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Staining
 

Staining

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Staining Staining Presentation Transcript

  • Gunjan Mehta,Dept. of Biotechnology, VSC, Rajkot. 1
  •  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
  •  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
  •  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
  • Method 1 Wipe the slide with dry cotton cloth Pass the slide through blue flame of Bunsen flame 16-12 timesMethod 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
  •  Should be ¾ or 7/8 inches 0.1 mm thickness Should be used only for wet preparations 6
  •  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
  •  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
  •  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
  •  Heat fixation  By passing the slide over flame Chemical fixation  Using ethanol  Methanol  Picric acid  Potassium permanganate  Formaldehyde vapour 10
  •  Three quick passes over the flame is sufficient for fixation Chemical fixation is just applying few drops of chemicals and allow to evaporate 11
  •  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
  •  Simple staining Differential staining Special staining 13
  • 14
  •  Monochrome staining / positive staining Negative / indirect staining 15
  •  Basic dyes are used for this Basic dyes are Positively charged These dyes get attached to negatively charged cytoplasm of microbial organism 16
  • 17
  •  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 18
  •  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 19
  •  Crystal violet 10 gm Absolute alcohol 100ml Distilled water 1 litre Dissolve the dye in alcohol Filter through filter paper Add to water 20
  •  Saturated solution of methylene blue in alcohol 300 ml KOH, 0.01% in water 1 litre Dissolve & mix thoroughly 21
  •  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. 22
  •  Dilute 1 volume of strong Carbol fuchsin with 10-20 volumes of distilled water. 23
  •  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 24
  •  Usual stains used are  Nigrosin  Indian ink 25
  •  Nigrosin 100gm Formalin 5ml Distilled water 100ml Dissolve nigrosin in formalin Add to Distilled water 26
  • 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. 27
  •  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 28
  • India ink NigrosinCryptococcus spore Cocci B. subtilis 29
  • 30
  • 31
  •  Gram’s method Acid fast staining 32
  • Hans Christian Gram 13-09-1853 to 14-11-1938.Danish bacteriologist. The work with Gram staininggained him international reputation 33
  •  Gram staining is a microbiological procedure that categorizes bacteria based on the physical and chemical structure of their outer surface 34
  •  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 35
  •  Insecond 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 36
  •  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 37
  • 38
  •  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 39
  •  Wash with water Cover with *Counter stain Wash with water Blot dry Observe under oil immersion  *Basic fuchsin  *Neutral red  *Safranine 40
  • 1 4 7 2 5 82 3 6 9 6 41
  • Figure 4.17.1
  • Figure 4.17.2
  • Figure 4.17.3
  • Figure 4.17.4
  •  Crystal violet 10 gm Absolute alcohol 100ml Distilled water 1 litre Dissolve the dye in alcohol Filter through filter paper Add to water 46
  •  Methyl violet 10 gm Absolute alcohol 100ml Distilled water 1 litre Dissolve the dye in alcohol Filter through filter paper Add to water 47
  •  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 48
  •  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 49
  •  Basic fuchsin 0.5 gm Distilled water 1 litre Dissolve the dye in water Recommended for general use  Apply for 10 -30 sec 50
  •  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 51
  •  Safranine 0.5 gm Distilled water 1 litre Dissolve the dye in water Recommended for general use  Apply for 10 -30 sec 52
  • 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 53
  •  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 54
  • GPC GPBGNC GNB GNB 55
  • Positivity of organism can loss Cell wall damage Over de-colorization Use of too old iodine Smear from old cultureNegative organism can appear positive When smear is too thick 56
  • 57
  •  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 58
  •  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 59
  •  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 60
  •  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 61
  •  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 62
  •  Wash off iodine with ethanol Pour fresh alcohol 30 sec Wash with water Pour neutral red 0.1% 2 min Wash & dry 63
  •  Cover slide with ammonium oxalate crystal violet 30 sec Wash with Lugol’s iodine 1% Coverwith fresh iodine Wash with iodine acetone 30 sec Wash with tap water Add Carbol fuchsin 30 sec 64
  •  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 65
  •  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 66
  •  Strong iodine solution  Iodine 10 gm  Potassium iodide 6 gm  Distilled water 10 ml  Ethanol 90% to 100 ml Strongiodine solution - 3.5 ml Acetone 96.5 ml Mix well before use 67
  • 68
  • Paul Ehrlich 14-03-1854 to 20-08-1915. German scientist in the fields ofhematology, Immunology, chemotherapy. Got Nobel prize for Medicinein 1908 69
