STAINING TECHNIQUES.pptx

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1. STAINING TECHNIQUES Presenter- Dr. Rahul Tiwari Dr. bhagyashree karekar Guide - Dr. vaishali wabale

2. INTRODUCTION Stain: A substance which binds to cell and imparts colour to the cell; used for biological purpose. Staining: It is a procedure in which coloured chemicals called dyes/stains are added to smears/films.

3. Why do we stain? To visualize the microscopic and semitransparent objects e.g. Bacteria, fungi, parasites, viruses To reveal their morphology. To study various internal eg . spores and external structures eg . capsules. To study classification and identification of microbes. To study chemical nature of the cells.

4. Vital TYPES OF STAINING Supravital Others Organisms are live Bacteria in living condition are studied after imparting colour e.g. Dilute methylene blue, crystal violet Organisms are killed Simple staining Negative staining Impregnation staining Differential staining Special staining Progressive staining Regressive staining Specific staining Metachromatic staining Bipolar staining Beaded staining

5. STAINS POSITIVE STAINS NEGATIVE STAINS ONLY THE CELLS/STRUCTURES STAINS ONLY THE BACKGROUND WILL TAKE THE STAIN.

6. POSITIVE STAINS SIMPLE DIFFERENTIAL SPECIAL MONOCHROMIC DYES(SINGLE COLOUR) WE CAN DETERMINE SIZE, SHAPE & ARRANGEMENT THROUGH THIS GRAM STAIN ACID FAST STAIN

7. SIMPLE/ MONOCHROME STAINING Simple stains: Watery solution of simple basic dye is used. e.g. methylene blue, crystal violet, dilute carbol fuschin . Reagents: 1) Loeffler’s methylene blue- Saturated solution of methylene blue in alcohol- 300ml KOH, 0.01% in distilled water- 1000m l 2) Polychrome methylene blue- Prepared by allowing Loeffler’s methylene blue to ripen 3) Dilute carbol fuschin

8. Mechanism: Surface of bacterial cells is acidic. Ionization of carboxyl group gives negative charge to cell surface. This combines with positively charged ion of basic dye; leads to staining. Observations: Bacteria appear blue/ violet/ pink depending on stain. Uses: 1 ) Loeffler’s methylene blue- To demonstrate morphology of , lymphocytes and other cells. 2) Polychrome methylene blue- To stain capsule of Bacillus anthracis . 3) Dilute carbol fuschin - To demonstrate Borrelia spp.

9. NEGATIVE/ INDIRECT STAINING Negative staining: Background stained; structure to be demonstrated is not stained. Reagents: 1) India Ink - Pelikan Drawin ink, 17 Black containing 0.3% tricresol as preservative 2) Modification of India Ink using 2% mercurochrome for demonstrating capsulated budding yeast cells of Cryptococcus neoformans 3) Nigrosin - Nigrosin 10g, Warm distilled water 100ml, formalin 40% 0.5ml as a preservative 4) Eosin- 1% solution

10. Observations: India Ink- Bacteria appear as clear transparent objects; Nigrosin - Bacteria appear as bright unstained objects . Uses: 1) India ink or Nigrosin 10%- To demonstrate capsules of Streptococcus pneumoniae , Klebsiella pneumoniae , Group B Streptococci, Haemophilus influenzae and Cryptococcus neoformans ; Demonstration of Spirochaetes by India ink . 2) To demonstrate bacterial motility. 3) Eosin- To demonstrate parasitic elements in stool.

11. Cryptococcus neoformans as seen in India ink staining

12. IMPREGNATION STAINING Impregnation staining: The structures too thin to be seen under ordinary microscope, can be visualized by increasing their thickness by impregnating silver. Reagents: Ammoniated silver solutions Silver nitrate- Most common Silver diamine , silver carbonate

13. Uses: To demonstrate Spirochaetes and bacterial flagella. e.g. Fontana’s method, Levaditi’s method.

14. Levaditi’s Method Used for staining of Spirochaetes in tissues. Fontana’s Method Reagents: Fixatives- Acetic acid, Formalin 40% Mordant- Phenol, Tannic acid Ammoniated silver nitrate Mechanism: Impregnation of silver salt on the surface of Spirochaetes increases their thickness Observations: Spirochaetes appear brownish black.

15. DIFFERENTIAL STAINING Gram staining Ziehl Neelsen staining Romanowsky staining

16. GRAM STAIN Discovered by Christian Gram in 1884: subsequently modified by Burke, Kopeloff and Beerman in 1922: further by Jensen, Weigert , Preston and Morrell in 1962

17. GRAM STAINING Differential staining: These differentiate two types of organisms. Observations: Gram positive- Resist decolourisation and retain primary stain; hence appear violet. Gram negative- Decolourised by organic solvents and take the counter stain; therefore appear red.

