PHARMACEUTICAL MICROBIOLOGYPHARMACEUTICAL MICROBIOLOGY

JIGAR SHAHJIGAR SHAH

INSTITUTE OF PHARMACYINSTITUTE OF PHARMACY

NIRMA UNIVERSITYNIRMA UNIVERSITY

Preparation of smear & Staining techniquesPreparation of smear & Staining techniques

��When specimen is to be fixed, a thin film of material containing the When specimen is to be fixed, a thin film of material containing the m.rganisms is spread over the surface of slide. This is smear.m.rganisms is spread over the surface of slide. This is smear.

��Fixing simultaneously kills the m.organisms and attaches them to the Fixing simultaneously kills the m.organisms and attaches them to the slide. It preserves various parts of microbes in their natural state slide. It preserves various parts of microbes in their natural state with only minimum distortion.with only minimum distortion.

��Staining simply means coloring the m.organisms with a dye that Staining simply means coloring the m.organisms with a dye that ��Staining simply means coloring the m.organisms with a dye that Staining simply means coloring the m.organisms with a dye that emphasizes certain structures.emphasizes certain structures.

��Air drying and flaming fix the m.organisms.Air drying and flaming fix the m.organisms.��Stains are salts Stains are salts –– a positive & a negative ion a positive & a negative ion –– one of which is one of which is

colored colored –– chromophore. In basic dye, it is in the positive ion; in chromophore. In basic dye, it is in the positive ion; in acidic dye, it is in the negative ion.acidic dye, it is in the negative ion.

��Bacterial cell has negative charge, so colored positive ion in a basic Bacterial cell has negative charge, so colored positive ion in a basic dye is attracted to the negatively charged bacterial cell.dye is attracted to the negatively charged bacterial cell.e.g. crystal violet, methylene blue, safranin, malachite greene.g. crystal violet, methylene blue, safranin, malachite green

Staining techniques…Staining techniques…

��AcidicAcidic dye’sdye’s negativenegative ionsions areare repelledrepelled byby thethe negativelynegatively chargedchargedbacterialbacterial surface,surface, soso thethe stainstain colorscolors thethe backgroundbackground..

��PreparingPreparing colorlesscolorless bacteriabacteria againstagainst aa coloredcolored backgroundbackground isis calledcallednegativenegative stainingstaining..

��ItIt isis valuablevaluable inin thethe observationobservation ofof overalloverall cellcell shapes,shapes, sizessizes andandcapsulescapsules becausebecause cellscells mademade highlyhighly visiblevisible againstagainst aa contrastingcontrasting darkdarkbackgroundbackground..

��SIMPLESIMPLE STAINSSTAINS::

�� ItsIts anan aqueousaqueous oror alcoholalcohol solutionsolution ofof aa singlesingle basicbasic dyedye..

�� ItIt isis useuse toto highlighthighlight thethe entireentire microorganismmicroorganism withwith cellularcellular shapesshapes andandbasicbasic structuresstructures..

�� Sometimes,Sometimes, aa chemical,chemical, mordantmordant isis addedadded toto thethe solutionsolution toto intensifyintensifythethe stain,stain, toto increaseincrease thethe affinityaffinity ofof aa stainstain forfor aa biologicalbiological specimenspecimenandand toto coatcoat aa structurestructure toto makemake itit thickerthicker andand easiereasier toto seesee..

Staining techniques…Staining techniques…

��DIFFERENTIAL STAINS:DIFFERENTIAL STAINS:

�� Two types 1) Gram Stain, 2) Acid fast stainTwo types 1) Gram Stain, 2) Acid fast stain

�� GRAM STAIN:GRAM STAIN:

�� It was developed in 1884 by the Danish bacteriologist Hans Christian It was developed in 1884 by the Danish bacteriologist Hans Christian Gram. It classifies bacteria into two large groups: Gram. It classifies bacteria into two large groups: GramGram--positivepositiveand and Gram negative.Gram negative.

�� A heat fixed smear is covered with a basic dye, crystal violet, primary A heat fixed smear is covered with a basic dye, crystal violet, primary �� A heat fixed smear is covered with a basic dye, crystal violet, primary A heat fixed smear is covered with a basic dye, crystal violet, primary stain.stain.

