Module STAINS IN BACTRIOLOGY - Copy.pptx

Good bacterilogy note

1. STAINS IN BACTERIOLOGY PRESENTED BY:DR RAVI SAINI GUIDE :- DR MUKESH KUMAR

2. STAINS USED IN BACTERIOLOGY HISTORY INTRODUCTION DYE &STAIN CLASSIFICATION STAINS FOR BACTERIA

3. HISTORY Natural stains like carmine and indigo used in 1850 for microbiology Aniline dyes were used in 1856

4. HISTORY CON.. Gerlach was a pioneer of histological staining and anatomical microphotography. In 1858 Gerlach introduced carmine mixed with gelatin as an histological stain. Father of staining

5. INTRODUCTION Bacteria are transparent and colorless , so they would be invisible to naked eye if observed under a microscope thus Bacteria should be stained with certain dyes in order to visualize bacterial cell or their internal structures using the light microscope.

6. INTRODUCTION CON.. Staining is an auxillary technique used in microscopy to enhance contrast in the microscopic image. A biological stain is a dye which impart a color and make microorganisms more clearly visible

7. INTRODUCTION CON… Dye – A colored substance -affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and may require a mordant to improve the fastness of the dye on the fiber. The first human-made (synthetic) organic dye, William Henry Perkin in 1856.

8. DYE &STAIN Dye :- A colouring agent that is used for general purposes is called a dye. Stain :- The one that is used for biological purposes is called a stain.

9. DYE Aromatic organic compounds Almost all biological useful dyes are derivatives of coal tar, the fundamental chemical structure of most dyes is the benzene ring Contain two groups - Chromophore - Auxochrome

10. CHROMOPHORE It is a group of atoms with in a dye molecule responsible for its color Most imp groups are – C=C,C=O,C=S,C=N,N=N,N=O,NO2 No. of these groups determine the intensity of color Benzene + Chromophore = Chromogen

11. CHROMOPHORE Auxochrome

12. AUXOCHROME An ionizing group which allow the dye molecule as a unit to have affinity for a compound It enables the dye to form a salt linkages with the ionizable radicals on protein, glycoprotein and lipoprotein on tissue or organism cellular components Auxochrome –OH,-NH2,-SO3,-COOH

13. DYE The dyes used in bacteriology have two features in common . They have chromophore groups, groups with double bonds, that give the dye its colour They can bind with cells by ionic, covalent or hydrophobic bonding .

14. TYPES OF DYES Basic dyes Acidic dyes Neutral dyes

15. TYPES OF DYES CON.. Basic dye s : In which chromogen is the positive ion ( cation ). Basic dye has the form: dye + Cl - Examples- Methylene Blue, basic fuchsin, crystal violet, malachite green, safranin . Acidic dyes: In which chromogen is the negative ion (anion). Acidic dye has the form: Na + dye - Examples include nigrosin and India ink , Rose Bengal and Acid fuchsin.

16. TYPES OF DYES CON… Neutral dyes: Both positively and negatively charged. Ex: Geimsa’s stain, Leishman’s stain, Wright’s stain

17. STAIN Stains are useful for the following reasons It makes the microscopic semi-transparent objects visible. To study the shape and size . To reveal the presence of various internal and external structures . To produce specific chemical and physical reaction.

18. CLASSIFICATION BASED ON STAINING PROCEDURE STAINS Simple stain Differential stain special stain

19. CLASSIFICATION CON… SIMPLE STAINING Methylene blue Dilute carbol fuchsin Polychrome methylene blue

20. CLASSIFICATION CON… DIFFERENTIAL STAINS Gram Staining Acid-fast staining SPECIAL STAINS Stains for Metachromatic granules Stain for spores Stain for capsules Stain for spirochetes Stain for flagella