18. Reagents for Gram Staining Primary stain - Crystal violet, methyl violet or gentian violet Mordant - Gram’s or Lugol’s iodine Decolouriser - Absolute alcohol, acetone or ethanol-acetone Counter stain- Safranine , neutral red

19. MORDANT A substance that increases binding between primary stain and cell cytoplasm. It act as the paste. It forms “dye iodide protoplasm complex” in gram positive, but no complex in gram negative. It forms insoluble compound with stain and helps to fix the colour to the cell. e.g. Iodine, Tannic acid, Salts of aluminium , Iron, Tin, Zinc, Phenol. It provides greater stability to the cell and does not allow the stain to get washed off.

20. DECOLORIZATION As due to complex formation, it resist purple colour in gram positive organisms. As their is no complex in gram negative organism, therefore colorless in gram negative.

21. SAFRANIN As gram positive are already purple, therefore they do not take safranin colour . And in gram negative as they are colorless, therefore they take pink/red colour of safranin .

22. WHAT IF ? MORDANT NOT ADDED ALL WILL BECOME PINK AS NO COMPLEX FORMATION ACETONE NOT ADDED AS NO COUNTERSTAIN, THEREFORE EVERYTHING BECOME PURPLE

23. MECHANISM CELL WALL PERMEABILITY THEORY According to this theory, the cell wall of Gram positive bacteria contain more peptidoglycan because of which it is thicker & stronger & impermeable as compared to Gram-negative bacteria. During staining with primary stain and mordant dye- iodine complex is formed within the cell. Under the action of this decolourizing agent, this complex diffuses freely out of the Gram-negative cell but not from the Gram positive cell because the Gram positive cell wall is less permeable due to its cell wall. In Gram-negative bacteria peptidoglycan is less and therefore their cell wall is relatively less strong and thin, hence dye-iodine complex diffuses out of cell freely and they take up the colour of the counter stain.

24. OTHER THEORIES RELATED TO GRAM STAINING Magnesium Ribonucleate Theory Lipid content Theory Acid Protoplasm Theory Isoelectric Point Theory

25. USES OF GRAM STAINING To differentiate between Gram positive and Gram negative bacteria E.g.: Gram positive cocci : Staphylococcus, Pneumococcus Gram positive bacilli : Clostridia, Corynebacteria , Bacillus spp Gram negative bacilli : E. coli, Klebsiella pneumonia Gram negative cocci : gonococci, meningococci To start empirical therapy To choose culture media for inoculation

26. ACID FAST STAINING Paul Ehrlich (1882)- Acid fast property Koch (1882) stained Mycobacterium tuberculosis Ziehl - Phenol as mordant Neelson - Mordant + Primary stain Paul Ehrlich

27. ZIEHL NEELSEN STAINING (ZN STAINING) Acid fastness depends on: Mycolic acid Integrity of cell wall It differentiates acid-fast organisms from non acid-fast organisms. Observations: Acid fast organisms- Resist decolourisation by acid and alcohol. Retain fuchsin ; appear bright red. Non acid fast organisms- Take the colour of methylene blue.

28. REAGENTS FOR ZN STAINING Primary stain- Carbol fuschin - Basic fuschin 10gm, Absolute alcohol 95%, 5% Phenol Decolouriser - Suphuric acid 25% - Sulphuric acid 250ml and distilled water 1000ml Counter stain- Methylene blue , Malachite green, Picric acid

29. PROCEDURE FOR ZIEHL-NEELSEN STAINING

30. Mycobacterium tuberculosis visualized by using Ziehl-Neelsen staining

31. Grading AS PER RNTCP RESULT GRADING NO. OF FIELDS EXAMINED >10 / oil immersion field + ve 3+ 20 1 – 10/oil immersion field + ve 2+ 50 10 – 99 / 100 oil immersion field + ve 1+ 100 1 – 9 / 100 oil immersion field + ve Scanty* 100 No AFB seen in 100 oil immersion fields - ve 1000 *Record actual no. of bacilli seen in 100 fields.

32. ACID FAST ORGANISMS Bacteria- Mycobacterium, Nocardia , Rhodococcus Parasites- Oocyst of Cryptosporidium, Cyclospora , Isospora ; Eggs of Taenia saginata Others- Spores, Spermatic head

33. Mechanism: Mycolic acid in the cell wall of acid fast organisms do not allow the stain to penetrate easily inside organisms. Here, carbol fuschin is applied with heat. Heat allows the dye to penetrate. When decolourised by acid, the dye does not come out as it is soluble in phenol and phenol is soluble in lipids; hence no decolourisation .