�� After some time, purple dye is washed off, smear is covered with After some time, purple dye is washed off, smear is covered with iodine, a mordant. When iodine is washed off, both bacteria appear iodine, a mordant. When iodine is washed off, both bacteria appear dark violet or purple.dark violet or purple.

�� Next, slide is washed with alcohol or an alcoholNext, slide is washed with alcohol or an alcohol--acetone solution, acetone solution, decolorizing agent, which removes purple from cells of some species decolorizing agent, which removes purple from cells of some species (gram negative) but not from others.(gram negative) but not from others.

Staining techniques…Staining techniques…��TheThe alcoholalcohol isis rinsedrinsed off,off, andand slideslide isis stainedstained withwith safranin,safranin, aa basicbasic redred

dye,dye, counterstaincounterstain.. TheThe smearsmear isis washedwashed again,again, blottedblotted drydry &&examinedexamined..

�� BacteriaBacteria thatthat retainretain purplepurple colorcolor afterafter thethe alcoholalcohol hashas attemptedattempted totodecolorizedecolorize themthem areare classifiedclassified asas gramgram--positivepositive;; bacteriabacteria thatthat loselose darkdarkvioletviolet colorcolor afterafter decolorizationdecolorization areare classifiedclassified asas gramgram negativenegative..

�� WHYWHY THISTHIS ISISHAPPEN?HAPPEN?WHYWHY THISTHIS ISISHAPPEN?HAPPEN?�� DifferentDifferent kindskinds ofof bacteriabacteria reactreact differentlydifferently toto thethe GramGram stain,stain, duedue toto

structuralstructural differencesdifferences inin theirtheir cellcell wallswalls whichwhich causecause retentionretention ororescapeescape ofof aa combinationcombination ofof crystalcrystal violetviolet –– iodineiodine complexcomplex (CV(CV--I)I)..

�� GramGram –– positivepositive bacteriabacteria havehave aa thickerthicker peptidoglycanpeptidoglycan (disaccharides(disaccharidesandand aminoamino acids)acids) cellcell wallwall thanthan gramgram--negativenegative bacteriabacteria..

�� CVCV--II complexcomplex formed,formed, isis largelarge enough,enough, cantcant bebe washedwashed outout ofof intactintactpeptidoglycanpeptidoglycan layerlayer ofof gramgram--positivepositive cellscells byby alcoholalcohol.. SoSo retainretain colorcolorofof crystalcrystal violetviolet dyedye..

Staining techniques…Staining techniques…�� Gram negative bacteria contain thin Gram negative bacteria contain thin peptidoglyacanpeptidoglyacan cell wall with a cell wall with a

layer of layer of lipopolysaccharidelipopolysaccharide..

�� The alcohol wash The alcohol wash distruptsdistrupts the outer the outer lipopolysaccharidelipopolysaccharide layer, and the layer, and the CVCV--I complex is washed out thorough thin layer of I complex is washed out thorough thin layer of peptidoglycanpeptidoglycan. So . So gramgram--negative cells are colorless and counterstained with negative cells are colorless and counterstained with safraninsafranin..

�� The Gram reaction of a bacterium can provide valuable information The Gram reaction of a bacterium can provide valuable information for treatment of disease. Gramfor treatment of disease. Gram--positive bacteria tend to killed easily positive bacteria tend to killed easily by by penicillinspenicillinsand and cephalosporinscephalosporins. Gram. Gram--negative bacteria are more negative bacteria are more by by penicillinspenicillinsand and cephalosporinscephalosporins. Gram. Gram--negative bacteria are more negative bacteria are more resistant because antibiotics can’t penetrate the resistant because antibiotics can’t penetrate the lipopolysaccharidelipopolysaccharidelayer.layer.

�� ACIDACID--FAST STAIN:FAST STAIN:

�� First red dye First red dye carbolfuchsincarbolfuchsin is applied to a fixed smear, heated & is applied to a fixed smear, heated & cooled, washed with water, Next apply acidcooled, washed with water, Next apply acid--alcohol, a alcohol, a decolorizerdecolorizer..