21. CLASSIFICATION CON… BASED ON REGRESSIVE OR PROGRESSIVE STAIN SLOW GIEMSA BASED ON LIVING AND NON LIVING VITAL STAIN INTRA VITAL STAIN SUPRA VITAL STAIN NEGATIVE STAINING INDIA INK NIGROSIN IMPREGNATION METHOD

22. SIMPLE STAINING PRINCIPLE: Bacterial cell surface is slightly negative so it tends to bind strongly to the cationic chromogen of basic dyes. To visualize morphological shapes and arrangements of bacterial cells. Stained with a single basic dye Provide the colour contrast but impart the same colour to all the organisms in a smear

23. SIMPLE STAINING CON.. Loeffler’s Methylene Blue Use:- Methylene blue staining is used to make out clearly the morphology of the organisms eg . H. influenzae in CSF, Gonococci in urethral pus

24. SIMPLE STAINING CON.. Dilute Carbol Fuchsin Made by diluting Z-N stain with 10- 15 times its volume of water Use:- To stain throat swab Vincent’s angina, ( Borrelia are better stained) Counter stain in Gram stain Demonstrate the morphology of Vibrio cholerae

25. SIMPLE STAINING CON.. Polychrome methylene blue Use : M’Fadyean’s reaction - B. anthracis

26. DIFFERENTIAL STAINING GRAM STAINING ACID- FAST STAINING

27. DIFFERENTIAL STAINING CON.. GRAM STAINING:- Principle

28. GRAM POSITIVE AND GRAM NEGATIVE BACTERIA

29. ORIGINAL FORMULATION OF GRAM

30. VARIOUS MODIFICATIONS OF GRAM STAINING Primary stain Mordent Decolourizer Counter stain Kopeloff & Beerman’s Methyl violet+ Sodium bicarbonate Iodine + NaOH Acetone Basic fuchsin Jensen’s Methyl violet. Lugol’s iodine(1%) Absolute Alcohol Neutral Red Preston&morrell’s Ammonium- oxyalate -crystal violet Lugol’s iodine(1%) Iodine-acetone Carbol fuchsin Weigert’s method - Gentian violet - Iodine - Aniline xylol carminic acid

31. Observation Gram positive or negative Morphology Arrangement

32. DIFFERENTIAL STAINING CON.. ZIEHL – NEELSEN STAIN Differentiates acid fast and non acid fast organisms Discovered by Ehrlich and modified by Ziehl - Neelsen

33. PRINCIPLE AFB contain mycolic acid (α-substituted β- hydroxy fatty acids ) in their cell wall Occur as esters attached to cell wall polysacharides Give them a coat impervious to dyes Heat / incorporation of detergent into stain is required for penetration Once stained cannot be decolorized by acid alcohol solvents Resistance to decolourisation is associated with the mycolic acid - arabinogalactan moieties

34. BASIC REQUIREMENTS

35. STAINING STEPS

36. MODIFICATIONS OF Z-N STAINING 5% sulphuric acid = M leprae 1% sulphuric acid = Nocardia, Cryptosporidium Isospora oocysts . 0.5% acetic acid = Brucella O.25% sulfuric acid = Spores

37. KINYOUN’S MODIFICATION By increasing the concentration of Carbol fuchsin and phenol the need for heating is avoided The primary stain consists of (4 g) Basic fuchsin (1g) (20ml) 95% Alcohol (10ml) ( 8g) Phenol (5g) (100) ml D/W Decolorizer is 1% acid alcohol

38. MICROSCOPICY P ink coloured slender rod shaped with curved ends bacilli seen against blue background .

39. RNTCP Grading for sputum smear Examination Result Grading > 10 AFB/oil immersion positive +++ 1-10 AFB/OIF positive ++ 10-100/100 OIF positive + 1-9 AFB /100 OIF scanty Record no. of AFB 0 AFB Negative No AFB seen

40. SPECIAL STAINING Staining of Volutin granules of Corynebacterium diphtheriae Al bert's stain Neissers stain Pander's stain

