KVV E-Modules

1. Capsule staining

d) Special staining techniques. They are designed for demonstration of either specific cell components like cell wall, capsule, flagella, endospore etc. or cytoplasmic components like lipid granules, PHB granules, nuclear material etc. These techniques are helpful for identification of unknown bacteria and therefore having great diagnostic values in medical microbiology. CAPSULE STAINING Some bacteria are surrounded by a viscous, slimy, gummy substance which form an envelope & which lies wall an external and which is non-ionic in nature to the cell wall is called capsule. Anatomically, they can be divided into 3 categories i) Microcapsules: They are at least 0.2 µm thick, can be easily demonstrated by light microscope & they have definite external surface. ii) Microcapsules: They are less than 0.2 µm thickness, can be detected immunologically. iii) slime layers: A viscous substance accumulates at the surface of micro-organisms. Formation of capsule is a hereditary property, also depends on environmental conditions. Functions of capsules. 1. They may block attachment of bacteriophages 2. Temporary protection against drying or desiccation as they are having ability to retain considerable amount of water. 3. Taxonomic importance 4. It may act as a reservoir of storage compounds so helpful during starvation. 5. Protection from phagocytosis 6. Site for disposal of waste material 7. To determine virulence of microorganisms Chemical composition & Growth 1. In capsular material 98 % of water & 2% of organic compounds in the form of polysaccharides or polypeptides are present 2. Capsule are not essential for bacterial growth & reproduction. Staining solution Role Maneval's solution: 5% phenol 25% acetic acid 30% ferric chloride 1% acid fuchsin 1% congo red Accentuator Changes color (congo red) of background to blue Mordant Stain to color the cell Red color initially for the background Results: Blue background, red coloured cells and colourless capsule. Staining methods Two types positive & negative 1) Positive: i) Method of white ii) Method of Moller iii) Method of Hiss 2) Negative: i) Maneval’s method ii) Dry India ink film method a) Maneval’s method Method of Maneval: (combination of positive and Negative stains): Place a drop of congo red on a slide at one end ↓ Mix a loopful of suspension in it ↓ Spread gently over a slide by using edge of another slide ↓ Allow to air dry at room temperature ↓ Flood the smear with Maneval’s stain for 2 minutes ↓ Decant the excess stain (Do not wash with water) ↓ Air dry and observe under oil immersion lense Results: Bacterial cell: Pink in colour Capsule: Colourless Background: Red/blue in colour Hiss' method (staining of capsule): The capsule has a very low affinity for dyes because of its non-ionic nature. Hiss' stain consists of gentian violet, a basic dye which stains the capsule as well as the bacterial cell violet in colour. On application of hypertonic solution (20% copper sulphate) osmotic difference is created which causes diffusion of stain towards the outer surface of the cell. On removal of copper sulphate solution, the dark colour of gentian violet becomes prominent, thereby showing a dark violet coloured cell surrounded by a light violet capsule on a light blue background. Procedure 1. Make a smear and air- dry it. 2. Flood the smear with Hiss' stain for about 30 seconds. 3. Gently heat the flooded smear till steam arises. 4. Wash off stain with 20% aqueous copper sulfate solution. 5. Blot and air dry. Do not wash. 6. Observe under oil immersion lens. The cells appear violet. The capsules appear light violet on a light blue background. Example encapsulated Capsule present bacteria 1. Klebsiella pneumoniae 2. Streptococcus pyogens 3. Enterobacter species 4. Bacillus anthrasis 5. Bacillus circulans 6. Bacillus megaterium 7. Rhizobium species 8. Azotobacter species 9. Leuconostoc mesenteroides 10. E-coli 11. Pseudomonas Spp 12. Xanthomonas spp 13. Cryptococcus spp

1. Capsule staining

Short notes Role of staining solutions in Maneval's method Hiss method of staining of capsule Composition of capsules in various bacteria Functions of capsule. One-line answers Capsules are required for bacterial growth & reproduction - True or false Answer- false Capsules can be stained by monochrome Staining technique - True or false Answer-False Capsule protects the bacterial cell from dehydration. Give reason Answer- because it contains 98% reason Capsule can be lost with repeated Subculturing Answer - True Why capsule is very difficult to stain? Answer -due to Tare due to nonionic nature Capsule staining is called relief staining Answer-True Bacterial film(smear) is heat fixed for Capsule staining. Answer -false What is need of staining? Define Stain. What are types of stains? MCQ Capsule staining is a type of ____________. a) Monochrome staining technique b) Negative staining technique c) Differential staining technique d) Special staining technique For capsule staining ____________ is used. a) Maneval's method b) Chance's method c)Dorner's method d) Bailey's method Congo red is __________. a) a basic stain b) an acidic stain c) a neutral stain d) a leuco compound _____________ is not encapsulated bacterium. a) Bacillus megaterium b) Azotobacter species c) Salmonella species d) Rhizobium species __________ staining is called relief staining a) Monochrome b) Negative c) differential

1. Capsule staining