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Edit Comment Close Premium member Presentation Transcript Staining part 1 : Staining part 1 Dr.Reena Gopinathan, 1st Year PG, DEPT. of Microbiology, Medical college, Thrissur. 1 Staining : Staining Staining is a technique used in microscopy to enhance contrast in the microscopic image Stains are dyes which are used to highlight microorganisms or biological tissues for viewing with the help of a microscope 2 Aims of Staining in microbiology : Aims of Staining in microbiology To increase visibility & contrast To study the morphology To detect extracellular and intracellular components of microbes To help in the primary level of diagnosis 3 Methods of making film/smear : Methods of making film/smear Film / smear preparations are made usually on 3x1’’ glass slides Slides should be superior quality and grease free If the grease free slide is not provided with, should clean the slides 4 Cleaning of slides : Cleaning of slides Method 1 Wipe the slide with dry cotton cloth Pass the slide through blue flame of Bunsen flame 16-12 times Method 2 Moisten the finger with water, rub it on the surface of fine sand soap Smear it over the slide Remove soapy film with clean cotton cloth 5 Cover slips : Cover slips Should be ¾ or 7/8 inches 0.1 mm thickness Should be used only for wet preparations 6 Making films : Making films Take a clean slide A target circle of approximately 1.5 cm diameter should be marked on the under surface of slide Place a loopful of liquid bacterial suspension on the circled area Spread the suspension with a Nichrome wire loop Allow the slide to air dry completely 7 Making smears from various specimens : Making smears from various specimens Purulent specimen Use sterile wire loop Make a thin smear If needed put a drop of saline Non Purulent specimen Make a smear from a drop of well mixed specimen Culture Emulsify a colony in sterile distilled water Use sterile wire loop Make a thin smear 8 Making smears from various specimens .. .. .. : Making smears from various specimens .. .. .. Sputum Use a broom stick Transfer a purulent part to the slide Spread evenly Swabs Roll the swab on a slide If needed put a drop of saline Faeces Use a broom stick Transfer a mucopurulent part to the slide Spread evenly 9 Fixing of slides : Fixing of slides Heat fixation By passing the slide over flame Chemical fixation Using ethanol Methanol Picric acid Potassium permanganate Formaldehyde vapour 10 fixation - procedure : fixation - procedure Three quick passes over the flame is sufficient for fixation Chemical fixation is just applying few drops of chemicals and allow to evaporate 11 fixation - uses : fixation - uses It kills bacteria rendering safe handling It prevents autolysis by inactivating the autolytic enzymes It increases the permeability of cells to stain It makes cells rigid It unfolds the globular proteins and exposing reactive groups & increasing affinity for stain 12 METHODS OF STAINING : METHODS OF STAINING Simple staining Differential staining Special staining 13 Simple staining : Simple staining 14 types : types Monochrome staining / positive staining Negative / indirect staining 15 Monochrome staining / positive staining - principle : Monochrome staining / positive staining - principle Basic dyes are used for this Basic dyes are Positively charged These dyes get attached to negatively charged cytoplasm of microbial organism 16 Negative / indirect stainingprinciple : Negative / indirect stainingprinciple Acidic dyes are used for this Acidic dyes are negatively charged These dyes get repelled by the negatively charged cytoplasm of microbial organism So the dye get gathered around the organism They give a contrast back ground Organism stands unstained 17 Monochrome staining : Monochrome staining The use of single stain to colour the bacteria is known as monochrome staining Usual stains used are Crystal violet Loeffler’s Methylene blue Dilute carbol fuchsin 18 Crystal violet : Crystal violet Crystal violet 10 gm Absolute alcohol 100ml Distilled water 1 litre Dissolve the dye in alcohol Filter through filter paper Add to water 19 Loeffler’s Methylene blue : Loeffler’s Methylene blue Saturated solution of methylene blue in alcohol 300 ml KOH, 0.01% in water 1 litre Dissolve & mix thoroughly 20 Carbol fuchsin - strong : Carbol fuchsin - strong Phenol 85 gm Basic fuchsin 15 gm Ethanol 250 ml Distilled water 1250 ml Mix Phenol and Basic fuchsin , heat gently to dissolve. Add ethanol and distilled water. Filter in to a bottle. 