fasting appears due to the presence of mycolic acid in their cell walls. Mycolic acid is a group of branched chain hydroxy lipids.

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It is most commonly used to identify M.tuberculosis and M.leprae, the pathogen responsible for tuberculosis and leprosy, respectively. These bacteria have cell walls containing lipids constructed from mycolic acids, a group of branched chain hydroxyl fatty acids, which prevent dyes readily binding to the cells.

However, M.tuberculosis and M.leprae can be stained by harsh procedures such as the Zeihl-Neelson method which uses heat and phenol to derive basic fuchsin into cells.

Once basic fuchsin has penetrated, M.tuberculosis and M.leprae are not easily decolorized by acidified alcohol (acid-alcohol) and thus are said to be acid-fast.

Non acid-fast bacteria are decolorized by acid-alcohol and thus are stained blue by methylene blue counterstain.

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MYCOLIC ACID IN M.Tuberculosis

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Procerdure • A clean sterile glass slide was taken.• A thin uniform smear was prepared in the slide

using a inoculation loop.• The smear was allowed to air dry and then

heat fixed.• The smear was flooded with carbol fuchsin

and heated until steam arises.• Preparation was allowed to stay for 5-7 min.

The stain must not be allowed to evaporate or dry in the slide. Pour more carbol fuchsin on slide is necessary.

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Procedure • It is then washed under a low steam

of running water.• The smear was decolorized with 20%

sulphuric acid.• The slide is then washed again under

running tap water.• Counter stain the smear with

methylene blue for the 2 min.• Washed under running tap water.• Slide was blot dried and examined

under microscope.

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Procedure

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Observation

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ZN methods for weakly acid-fast organisms

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 nocardiae can be stained by ZN stain and decolorized with 1% sulphuric acid to demonstrate the acid-fastness of the clubs.

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ZN methods for weakly acid-fast organisms

3. Brucella differential stain. Brucella abortus in infected tissue or exudate may be distinguished from the latter by its weakly acid-fast reaction. Stain with dilute (1-in-10) carbol fuchsin, without heating, for 15 min. Decolorize with 0.5% acetic acid solution for 15 seconds, wash thoroughly with tap water and counter stain with Loeffler’s methyene blue for 1 min.

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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 mainly of polyphosphate, RNA and protein.

• 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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Volutin granules

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PrincipleAlbert’s stain contains cationic dyes like toludine

blue and malachite green.Due to the highly acidic nature of the granules,

they can be selectively stained by acidified basic dyes.

The toludine blue preferentially stain volutin granules while malachite green stains the cytoplasm.

Later due to application of Albert’s iodine, the dye molecule are fixed by precipitation.

Well developed granules of volutin (polyphosphate) may be seen in unstained wet preparations as round refractile bodies within the bacterial cytoplasm

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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 3-5 min.• The slide was then washed under running tap

water.• Flood the slide with Albert’s Iodine and

allowed to react about 1 min.• Slide was then washed and blot dried.• The slide was observed under oil immersion

objective of a microscope.

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Observation

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SPORE STAINING • The morphology of bacterial endospores is best

observed in unstained wet films under the phase contrast microscope, where they appear as large, refractile, oval or spherical bodies within a bacterial 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 is unstained and appears as clear area in the organism.

• This is the way in which spores are most commonly observed.

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Spore staining• If desired , however , it is possible by

vigorous staining procedures to introduce dye into the substance of the spore.

• When thus stained , the spores tends to retain the dye after treatment with decolorizing agents, and in this respect behaves similarly to the tubercle bacillus, but is more weakly acid-fast.

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Spore of a mold in bread

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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 resistance by spores is due to the high content calcium- dipicolinic acid.

• The spores are differentially stained using special procedures that help dye to penetrate the spore wall.

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Principle • 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 safranine and appears red.

• A modified Ziehl-Neelsen stain in which weak, 0.25% sulphuric acid is used as decolorizer, yields red spores in blue-stained bacteria. Lipid granules also stain red, appearing like small spherical spores.