41. Albert’s stain PRINCIPLE: - On staining with Albert’s stain the granuls of Corynebacterium diphtheria take up a bluish purple colour and hence they are called metachromatic granules     Albert stain I Toluidine blue 0.15 gm Malachite green 0.20 gm Glacial acetic acid 1.0 ml Alcohol(95%) 2.0 ml Distilled water 100 m Albert stain II Iodine 2.0 gm Potassium iodide 3.0 gm Distilled water 300 ml

42. ALBERT’S STAIN Corynebacterium diphtheriae Stained by Albert’s stain Green colored bacilli showing " Chinese-letter" arrangement at angles to each other containing bluish-black metachromatic granules of cells.

43. SPECIAL STAINING CON.. Spores staining Principle:- Malachite green stain - Malachite green (5%) for 1 min - Safranine (0.5%) or Basic fuchsin (0.05%) for 30 sec Modified ZN stain can also be used 0.25% sulphuric acid is used as decolourizer , yields red spores in blue – stained bacteria.

44. SPECIAL STAINING CON.. CAPSULE Principle:- The capsule is non-ionic, so that the dyes commonly used will not bind to it. Two dyes, one acidic and one basic, are used to stain the background

45. NEGATIVE STAINING:- Organisms are not stained, only the background is stained Capsule appear as clear halo stained by Grams or Leishman stain India ink Nigrosin Capsulated Bacteria Streptococcus pneumoniae , Haemophillus influenzae , Anaerobic GNB. Bacillus anthracis .

46. SPECIAL STAINING CON.. IMPREGNATION METHOD PRINCIPLE:- Structures too thin to be seen under the ordinary microscope may be rendered visible if they are thickened by impregnation of Silver on the surface. Such method are used:- Demonstration of spirochetes Bacterial flagella

47. FLAGELLAR STAIN LIEFSON'S FLAGELLAR STAIN Basic fuchsin Tannic acid Phenol RYU’S STAIN Crystal violet Phenol Tannic acid SILVER IMPREGNATION SPECIAL STAINING CON..

48. SPECIAL STAINING CON.. STAINING OF SPIROCHAETES The three important groups of spirochaetes are 1) Treponema 2) Leptospira 3) Borrelia They are not easily stained The larger spirochetes, e.g. borreliae , stain by ordinary methods, including Gram’s , Leishman’s and Giemsa’s . The smaller ones, e.g. treponemes and leptospires , are too thin to be demonstrated by ordinary stains.

49. SPECIAL STAINING CON.. SPIROCHAETES STAIN STEINER & STEINER METHOD PRINCIPLE: The bacteria absorb the silver from the silver solution, with the aide of a reducing agent, the silver is then transformed to a visible metallic state

50. Silver impregnation method Fontana (for films) Leavaditi (for tissue sections) Leptospires from a culture stained by modified Fontana silver examination by dark- field microscopy .

51. SPECIAL STAINING CON.. RICKETTSIAL STAIN SLOW GIEMSA Rickettsiae stain a bluish purple colour.

52. SPECIAL STAINING CON.. STAIN FOR MYCOPLASMA Mycoplasmal microcolonies DIENE'S STAIN contains methylene blue, maltose and azure , used for staining mycoplasmal microcolonies

53. SPECIAL STAINING CON.. STAINS FOR RICKETTSIA Castaneda's stain :-A technique for demonstrating chlamydial elementary bodies, using formol blue and safranine Macchiavello & Gimenez stain:- A basic fuscin solution for staining chlamydial elementary bodies, Rickettsiae

54. VITAL STAIN A vital stain is a stain that can be applied on living cells without killing them Supravital stain   a stain introduced in living tissue that has been removed from the body, but before cessation of the chemical life of the cells

55. INTRAVITAL STAINING The stain may be injected into a living animal and the stained tissue removed and examined Vital stains include trypan blue, vital red, and Janus green, the latter being especially suitable for observing mitochondria