21 Carbol fuchsin - weak : Carbol fuchsin - weak Dilute 1 volume of strong Carbol fuchsin with 10-20 volumes of distilled water. 22 Procedure Monochrome staining : Procedure Monochrome staining Cover the fixed smear with stain If Crystal violet keep stain for 2-6 sec If Methylene blue 3-5 min If Dilute Carbol fuchsin 15-30sec Wash under running water Blot dry Examine under oil immersion 23 Negative / indirect staining : Negative / indirect staining Usual stains used are Nigrosin Indian ink 24 Nigrosin : Nigrosin Nigrosin 100gm Formalin 5ml Distilled water 100ml Dissolve nigrosin in formalin Add to Distilled water 25 India ink : India ink A dense homogenous India ink free from large particles is to mix with quarter of its volume of grade 12 ballotini glass beads 0.22 mm and shake well for one hour in a Mickle tiss disintegrator. This ink may be improved by evaporation to concentrate. 26 Negative staining - procedure : Negative staining - procedure Wipe the slide clean Place a large drop of stain on the slide Emulsify small portion of bacterial culture Place a cover slip Look under high power objective 27 Negative staining : Negative staining 28 India ink Nigrosin Cryptococcus spore B. subtilis Cocci Differential staining : Differential staining 29 Differential staining : Differential staining Gram’s method Acid fast staining 30 Salutes .. .. .. : Salutes .. .. .. Hans Christian Gram 13-09-1853 to 14-11-1938. Danish bacteriologist. The work with Gram staining gained him international reputation 31 Gram’s staining : Gram’s staining Gram staining is a microbiological procedure that categorizes bacteria based on the physical and chemical structure of their outer surface 32 Gram’s method - principle : Gram’s method - principle The cell wall of gram positive bacteria contains high amount of Peptidoglycan and negligible amount of lipids The cell wall of gram negative bacteria have very negligible Peptidoglycan and high amount of lipids The primary basic Stain, stains both kind of cells 33 Gram’s method – principle .. : Gram’s method – principle .. In second step when iodine add, it act as a mordant and form crystal violet iodine Peptidoglycan complex in gram positive bacteria This complex is not formed in gram negative bacteria as Peptidoglycan is much less in their cell wall 34 Gram’s method – principle .. : Gram’s method – principle .. In the next step, application of decolorizer extract the lipids from the gram negative bacteria Removal of lipid makes the cell porous Porosity allows the release of primary stain from cell Gram negative cells become colourless at this step This colourless bacteria took the counter stain 35 Slide 36: 36 Gram’s method – procedure : Gram’s method – procedure Cover the smear with *Basic pararosaniline violet dye for1min Wash with water Cover with Gram’s iodine for 1 min Wash with water Decolourise with acetone for 2 sec *Crystal violet *Methyl violet *Gentian violet 37 Gram Staining contd : Gram Staining contd Wash with water Cover with *Counter stain Wash with water Blot dry Observe under oil immersion *Basic fuchsin *Neutral red *Safranine 38 Slide 39: 39 2 9 8 7 6 1 2 3 4 5 6 Crystal violet : Crystal violet Crystal violet 10 gm Absolute alcohol 100ml Distilled water 1 litre Dissolve the dye in alcohol Filter through filter paper Add to water 40 Methyl violet : Methyl violet Methyl violet 10 gm Absolute alcohol 100ml Distilled water 1 litre Dissolve the dye in alcohol Filter through filter paper Add to water 41 Gentian violet : Gentian violet Crystal violet 5 gm Methyl violet 5gm Absolute alcohol 100ml Distilled water 1 litre Dissolve the dye in alcohol Filter through filter paper Add to water 42 Lugol’s iodine : Lugol’s iodine Iodine 5 gm Potassium iodide 10 gm Distilled water 100 ml