34. MODIFICATIONS OF ZN STAINING Modification Particulars Lepra bacilli Decolouriser - 5% Sulphuric acid Cryptosporidium spp Decolouriser - 1% Sulphuric acid Nocardia spp Decolouriser - 1% Sulphuric acid Actinomycetes Decolouriser - 1% Sulphuric acid Bacterial spores Decolouriser - 0.25-0.5% Sulphuric acid Cryptosporidium spp Kinyoun’s cold method. No intermittent heating. Decolouriser - Acid-alcohol 3ml HCl + 97ml of 95% alcohol

35. FLUOROCHROME STAINING FOR AFB A fluoroscent dye Auramine O is substituted for the hot carbol fuchsin Method - Auramine phenol (0.3%) for 10 min - Acid alcohol (1%) for 5 min - Potassium permanganate (0.1%) for 15 sec Examine under fluorescent microscope

36. Advantages - Can be examined under low magnification - Shorter time - Rapid screening Disadvantages - Need for Fluorescent microscope

37. ROMANOWSKY STAINING Permanent staining method to demonstrate haemoparasites . It is a type of differential staining. Romanowsky stain- Watery solutions of eosin + zinc-free methylene blue in methyl alcohol.

38. MODIFICATIONS OF ROMANOWSKY STAINING Leishman’s stain Wright stain Giemsa stain Field’s stain Jenner’s stain Jaswant Singh-Bhattacharya (JSB) stain

39. Leishman’s stain Leishman’s powder- 150mg Methanol- 100ml Observations: -Cytoplasm of WBCs & protozoal parasites appear blue -Nuclei, parabasal body & flagella appear red -RBCs appear pink Granules of eosinophils appear pink Wright stain Wright stain powder- 0.3gm Gycerol - 3ml Absolute methanol- 7ml Observations: Same as Leishman’s stain Stained thin blood smear using Leishman’s stain Unstained thin blood smear

40. Giemsa stain reagents: Giemsa stain powder- 0.75gm Gycerol - 25ml Methanol- 75ml Observations: Same as Leishman’s stain 1.Rapid method- To demonstrate blood parasites 2. Modified rapid method- Heating for fixation of smear instead of methanol; -To demonstrate spirochaetes 3. Slow method - Excellent stain for inclusion bodies of small pox GIEMSA STAIN

41. Giemsa stain Plasmodium vivax in Giemsa -stained thin blood smear with all developmental stages present in peripheral blood. (A) Growing amoeboid trophozoite in enlarged red blood cell (RBC) with eosinophilic stippling ( schuffner's dots). (B) Immature schizonts with clumps of brown pigment almost fill the enlarged RBCs. (C) Mature schizont with merozoites (about 14) and clumped pigment. (D) Macrogametocyte with diffuse brown pigment and eccentric compact chromatin.

42. Jenner’s stain Jenner’s stain powder- 5gm Methanol- 1000ml JSB stain JSB A Eosin -2.5gm Distilled water- 100ml JSB B Methylene blue- 2.5gm Distilled water- 100ml Field’s stain Solution A Methylene blue 0.4gm Buffered water- 250ml Solution B Buffered water Distilled water- 1000ml Solution C Eosin- 0.5gm Buffered water- 250ml

43. STAINING OF SPORES Modified ZN staining Observations: Spores appear bright red Bacterial body appears blue Schaeffer and Fulton’s staining 5% aqueous solution of malachite green Counter staining with 0.55 safranine or 0.05% fuschin basic Observations: Spores appear green Bacterial body appears red Dorner’s method 3-4 drops of freshly filtered Kinyoun’s carbol fuschin Hold test tube in boiling water for 5-10 min Loopeful of sample from test tube + a drop of 10% aqueous solution of nigrosin Observations: Spores appear pink Bacterial body appears colourless Background appears dark grey

44. A Schaeffer and Fulton stained preparation of  Bacillus subtilis  showing endospores as green and the vegetative cell as red

45. STAINING OF SPORES Modified Dorner’s method Apply carbol fuschin steam for 5-7 min Blow dry and cover with thin film of nigrosin Observations: Spores appear pink Bacterial body appears colourless Background appears dark grey Wirtz and Conklin method 5% aqueous solution of malachite green And steaming for 3-6 min Counter staining with 0.5% safranine Observations: Spores appear green Bacterial body appears pink Abbott stain Apply saturated alcoholic solution of methylene blue Heat till stain boils Cover the film with saturated alcoholic solution of eosin Y and nine parts of distilled water for 5-10 sec Observations: Spores appear blue Bacterial body appears pink

46. STAINING FOR VOLUTIN CONTAINING GRANULES Best seen in young culture on enriched media Granules are metachromatic Neisser’s , Albert’s or Ponder’s method Albert- Laybourn method is commonly used - Sol A ( Toluidine blue,Malachite green) - Sol B (Albert’s iodine) Observations: -Granules stain bluish black - Bcterial body appears green.