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ProcedureFilms are dried and fixed with minimal flaming.1. Place the slide over a beaker of boiling

water , resting it on the rim with the bacterial film uppermost.

2. When , within several seconds , large droplets have condensed on the underside of the slide , flood it with 5% aqueous solution of malachite green and leave to act for 1 min while the water continues to boil.

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Procedure3. Wash in cold water.4. Treat with 0.5% safranine or 0.05%

basic fuchsin for 30 seconds.5. Wash and dry.This method colors the spores green

and the vegetative bacilli red. Lipid granules are unstained.

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Observation

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CAPSULE STAININGSome bacteria secrete a prominent slimy or gummy

material on their surface usually polysaccharide make up the capsule. It is not essential for life, but may serve as a reserve food. It provides protection against dehydration and also against phagocytosis. The chemical composition of capsule varies according to organism but usually consist of polysaccharides e.g., glucose, galactose, amino sugars.

Capsule are colorless and have low refractive index and so are difficult to observe without a special staining technique. Moreover the capsule is non-toxic, hence cannot be stained in the usual manner. Techniques like negative staining can be used to demonstrate the capsule.

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Principle Congo red is a negative stain. It stains

the back ground leaving the capsule unstained. Acid fuschin fixes congo red and reacts with it to give a blue back ground. Acid fuschin also stains the organism pink, leaving the capsule colorless.

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Procedure • Placed a drop of Manevali’s solution I on a clear

glass slide.• Sterilised the nichrome loop, cooled it and just

touched to the growth of organism on the slant.• Mixed the culture on loop with a drop of

Manevali’s solution I on the slide.• The drop was spread into a thin film. • The film was allowed to dry completely.• The smear was covered with Manevali’s

solution II and allowed to react for 1 min.• The excess stain was discarded and dried in air.• Observe under oil immersion objective.

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Observation

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FLAGELLAR STAININGFlagellar staining provides

taxonomically valuable information about the presence and distribution pattern of flagella on prokaryotic cells. Bacterial and archaeal flagella are fine, threadlike 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 with the light microscope, their thickness is increased by coating them with mordants such as tannic acid and potassium alum, and then staining with pararosalineor basic fuchsin.

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Flagella

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Procedure • Grow bacteria for 16 – 24 hrs on a

non-inhibitory medium , e.g. tryptic soy agar or blood agar.

• Touch a loopful of water onto the edge of a colony and let motile bacteria swim into it.

• Then transfer the loopful into a loopful of water on a slide to get a faintly turbid suspension and cover it with a cover-slip.

• The bacterial suspension is thus prepared with a minimum of agitation , which would detach the flagella.

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Procedure • After 5-10 min , when many bacteria

have attached to the surfaces of the slide and cover-slip , apply two drops of Ryu’s stain to the edge of the cover-slip and leave the stain to diffuse into the film.

• Examine with the microscope after standing 5-15 min at ambient temperature.

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Observation

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PERIODIC ACID-SCHIFF (PAS) METHOD FOR FUNGI IN TISSUE

SECTIONSThe polysaccharide constituents of

bacteria and fungi are oxidized by periodate to form polyaldehydes which yield red-colored compounds with Schiff’s fuchsin-sulphite; the proteins and nucleic acids remain uncolored. The method may be used to reveal fungal elements in sections of infected animal tissue; the fungi stain red, while the tissue material, except glycogen and mucin, fails to take the stain.

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Procedure • Bring sections to distilled water.• Treat for 5 min with a freshly prepared

1% solution of periodic acid in water.• Wash in running tap water for 15 min

and rinse in distilled water.• Stain with fuchsin-sulphite for 15 min.• Wash two or three times with sulphite

wash solution. Wash with water.

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Procedure • Wash in running tap water for 5 min

and rinse in distilled water.• Counterstain with dilute aqueous

malachite green or with0.1 % light green in 90 % alcohol for 1 min.

• Dehydrate rapidly in absolute alcohol , clear in xylene and mount in Canada balsam.