Dissolve Iodine & Potassium iodide in some water. Add remaining water. For use dilute 1/5 with Distilled water 43 Basic fuchsin : Basic fuchsin Basic fuchsin 0.5 gm Distilled water 1 litre Dissolve the dye in water Recommended for general use Apply for 10 -30 sec 44 Neutral red : Neutral red Neutral red 1 gm 1% Acetic acid in water 2ml Distilled water 1 litre Dissolve dye in water Add Acetic acid Recommended for Gonococci & other intracellular gram negative bacteria Apply for 2-4 min 45 safranine : safranine Safranine 0.5 gm Distilled water 1 litre Dissolve the dye in water Recommended for general use Apply for 10 -30 sec 46 Gram Staining - Results : Gram Staining - Results Report should include Number of bacteria Gram reaction of bacteria Morphology Presence and number of pus cells Presence of yeast cells Presence of epithelial cells 47 Gram Staining - Results : Gram Staining - Results When reporting Gram’s staining, consider the morphology and colour of organism. Spherical purple organism - report as GPC Rod shaped purple organism - report as GPB Spherical pink/red organism - report as GNC Rod shaped pink/red organism -report GNB 48 Slide 49: 49 GPC GPB GNC GNB GNB Variations in gram reactions : Variations in gram reactions Positivity of organism can loss Cell wall damage Over de-colorization Use of too old iodine Smear from old culture Negative organism can appear positive When smear is too thick 50 modifications : modifications 51 Modifications of gram’s staining : Modifications of gram’s staining Kopeloff & Beerman’s Gram method for films Kopeloff & Beerman’s Gram method for sections Jensen’s Gram method for smears Preston & Morrel’s Gram method Quick gram method for single slide 52 Kopeloff & Beerman’s Gram method for films : Kopeloff & Beerman’s Gram method for films Apply methyl violet for 5 min Wash with iodine solution Cover slide with fresh iodine solution - 2 min Decolorize with acetone - 2-3 sec Apply basic fuchsin - 30 sec Wash under tap - 5 sec Blot dry with out rubbing Complete drying by waving in warm air 53 Kopeloff & Beerman’s Gram method for sections : Kopeloff & Beerman’s Gram method for sections Remove paraffin wax with xylene Remove xylene by flooding with absolute alcohol Flood with 50% alcohol Apply methyl violet for 5 min Cover slide with fresh iodine solution - 2 min Decolorize with acetone for 2-3 sec Apply basic fuchsin for 30 sec 54 Kopeloff & Beerman’s Gram method for sections … : Kopeloff & Beerman’s Gram method for sections … Wash under tap 5 sec Blot dry without rubbing Dehydrate quickly by flooding with 95% alcohol Drain and add 50% alcohol Immerse slide in xylene Wipe away excess xylene Mount No.1 cover slip in Canada balsam or DPX 55 Jensen’s Gram method for smears : Jensen’s Gram method for smears Recommended for smears of gonococci and meningococci Cover the slide with 0.5% methyl violet 30 sec Pour Lugol’s iodine 1% to wash away stain Cover with fresh iodine - 30 sec 56 Jensen’s Gram method for smears …. : Jensen’s Gram method for smears …. Wash off iodine with ethanol Pour fresh alcohol 30 sec Wash with water Pour neutral red 0.1% 2 min Wash & dry 57 Preston & Morrel’s Gram method : Preston & Morrel’s Gram method Cover slide with ammonium oxalate crystal violet 30 sec Wash with Lugol’s iodine 1% Cover with fresh iodine Wash with iodine acetone 30 sec Wash with tap water Add Carbol fuchsin 30 sec 58 Quick gram method for single slide : Quick gram method for single slide Held the fixed slide with forceps through out the staining Flood the slide with basic stain Allow to act for 5 sec Tip off stain Flood the tilted slide with iodine solution for 5 sec Flood the tilted slide with acetone for 2 sec Flood the tilted slide with counter stain for 5 sec 59 Ammonium oxalate crystal violet stain : Ammonium oxalate crystal violet stain Solution A crystal violet 10 gm Ethanol 95% 100 ml Mix and dissolve Solution B Ammonium oxalate 1% aqueous solution Mix 20 ml of solution A and 80 ml of Solution B 60 Acetone-iodine solution : Acetone-iodine solution Strong iodine solution Iodine 10 gm Potassium iodide 6 gm Distilled water 10 ml Ethanol 90% to 100 ml Strong iodine solution - 3.5 ml Acetone 96.5 ml Mix well before use 61 Acid-Fast staining : Acid-Fast staining 62 Salutes.. .. .. : Salutes.. .. .. Paul Ehrlich 14-03-1854 to 20-08-1915. German scientist in the fields of hematology, Immunology, chemotherapy. Got Nobel prize for Medicine in 1908 63 Acid-Fast staining : Acid-Fast staining Is a differential staining developed in 1882 by Paul Ehrlich. Improved by Ziehl and Neelsen 1885 It is an important diagnostic procedure for identification of Mycobacterium and Nocardia. 