Capsule staining

Capsule is an layer around the bacteria cell which gives bacteria the protection and pathogenicity. Staining such an layer is difficult with the normal stains so it is necessary to stain the background and the cell itself which makes the capsule appear colourless.

1. Presented by Nagendra P 16PBT2004 Capsule Staining 1

2. Know the bacterial cell 2

3. Did you notice Capsule ? Some bacteria have a layer of material lying outside the cell wall . When the layer is well organized and not easily washed off, it is called a capsule . Capsules, usually are composed of polysaccharides , known as Capsular Polysaccharides(CPS) but they may be constructed of other materials . For example, Bacillus anthracis has a capsule of poly-D-glutamic acid. Capsules are clearly visible in the light microscope when negative stains or special capsule stains are employed. 3

4. What does these capsules do ? H elp bacteria resist phagocytosis. P rotect bacteria against desiccation. Provide a food reserve when certain organic compounds are in excess. A virulence determinant of pathogenic microbes . E.g. Streptococcus pneumonia Prevention of complement-mediated bacterial cell lysis . Protection of anaerobes from oxygen toxicity. They exclude bacterial viruses and most hydrophobic toxic materials such as detergents. It also aids bacterial attachment to surfaces of solid objects in aquatic environments or to tissue surfaces in plant and animal hosts. 4

5. Few Strains having Capsules Capsules are widely distributed and are found in such diverse pathogens as Escherichia coli, Neisseria meningitidis , Actinobacillus pleuropneumoniae , Sinorhizobium meliloti , as well as in important Gram-positive pathogens including Staphylococcus aureus and Streptococcus pneumoniae . 5

6. Staining Staining -It’s a process of adding a dye to a microbial culture. Simple Staining (shapes and arrangements ) Differential Staining (Gram reactions) Special Staining (Capsule, flagella, spores ) Dye-Is a  colored substance that has an  affinity to the substrate to which it is being applied . Basic dye—possess a positive charge ( methylene blue, basic fuchsin , crystal violet, safranin , malachite green) Acidic dye—possess a negative charge ( eosin, rose bengal , and acid fuchsin ) 6

7. Why Capsule Staining ? Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces. Because most capsule materials are water soluble, simple stains will not adhere to them. Since the capsule is a major virulence factor in the major disease-causing bacteria, it is essential to identify the strain. 7

8. Principle of Capsule staining Negative staining methods contrast a translucent, darker colored, background with stained cells but an unstained capsule. The background is formed with  india ink or nigrosin or congo red . Next counterstaining with dyes like  crystal violet or methylene blue , bacterial cell wall takes up the dye. Capsules appear colourless with stained cells against dark background. Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by heating. A drop of serum can be used during smearing to enhance the size of the capsule and make it more easily observed with a typical compound light microscope. 8

9. Procedure Place a small drop of a  negative stain   on the slide.  Using sterile technique, add a  loopful of bacterial culture to slide, smearing it in the dye. With an other slide, drag the ink-cell mixture into a thin film along the first slide and let stand for 5-7 minutes.   Allow to air dry (do not heat fix). Flood the smear with  crystal violet stain  (this will stain the cells but not the capsules) for about  1 minutes . Drain the crystal violet by tilting the slide at a 45 degree angle and let stain run off until it air dries . Examine the smear microscopically (100X) for the presence of encapsulated cells as indicated by clear zones surrounding the cells. 9

10. Non Capsulated Bacterium 10

11. Quality Control Positive control:   Klebsiella pneumoniae  (ATCC e13883) Negative control:   Alacilgenes denitrificans  (ATCC 15173) 11

12. Uses of Capsule Staining Detection of Anthrax 12

13. References http://homeinsurancequotations.com/post/bacterial-cell-diagram-and functions.html http:// www.microbiologyinfo.com/capsule-staining-principle-reagents-procedure-and-result http:// microbeonline.com/bacterial-capsule-structure-and-importance-and-examples-of-capsulated-bacteria https://www.ncbi.nlm.nih.gov/books/NBK310485/#annex1.s68 http://textbookofbacteriology.net/Anthrax_3.html Willis LM, Whitfield C. Structure, biosynthesis, and function of bacterial capsular polysaccharides synthesized by ABC transporter-dependent pathways. Carbohydrate research. 2013 Aug 30;378:35-44 . Rautemaa R, Meri S. Complement-resistance mechanisms of bacteria. Microbes and infection. 1999 Aug 31;1(10):785-94. Microbiology; 5th Edition; Lansing M. Prescott; October 2002. 13

14. Thank YOU 14