�� The acidThe acid--fast bacteria retain red color because fast bacteria retain red color because carbolfuchsincarbolfuchsin is more is more soluble in cell wall lipids than in acidsoluble in cell wall lipids than in acid--alcohol. alcohol.

Staining techniques…Staining techniques…�� In non acid fast bacteria, cell walls lack lipid, carbolfuchsin is In non acid fast bacteria, cell walls lack lipid, carbolfuchsin is

removed, cells stained with methylene blue, counterstain.removed, cells stained with methylene blue, counterstain.

�� Microbiologist use this stain to identify all bacteria in the genus Microbiologist use this stain to identify all bacteria in the genus Mycobacterium. Mycobacterium tuberculosis (tuberculosis), and Mycobacterium. Mycobacterium tuberculosis (tuberculosis), and Mycobacterium Leprae (leprosy).Mycobacterium Leprae (leprosy).

Staining techniques…Staining techniques…�� SPECIAL STAINS:SPECIAL STAINS:

�� They are used to color and isolate specific parts of microorganisms, They are used to color and isolate specific parts of microorganisms, such as such as endosporesendospores, flagella, and to reveal the presence of capsules., flagella, and to reveal the presence of capsules.

�� Negative staining for capsules:Negative staining for capsules:

�� Capsule staining is more difficult because capsular materials are Capsule staining is more difficult because capsular materials are soluble in water and may be removed during vigorous washing.soluble in water and may be removed during vigorous washing.

�� To demonstrate presence of capsules, mix bacteria in a fine colloidal To demonstrate presence of capsules, mix bacteria in a fine colloidal suspension of colored particles solution (suspension of colored particles solution (nigrosinnigrosin), to provide a dark ), to provide a dark background and stain the bacteria with simple stain, such as background and stain the bacteria with simple stain, such as safraninsafranin. . So due to its chemical composition, capsules do not accept dye, and So due to its chemical composition, capsules do not accept dye, and appear as halos surrounding each stained bacterial cell.appear as halos surrounding each stained bacterial cell.

�� EndosporeEndospore (Spore) staining:(Spore) staining:

�� An An endosporeendospore, a dormant structure, is formed within a cell that , a dormant structure, is formed within a cell that protects a bacterium from adverse environmental conditions.protects a bacterium from adverse environmental conditions.

Staining techniques…Staining techniques…�� Endospores cannot be stained by ordinary methods, because the dyes Endospores cannot be stained by ordinary methods, because the dyes

do not penetrate the wall of the endospore.do not penetrate the wall of the endospore.

�� The most commonly used endospore stain is the SchaefferThe most commonly used endospore stain is the Schaeffer--Fulton Fulton endospore stain.endospore stain.

�� Malachite green, primary stain, is applied to a heatMalachite green, primary stain, is applied to a heat--fixed smear and fixed smear and heated for 5 minutes.heated for 5 minutes.

�� Heating helps stain to penetrate the endospore wall.Heating helps stain to penetrate the endospore wall.

�� Preparation is washed with water to remove primary stain from all the Preparation is washed with water to remove primary stain from all the cells parts except endospore.cells parts except endospore.

�� safranin, counter stain, is applied to smear to stain portions of the cell safranin, counter stain, is applied to smear to stain portions of the cell other than endospores.other than endospores.

�� Endospore appear green within red or pink cells.Endospore appear green within red or pink cells.

Staining techniques…Staining techniques…�� Flagella Staining:Flagella Staining:

�� Bacteria flagella are structures of locomotion too small to be seen with Bacteria flagella are structures of locomotion too small to be seen with a light microscope without staining,a light microscope without staining,

�� A tedious and delicate staining procedure uses a mordant and the stain A tedious and delicate staining procedure uses a mordant and the stain carbolfuchsin to build up the diameters of the flagella.carbolfuchsin to build up the diameters of the flagella.

�� Microbiologists use the number and arrangement of flagella as Microbiologists use the number and arrangement of flagella as diagnostic aids.diagnostic aids.diagnostic aids.diagnostic aids.