64 Acid-Fast Staining - PRINCIPLE : Acid-Fast Staining - PRINCIPLE The cell wall of Mycobacterium & Nocardia is composed of unique type of lipids. One of which, a fatty acid called Mycolic acid This impact a waxy nature to the cell wall It is responsible for the characteristic feature of acid-fast staining 65 Ziehl Neelsen method for tubercle bacillus : Ziehl Neelsen method for tubercle bacillus Flood the slide with strong Carbol fuchsin. Heat until steam arise. Care not to boil. Keep for 5-7 minutes Wash under running water Decolorize with 20% H2SO4 for 1 minute Counter stain with methylene blue / malachite green for 1 minute Wash and stand on to drain. Do not blot. 66 Slide 67: 67 1 2 3 4 5 6 7 8 9 Acid-Fast Staining : Acid-Fast Staining The results: - Acid-fast cells retain the primary dye and appear pink - Non Acid-fast cells appear blue/green 68 AFB Ziehl Neelsen SMEAR EVALUATIONAND AFB REPORT : Ziehl Neelsen SMEAR EVALUATIONAND AFB REPORT 69 Ziehl Neelsen CARBOL FUCHSIN : Ziehl Neelsen CARBOL FUCHSIN Basic fuchsin 5gm Phenol 25 gm Alcohol 95% 50 ml Distilled water 500ml Dissolve fuchsin in phenol Add alcohol, mix thoroughly Add distilled water 70 Sulphuric acid 20% : Sulphuric acid 20% Concentrated sulphuric acid 250 ml Distilled water 1 litre Pour water into a large flask Place the flask in 5-8 cm of cold water in the sink Add acid very slowly through the side of the flask Mix gently 71 Loeffler’s Methylene blue : Loeffler’s Methylene blue Saturated solution of methylene blue in alcohol 300 ml KOH, 0.01% in water 1 litre Dissolve & mix thoroughly 72 Malachite green : Malachite green Malachite green 5 gm Distilled water 500ml Dissolve the dye into water to get stock solution Working solution Stock solution 40ml Distilled water 360ml 73 modifications : modifications 74 Ziehl Neelsen method for tissue sections : Ziehl Neelsen method for tissue sections Treat slide with xylene Wash well with alcohol & then with water Flood the slide with strong Carbol fuchsin. Heat until steam arise. Care not to boil. Keep for 5-7 minutes Wash under running water Decolorize with 25% H2SO4 for 1 minute 75 Ziehl Neelsen method for tissue sections CONTD…. …. : Ziehl Neelsen method for tissue sections CONTD…. …. Counter stain with methylene blue for 1 minute Gently blot slide Flush with absolute alcohol Wipe with fresh paper Clear in xylene Mount in Canada balsam or DPX 76 Ziehl Neelsen method for WEAKLY ACID FAST ORGANISMs : Ziehl Neelsen method for WEAKLY ACID FAST ORGANISMs For Leprosy bacilli use 5% H2SO4 For Actinomycetes & Nocardia use 1% For Brucella abortus Stain with dilute carbol fuchsin without heating Decolourise with 0.5% acetic acid 15 sec Counter stain with Loffler’s methylene blue 1 min 77 Special staining : Special staining 78 Special staining : Special staining Spore staining Flagella staining Staining for metachromatic granules Leishman’s stain Giemsa’s stain Staining for spirochaetes 79 Spore staining : Spore staining 80 Spore staining : Spore staining Modified Ziehl Neelsen method Malachite green stain 81 Modified Ziehl Neelsen method : Modified Ziehl Neelsen method Flood the slide with strong Carbol fuchsin. Heat until steam arise. Care not to boil. Keep for 5-7 minutes Wash under running water Decolourise with 0.25% H2SO4 for 1 minute Counter stain with methylene blue for 1 minute Wash and stand on to drain. Do not blot. Pink spores on blue stained bacteria 82 Slide 83: 83 Malachite green stain : Malachite green stain Place the slide over a beaker of boiling water When there is condensation under the slide, flood with 5% aq.solution of Malachite green stain 1min Wash in tap water Add 