47. Corynebacterium diphtheriae

48. STAINS FOR FUNGI Lactophenol Cotton Blue Calcofluor White- Binds to cellulose and chitin Can be subsequently stained by Periodoc acid schiff (PAS) or Gomor i’s Methenamine stain (GMS) Rapid detection in wet mount (KOH ),smears and tissue sections. Fluorescent -Antibody staining: This stain is important in detection of fungi or their antigens in tissues, pus, blood or other specimens in various fungal infections where organisms are scanty in number.

49. PERIODIC ACID SCHIFF The polysaacharides of fungi and bacteria are oxidized by periodic acid to yield large number of aldehyde groups because of which they stain intense red or magenta in colour with Schiff’s reagent. Glucan and mannan in fungal cell wall are oxidized to aldehydes .

50. Esophageal candidiasis , PAS stain

51. To stain fungi , specially used for Pneumocystis jirovecii The cell walls are stained brown to black. GOMORIMETHAMINE SILVER STAIN Pneumocystis jiroveci

52. STAINING PROCEDURES IN PARASITOLOGY Temporary Preparations Permanent Preparations Wet Mounts 1) Modified Ziehl-Neelsen Technique 2) Hot Modified Acid-fast 1) Saline preparation Staining Method 2) Iodine Preparation 3) Kinyoun’s Cold Stain 3) Lactophenol cotton blue (LPCB) mount Method 4) Wet mount using capillary blood 4) Safranine-methylene Blue (detection of microfilaria and trypanosoma ) Technique 5) Romanowsky’s Stain 6) Hematoxylin Stain

53. Cyst of Entamoeba histolytica o n wet mount Lefts side: cyst stained with iodine Right side: unstained preparation. [ Arrow - notice the chromatid body with blunt, rounded ends ]

54. WET MOUNTS Saline Preparation: Observations: It is an unstained preparation: Parasitic elements are not stained and they appear colourless . As parasitic elements are live in this preparation: Live motile trophozoites can be seen Helminthic eggs Helminthic larvae Iodine Preparation : Observations: Cytoplasm of the cysts appear yellow or light brown Nuclei appear dark brown The peripheral chromatid bodies appear light yellow Glycogen mass in young cysts appears dark brown with iodine. LPCB: Observations: Cytoplasm appears deep blue in colour . - Nucleus / nuclei appear blue in colour - Protozoal cysts and helminthic eggs are stained deep blue

55. Modified Kinyoun’s acid fast – stained smear demonstrating oocyst of Isospora belli with a sporont (A) , an Isospora belli oocyst with two sporoblasts and a Cyclospora cayetanensis oocyst with a sporont .

56. USES OF WET MOUNTS Saline Preparation: To demonstrate: 1) Motile trophozite stage of protozoan parasites 2) Helminthic eggs 3) Helminthic larvae 4)Cysts of protozoa Iodine Preparation: To demonstrate: 1) To stain glycogen and nuclei of cysts specifically used for demonstration of cystic stage 2) Trophoite stage 3) Eggs and larvae can also be demonstrated but as iodine stains non-bile stained eggs also, hence less important as far as differentiation of bile stained and non-bile stained eggs are concerned LPCB: To demonstrate: The internal structures of trophozoites , cysts, and ova, thus facilitating their recognition and identification in the stool specimens Intestinal coccidian sparasites such as Cyclospora and Isospora in stool specimens

57. PERMANENT PREPARATIONS Permanently stained preparations are occasionally required for the following purposes: 1)For identification of: - oocysts of cryptosporidium - protozoan trophozoites - protozoan cysts 2) Keeping a permanent cord 3) For sending to a reference laboratory for an expert opinion.

58. Stain for viruses Seller stain: To differentiate rabies inclusions from other intracellular inclusions. Negri bodies appear magenta in colour and have small (0.2 µm to 0.5 µm), dark-blue interior basophilic granules .

