B A C T E R I A L S T A I N I N G

1. 1 BACTERIAL STAINING basicmethods

2. 3 Why we should beStain Bacteria Bacteria have nearly the same refractive index as water, therefore, when they are observed under a microscope they are nearly invisible to the naked eye. Different types of staining methods are used to make the cells and their internal structures more visible under the light microscope.

3. 6 What is aStain • A stain is a substance that adheres to a cell, • giving the cellcolor. • The presence of color gives the cells significant contrast so are much morevisible. • Different stains have different affinities for different organisms, or different parts of organisms • They are used to differentiate different types of organisms or to view specific parts oforganisms

4. 7 StainingTechniques • Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains anddyesare frequently used inbiology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes.

5. 8 Smearing out ofthe sample

6. 9 Fixation • Fixation–whichmay itself consist of several steps–aims to preserve the shape of the cells or tissue involved as much as possible. Sometimes heat fixation is used to kill, adhere, and alter the specimen so it will accept stains

7. 10 Simplestaining • simplest, the actual staining process may involve immersing the sample (before or after fixation and mounting) in dye solution, followed by rinsing and observation. • The stain can bepoured • drop by drop on theslide

8. 11 Simplestaining • Methylene blue, Basicfuchsin • Provide the color contrast but impart the same color to all the organisms in asmear • Procedure: • A bacterial smear is prepared, air-dried and heat-fixed. • A Heat-fixed smear is flooded with either one of the basic stain and allowed to react for 1-2 minutes and then washed under running tap water. • Air dried and focused with 10x,45x & 100x.

9. 12 • Results: • Morphology – spherical / rod. • Arrangement – cocci – clusters/chains.

10. 13 SimpleStains

11. 15 Simple Staining Easier to Perform But hasLimitations • Simple easy to use; single staining agent used; using basic and aciddyes. • Features of dyes: give coloring of microorganisms; bind specifically to various cellstructures

12. 16 DifferentialStains Differential Stains use two or more stains and allow the cells to be categorized into various groups ortypes. Both techniques allow the observation of cell morphology, or shape, but differential staining usually provides more information about the characteristics of the cell wall(Thickness).

13. 17 Gramstaining • Named after Hans Christian Gram, differentiates between Gram- positive purpleand Gram-negative pink stains and is used to identify certainpathogens.

14. 18

15. 19 Gram staining -Principles • Gram staining is used to determine gram status to classify bacteria broadly. It is based on the composition of their cell wall. Gram staining uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria. Gram status is important in medicine; the presence or absence of a cell wall will change the bacterium's susceptibility to someantibiotics. • Gram-positive bacteria stain dark blue or violet. Their cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria

16. 20 Gram StainingSteps Crystal violet acts as the primary stain. Crystal violet may also be used as a simple stain because it dyes the cell wall of anybacteria. Gram’s iodine acts as a mordant (Helps to fix the primary dye to the cell wall). Decolorizer is used next to remove the primary stain (crystal violet) from Gram Negative bacteria (those with LPS imbedded in their cell walls). Decolorizer is composed of an organic solvent, such as, acetone or ethanol or a combination ofboth.) Finally, a counter stain (Safranin), is applied to stain those cells (Gram Negative) that have lost the primary stain as a result ofdecolorization

17. 21 Stains differentiatesdifferent groups ofBacteria • To distinguish different kinds of bacteria into separate groups based onstaining properties • Two types:Gram stain & Acid-fast stain.

18. 22 ramStain Differential Stains:G

19. 23 Gram Stainingtechnique

20. 24 Dr.T.V.RaoMD Gram StainingProcedure

21. 26 Structure and Reactivity to Gram Staining.

22. 28 Cell structure differentiates Gram positive from GramNegative

23. 29 Gm+ve cocci & Gm-vebacilli

24. 37 ACIDFAST STAINING

25. 39 Acid-FastStain • Acid-fast cells contain a large amount of lipids and waxes in their cellwalls • ▫ primarilymycolic acid • Acid fast bacteria are usually members of the genus Mycobacterium or Nocardia • ▫ Therefore, this stain is important to identify Mycobacterium or Nocardia

26. 40 Ziehl-Neelsenstain • Ziehl-Neelsen staining is used to stain species of Mycobacterium tuberculosis that do not stain with the standard laboratory staining procedures like Gram staining. • The stains used are the red colored Carbol fuchsin that stains the bacteria and a counter stain like Methylene blue or Malachitegreen.

27. 41 Acid-FastOrganisms • Primary stain binds cell wall mycolic acids • Intense decolorization does not release primary stain from the cell wall ofAFB • Colorof AFB-based on primarystain • Counterstain provides contrasting background

28. 42 Dr.T.V.RaoMD AFB StainingMethods • Zeihl Neelsen’s-hot stain • Kinyoun’s-cold stain • Modifications

29. 43 Procedure • Strong carbolfucshin-heat till steam rises – allow 5-10 min to act (alternately leave it 10-15 min – cold staining method) – wash. • 2. Decolorise with acid-alcohol mixture till get a faint pink colour in the smear (take 3-5 min) – wash. • 3. Methylene blue/Malachite green – 2 min – wash. • 4. Allow to dry and focuss under microscope.

30. Results • Pink bacilli – Acid fast bacteria/bacilli Eg., M.tuberculosis – long slender bacilli. • M. leprae – short thick bacilli. • Blue colored bacteria – Non-acid fast Eg., Epithelial cells, pus cells, other bacteria.

31. 1 2 3 Dr.T.V.RaoMD 4 5 6 7 Ziehl-Neelsen stain

32. 46 How the Acid fastbacteria appear

33. SPECIAL STAIN: Used to stain special structures of bacteria– capsule, spores, flagella, metachromatic granules.

34. CAPSULE STAIN:Negative stain:1.Drop of Nigrosin ink+ indian ink2. Bacterial culture ( 1-2 colonies)3. Spread evenly and air-dry.4. Look for unstained structures against stained background.

35. CAPSULE STAIN BY NIGROSIN INK (BLACK)

36. CAPSULE STAIN- INDIAN INK(BLUE)

37. SPORE STAIN:1. Malachite green- 2 min- heat stain till steam rises -2 min - wash.2. Counterstain with safranin –1 min- wash.3. Dry the slide and examine.Spore forming bacteria:Eg., Clostridium species. Bacillus species – Eg. B. anthracis

38. Spore stain

39. FLAGELLAR STAIN – SILVER STAIN:This stain increases the thickness of flagella – thus easy to see under light microscope.

40. Metachromatic granule staining:* To demonstrate polar granules of Corynebacteriumdiphtheriae.* Take up the stain of methylene blue – but appears bluish black – hence granules called metachromatic granules.* Bacilli stains blue not bluish black.