0.5% safranine / 0.05% basic fuchsin 30 sec Wash & dry Spores appears green & bacterium red / pink 84 Malachite green stain : Malachite green stain spore 85 Flagella staining : Flagella staining 86 Flagella staining : Flagella staining Grow bacteria for 16-24 hr on a non inhibitory medium Touch a loopful of water on to the edge of a colony and let motile bacteria swim into it Transfer the loopful in to a loopful of water on a slide Cover with a cover slip [ to detach flagella ] After 10 min apply 2 drops of Ryu's stain to the edge of cover slip, stand for 5-15 min 87 Ryu’s flagella stain : Ryu’s flagella stain Solution A Tannic acid powdered 10 gm Phenol 5 % aq. Solution 50 ml Aluminum potassium sulphate 50 ml Solution B Crystal violet 12 gm Ethanol saturated solution 100 gm 88 Ryu’s flagella stain .. .. .. : Ryu’s flagella stain .. .. .. Mix 10 parts of Solution A one part of Solution B Store at ambient temperature Mixture is stable indefinitely Does not require filtration Allow to stabilize by standing Fresh stain is potent 89 Flagella staining : Flagella staining 90 flagella staining for spirochaetes : staining for spirochaetes 91 Staining for spirochaetes : Staining for spirochaetes Larger spirochaetes stain by ordinary methods Smaller spirochaetes are best observed under dark ground microscope If permanent preparation of small spirochaetes are required use Fontana or Levaditi methods 92 Fontana staining - films : Fontana staining - films Treat the film with fixative three times,30 sec each Wash with absolute alcohol - 3 min Drain off excess alcohol Pour on the mordant Heat for 30 sec until steam arises Wash in distilled water and dry the slide 93 Fontana staining.. .. .. : Fontana staining.. .. .. Treat with ammoniacal silver nitrate Heat till steam arise Keep for 30 sec [till the film becomes brown] Wash well with distilled water, dry and mount in Canada balsam The spirochaetes are stained brownish – black on a brownish – yellow background 94 Fontana’s stain : Fontana’s stain Fixative Acetic acid 1ml Formalin [40%] 2ml Distilled water 100ml Mordant Phenol 1gm Tannic acid 5gm Distilled water 100ml Ammoniacal silver nitrate 95 Fontana’s stain … … … : Fontana’s stain … … … Add 10% ammonia to 0.5% solution of silver nitrate in distilled water until the precipitate formed just dissolves. Add more silver nitrate solution drop by drop until the precipitate returns and does not re-dissolve. 96 spirochaetes : spirochaetes 97 Levaditi’s staining - tissues : Levaditi’s staining - tissues Fix 1mm thick tissue in 10% formalin for 24 hrs Wash the tissue for 1 hr in water Place in 96 -98 % alcohol for 24 hrs Place the tissue in 1% solution of silver nitrate for 2hrs There after at about 500C for 4-6 hrs Rapidly wash the tissue in 10% pyridine solution 98 Levaditi’s staining.. .. .. : Levaditi’s staining.. .. .. Transfer to the reducing fluid, keep for 2 days at room temperature in dark Wash and dehydrate the tissue with alcohol and embed in paraffin Cut thin sections, remove paraffin using xylene, mount in Canada balsam 99 Staining for meta chromatic granules : Staining for meta chromatic granules Fix the dried smear using alcohol Cover the smear with the toluidine blue malachite green for 3-5 min Wash with distilled water Cover smear with Albert’s iodine for 1 min Wash with distilled water Air dry 100 Albert’s stain : Albert’s stain Malachite green 0.2 gm Toluidine blue 0.15 gm Ethanol 95% 2 ml Glacial acetic acid 1ml Distilled water 100 ml Dissolve the dyes in ethanol. Mix the acid & water and allow to stand for 24 hours and filter 101 Leishman’s stain - film : Leishman’s stain - film Pour the undiluted stain on the unfixed film and allow it to act for 1 min Add double volume of distilled water to the slide Allow to stand for 12 min Flood gently with distilled water Keep for 30 sec Remove excess water by blotting Air dry 102 Leishman’s stain - section : Leishman’s stain - section Treat the section with xylene Treat with ethyl alcohol Treat with distilled water Drain & stain for 5-10 min with 1 part of Leishman’s stain and 2 parts distilled water Wash with distilled water Blot, dehydrate with a few