59. Giemsa stain: To demonstrate Tzanck cells in the infections caused by herpes simplex virus. To demonstrate multinucleate giant cells in the infections caused by herpes-zoster virus

60. IMMUNOFLUORESCENCE STAINING Fluorescence is the property of absorbing the light rays of one particular wave length and emitting the light rays of different wave length. Fluorescent dyes can absorb ultraviolet(UV) light and can convert or reflect back them into visible light. Fluorescent dyes such as flourescein isothiocyanate (FITC), tetramethyl rhodamine , dansyl and phycoerythrincan be used to detect infected cells in exudates, secretions and tissues. FITC is he most commonl used. It emits a greenish / yellowish light. Rhodamine emits red / orange light and dansyl emits yellow light.

61. Commonly used for the detection of different bacteria, viruses, fungi and parasites in tissues, blood, CSF, urine, faeces and other body fluids. Also used for the detection of other antigens( Ags ) such as tumour Ags , organ and tissue Ags , blood cells, enzymes, hormones, etc. It is most commonly used in the diagnosis of viral infections such as rabies, measles, mumps, etc. USES OF IMMUNOFLUORESCENCE STAINING

62. Photomicrograph of a histological section of human skin prepared for  direct immunofluorescence  using an anti- IgA antibody. The skin is from a patient with  Henoch–Schönlein purpura . IgA deposits are found in the walls of small superficial capillaries (yellow arrows). The pale wavy green area on top is the epidermis, the bottom fibrous area is the dermis.

63. THANK YOU

64. Modification Particulars Use Burke’s Decolouriser - Acetone/ Acetone-ether mixture Kopeloff and Beermann’s Primary stain- Methyl violet Decolouriser - Acetone Counter stain- Basic fuschin For general use Jensen’s Primary stain- Methyl violet Counter stain- Neutral red For gonococci and meningococci Weigert’s Primary stain- Carbol gentian violet Decolouriser - Aniline xylol For tissue sections Preston and Morrell’s Primary stain- Crystal violet/ Ammonium oxalate Mordant- Lugol’s iodine Decolouriser - Iodine-acetone Counter stain- Dilute carbol fuschin For routine staining Hucker’s Primary stain- Methyl violet/ Crystal violet Decolouriser - Acetone Counter stain- Safranine / Neutral red Brown and Brenn Primary stain- Methyl violet/ Crystal violet Decolouriser - Acetone and 95% ethanol Counter stain- Safranine / Dilute carbol fuschin

65. Technical Report Published: 09 July 2020 Detection of SARS-CoV-2 in formalin-fixed paraffin-embedded tissue sections using commercially available reagents Alejandro Best Rocha,  Edana Stroberg , Lisa M. Barton, Eric J. Duval,  Sanjay Mukhopadhyay , Nicole Yarid , Tiffany Caza , Jon D. Wilson,  Daniel J. Kenan ,  Michael Kuperman ,  Shree G. Sharma &  Christopher P. Larsen  Laboratory Investigation   volume 100 , pages1485–1489(2020) Cite this article 6209  Accesses 7  Citations 62  Altmetric Metrics

66. BRUCELLA DIFFERENTIAL STAIN Reagents: Primary stain- 1:10 diluted carbol fuschin Heating is not required. Decolouriser - Acetic acid 0.5% Counter stain- Methylene blue Uses: To demonstrate Brucella abortus in infected tissues or exudates. To demonstrate Coxiella burnetii and Chlamydia in tissue sections.

67. Observations: Brucella abortus appears red or orange.

68. Cover the whole slide with carbol fuchsin and heat gently from below until steam rises Allow it to act for 5-10 minutes with intermittent heating (Heat and phenol from carbol fuchsin help the primary stain to penetrate through the cell wall ) Wash slide with tap water Use of 25% H2SO4 for 2-4 minutes as a decolourizer for M. tuberculosis is recommended by RNTCP Wash slide with tap water Counter stain with methylene blue or malachite green for 1-2 minutes Wash slide with tap water Air dry the slide before observing under oil immersion objective Observe under 100x after putting a drop of cedarwood oil

69. Schaeffer procedure for staining of spores

70. Examples of endospore stain positive organisms Clostridium perfringens C. tetani C. botulinum Bacillus cereus Bacillus anthracis  

71. Most kits use Fluorescein isothiocyanate (FITC) or Tetramethyl rhodamine isothioyanate as a dye Ethidium bromide (fluorescent indicator dye) in PCR SYBR dye binds to dsDNA and increases the fluorescence In Micro-array technology Cyanine 3 and Cyanine 5