  •  Isa differential staining developed in 1882 by Paul Ehrlich. Improved by Ziehl and Neelsen 1885 Itis an important diagnostic procedure for identification of Mycobacterium and Nocardia. 70
  •  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 71
  •  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. 72
  • 1 4 72 5 83 6 9 73
  •  The results: - Acid-fast cells retain the primary dye and appear pink - Non Acid-fast cells appear blue/green AFB 74
  •  Cells that retain a basic stain (carbolfucsin) in the presence of acid-alcohol are called acid- fast. Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with methylene blue) to see them. • used to identify bacteria in the genus Mycobacterium (two members are pathogens which cause tuberculosis and leprosy). Figure 3.11
  •  Endospores are stained with the primary stain malachite green. The vegetative cells (actively growing cells) are counterstained with safranin (pink cells). • used to identify bacteria in the genera : 1. Clostridium: members are pathogens which cause gangrene, tetanus, and botulism. 2. Bacillus: one member is a pathogen that causes anthrax. Figure 3.11
  • Figure 4.21
  • No. of AFB Oil immersion Report field0 300 AFB not seen1-2 300 Doubtful , repeat1-9 100 Scanty exact bacilli1-99 100 1+1-9 Each field 2+10/ more Each field 3+ 78
  •  Basic fuchsin 5gm Phenol 25 gm Alcohol 95% 50 ml Distilled water 500ml Dissolve fuchsin in phenol Add alcohol, mix thoroughly Add distilled water 79
  •  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 80
  •  Saturated solution of methylene blue in alcohol 300 ml KOH, 0.01% in water 1 litre Dissolve & mix thoroughly 81
  •  Malachite green 5 gm Distilled water 500ml Dissolve the dye into water to get stock solution Working solution  Stock solution 40ml  Distilled water 360ml 82
  • 83
  •  Treatslide 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 84
  •  Counter stain with methylene blue for 1 minute Gently blot slide Flushwith absolute alcohol Wipe with fresh paper Clearin xylene Mount in Canada balsam or DPX 85
  •  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 86
  • 87
  •  Spore staining Flagella staining Staining for metachromatic granules Leishman’s stain Giemsa’s stain Staining for spirochaetes 88
  • 89
  •  Modified Ziehl Neelsen method Malachite green stain 90
  •  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 91
  • 92
  •  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 93
  • spore 94
  • 95
  •  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 Ryus stain to the edge of cover slip, stand for 5-15 min 96
  •  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 97
  •  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 98
  • flagella 99
  • 100
  •  Largerspirochaetes stain by ordinary methods Smaller spirochaetes are best observed under dark ground microscope Ifpermanent preparation of small spirochaetes are required use Fontana or Levaditi methods 101
  •  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 102
  •  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 103
  •  Fixative  Acetic acid 1ml  Formalin [40%] 2ml  Distilled water 100ml Mordant  Phenol 1gm  Tannic acid 5gm  Distilled water 100ml Ammoniacal silver nitrate 104
  •  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. 105
  • 106
  •  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 107
  •  Transferto the reducing fluid, keep for 2 days at room temperature in dark Washand dehydrate the tissue with alcohol and embed in paraffin Cutthin sections, remove paraffin using xylene, mount in Canada balsam 108
  •  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 109
  •  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 110
  •  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 111
  •  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 112
  •  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 113
  • 114
  •  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 115
  •  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 116
  •  Fixthe 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 117
  •  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. 118
  •  Stock solution 1 part 0.01 M-Phosphate buffer [ ph 7 ] 10 parts Mix and allow to stand 119
  • Plasmodium vivax Plasmodium falciparum 120
  • Trypanosoma cruzi Trypanosoma brucei 121
  •  Auramine – phenol technique Acridine – orange technique 122
  •  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 123
  •  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 124
  •  Potassium permanganate 2 gm Distilled water 2 liters Add Potassium permanganate to distilled water Shake to dissolve Keep in stoppered bottle 125
  •  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 126
  • Cryptococcus spore Tubercle bacilli 127
  •  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 128
  •  Acridine – orange 0.13gm Glacial acetic acid 10 ml Distilled water 490 ml Mix well filter 129
  •  Absolute ethanol 5ml Saline 245 ml Fill the flask with saline Add Ethanol 130
  •  Sodium chloride 8.5 gm Distilled water 1000ml Mix well Filter 131
  •  T.vaginalis - orange red with yellow green nucleus Yeast cells – orange Bacteria – orange Pus cells – yellow green Epithelial cells – yellow green 132
  • Trypanosoma brucei Clostridium tetani 133
  • Thanks…. 134