drops of absolute alcohol, clear in xylene and mount in Canada balsam 103 Leishman’s stain : Leishman’s stain Leishman’s powder 0.15 gm Methanol 100ml The powder is ground in a mortar with a little methanol The residue of undissolved stain is allowed to settle Fluid decanted in to a bottle Residue in the mortar is treated with more methanol Repeat till stain goes into solution 104 Giemsa’s stain : Giemsa’s stain 105 Giemsa’s stain – rapid method : Giemsa’s stain – rapid method Fix film in methanol for 3 min Stain in a mixture of 1 part stain and 10 parts buffer solution for 1 hr Wash with buffer solution 30 sec This is excellent for malaria parasites and trypanosomes 106 Giemsa’s stain – rapid method with heat : Giemsa’s stain – rapid method with heat Fix preparation with absolute alcohol 15 min Prepare fresh solution of 10 drops of Giemsa’s solution with 10 ml of buffer solution Cover the fixed film with the above solution Heat till steam arise Allow to cool for 15 sec Pour off and replace with fresh stain, heat again Repeat 5 times, wash, dry & mount 107 Giemsa’s stain –slow method : Giemsa’s stain –slow method Fix the film in methanol for 3 min Mix 1 ml stain with 20 ml buffer solution in a petri dish Place a piece of thin glass rod in the stain in the dish Place the fixed slide downwards in the stain with the help of glass rod Leave for 24 hrs Wash with buffer solution Dry and mount 108 Giemsa stain – stock solution : Giemsa stain – stock solution Giemsa stain powder 1 gm Glycerol 60 ml Absolute methanol 60 ml Heat glycerol to 55-600C in a water bath. Add the stain powder and mix thoroughly. Incubate the mixture at the same temperature for 2 hours. Cool and add methanol. Keep to mature for about 2 weeks. 109 Giemsa stain .. .. .. : Giemsa stain .. .. .. Stock solution 1 part 0.01 M-Phosphate buffer [ ph 7 ] 10 parts Mix and allow to stand 110 Giemsa stain : Giemsa stain Plasmodium vivax Plasmodium falciparum 111 Giemsa stain : Giemsa stain Trypanosoma cruzi Trypanosoma brucei 112 Fluorochrome technique : Fluorochrome technique Auramine – phenol technique Acridine – orange technique 113 Auramine – phenol technique : Auramine – phenol technique Cover smear with auramine phenol 10 min Wash with tap water Decolourise with 1% acid alcohol Wash with tap water Add 0.1% potassium permanganate stain 15 sec Wash with tap water Dry- do not blot Exam by fluorescence microscopy 114 Auramine phenol stain : Auramine phenol stain Auramine O powder 3 gm Crystalline Phenol 30 gm Distilled water 1 liter Dissolve phenol in water with gentle heat Add auramine slowly Shake vigorously until dissolved Filter & store in stoppered bottle 115 Potassium permanganate stain : Potassium permanganate stain Potassium permanganate 2 gm Distilled water 2 liters Add Potassium permanganate to distilled water Shake to dissolve Keep in stoppered bottle 116 1% acid alcohol decolorizer : 1% acid alcohol decolorizer Conc.HCl 20 ml Methylated spirit 1980 ml Pour Methylated spirit into large flask Place flask in cold water Add Conc.HCl and cover the top Leave for 10 min Keep in stoppered bottle 117 Fluorochrome staining : Fluorochrome staining Cryptococcus spore Tubercle bacilli 118 Acridine – orange technique : Acridine – orange technique Cover the unfixed dried smear with acridine orange acid stain for 5 – 10sec Wash off stain Decolorize with alcohol saline solution for 5-10 sec Rinse with saline Put a cover glass Examine by fluorescence microscopy 119 Acridine – orange stain : Acridine – orange stain Acridine – orange 0.13gm Glacial acetic acid 10 ml Distilled water 490 ml Mix well filter 120 Alcohol saline solution : Alcohol saline solution Absolute ethanol 5ml Saline 245 ml Fill the flask with saline Add Ethanol 121 Saline : Saline Sodium chloride 8.5 gm Distilled water 1000ml Mix well Filter 122 result : result T. vaginalis - orange red with yellow green nucleus Yeast cells – orange Bacteria – orange Pus cells – yellow green Epithelial cells – yellow green 123 Acridine – orange stain : Acridine – orange stain Trypanosoma brucei Clostridium tetani 124 Slide 125: 125 Thanks…. 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