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Observation

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LEISHMAN’S STAINDry unfixed films are used. The stain is first used

undiluted , and the methyl alcohol fixes the film.

The stain is then diluted with distilled water, and the staining properly carried out.

• Pour the undiluted stain on the unfixed film and allow it to act for 1 min.

• By means of a pipette and rubber teat, add double the volume of distilled water to the slide, mixing the fluids alternately sucking them in the pipette and expelling them.

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Leishman’s stain• Allow the diluted stain to act for 12

min.• Flood the slide gently with distilled

water , allowing the preparation to differentiate in the distilled water until the film appears bright pink – usually about 30 seconds.

• Remove the excess water with blotting paper and dry in air.

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Observation

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GIEMSA STAINThis consists of a number of

compounds made by mixing different proportions of methylene blue and eosin. These have been designated Azur I, Azur II and Azur II-eosin. It is an advantage of Giemsa’s stain over Leishman’s that fixation is by alcohol instead of by undiluted stain which, particularly in the tropics, may deposit precipitate on the film.

Rapid and slow methods are preferred for staining and multiple films of malaria blood and tissue aspirates.

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Procedure 1. Fix films in methyl alcohol for 3 min.2. Stain in a mixture of 1 part stain and 10 parts

buffer soution pH 7.0 for 1 hr.3. Wash with buffer solution, allowing

preparation to differentiate for about 30 seconds.

4. Blot and allow to dry in air.This method of staining gives excellent results

with thin blood films for malaria parasites, Schuffner’s dots being well defined. Trypanosomes are also well demonstrated.

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Observation

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STAINING FOR SPIROCHAETES

The large spirochaetes borreliae stain by ordinary method including Gram’s (giving a negative reaction), Leishman’s and Giemsa’s. The smaller ones, e.g, treponemes and leptospires, are too thin to be demonstrated by ordinary stains. They are best observed in unstained wet films under the dark-ground microscope where their bright appearance and motility draw attention to them.

If a permanent preparation of small spirochaetes is required, use may be made of a silver impregnation method which artificially thickness them with a deposit of silver. The classical method are Fontana’s (for films) and Levaditi (for sections). Faulkener and Lille’s modified silver methods has been recommended for demonstrating leptospires in sections of tissue.

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Procedure • Treat the film three times, 30

seconds each, with a fixative.• Wash off the fixative with absolute

alcohol and allow the alcohol to act for 3 min.

• Drain off the excess alcohol and carefully burn off the remainder until the film is dry.

• Pour on the mordant, heating till steam rises, and allow it to act for 30 seconds.

• Wash well in distilled water and again dry the slide.

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Procedure • Treat with ammoniated silver nitrate, heating

till steam rises, for 30 seconds, when the film becomes brown in color.

• Wash well in distilled water, dry and mount in Canada balsam.

It is essential that the specimen be mounted in balsam under a cover-slip before examination, as some immersion oils cause the film to fade at once.

The spirochaetes are stained brownish-black on a brownish-yellow background

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Observation

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LACTOPHENOL COTTON BLUE STAINING

Lactophenol cotton blue stain is used for making a semi permanent microscopic preparation of fungi. It stains the fungal cytoplasm and provides a light blue background against which walls of the hyphae can readily be seen. It contains four constituents, phenol serves as fungicidal agent, lactic acid acts as a clearing agent, cotton blue stains the cytoplasm of fungus and glycerine which gives the semi permanent preparation. A permanent preparation is made by incorporating polyvinyl alcohol in place of glycerine.

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Procedure A drop of lactophenol cotton blue was placed on

a clean glass slide.A small part of fungal colony was taken from

Potato Dextrose Agar (PDA) plate using a sterile needle and it was placed on the drop of lactophenol cotton blue that had been taken on the glass slide. The fungal elements were spread with sterile needle.

A coverslip was placed over the specimen taking care to avoid the formation of air bubbles.

Excess stain was wiped off. The slide was kept for 5-10 min and was observed first under 10X then under 45X objective.

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Observation

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