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Capsule staining
- 1. CAPSULE STAINING Presented by- SushantBalasaheb Jadhav Roll no. – 18PBT206 M. Tech. Pharmaceutical Biotechnology Institute of Chemical Technology, Mumbai
- 2. What is aCapsule? • A well organized layer of material lying outside the cell wall referred to as a capsule (or K antigen) • Capsules are well organized layers that cannot be easily washed off • A capsule is composed of capsular polysaccharides (CPS) which are polysaccharides • They may also be composed of a number of other materials such as poly-D-glutamic acid. 2
- 3. Functions of Capsule •Capsules are very important in that they are a major virulence factor • They protect the bacteria from phagocytic actions • Protecting bacteria from desiccation • Providing a food reserve • Protecting anaerobes from oxygen toxicity • To help bacteria attach onto surfaces • They exclude bacterial viruses and most hydrophobic toxic materials such as detergents 3
- 4. Strains having Capsule •Streptococcus pneumonia • Haemophilus influenza • Neisseria meningitidis • Klebsiella pneumonia • Group B streptococci • Actino bacillus • Pleuro pneumonia • Sinorhizobium meliloti • Staphylococcus aureus 4
- 5. Why Capsule Staining? • Most capsule materials are water soluble, simple stains will not adhere to them • Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces • Capsule stain is a type of differential stain which involves the use of two stains; primary stain and the counterstain • It becomes necessary to stain the background using an acidic stain while the cell is stained using a basic stain • Since the capsule is a major virulence factor in the major disease-causing bacteria, it is essential to identify the strain 5
- 6. Methods for CapsuleStaining •India ink method •Anthony's stain method 6
- 7. India Ink Method Requirements •India ink • Crystal violet • Glass slide • Inoculating loop 7
- 8. India Ink MethodCont. Procedure • Place a drop of India ink onto a clean glass slide • Using a sterile loop, obtain a sample and place it onto the slide and mix it with the drop of India ink • Obtain another sterile glass slide and having laid it at an angle on one end of the first slide, spread out the drop into a film to have a thin layer of the smear-ink mixture • Allow the slide to stand for about 5 minutes (air- dry) • Saturate the slide with crystal violet for a minute and then tilt the slide to drain excess dye • Allow the slide to dry (air-dry) • View the slide using 100x 8
- 9. India Ink MethodCont. • India ink is often used as the negative stain • Other stains that can also be used include: Congo red Nigrosin Eosin • Because of its acidic nature, India ink (or Congo red, nigrosin) stains the background dark 9
- 10. India Ink MethodCont. • Crystal violet is used for number of reasons including: To act as a fixative Increase penetration power Stain the cells (being a basic dye) Decrease pH of smear • The capsule will be clear against a dark background given that it takes no stain • Nigrosin is recommended to India ink because it gives a more even background and spreads more easily 10
- 11. India Ink MethodCont. 11
- 12. Anthony's Stain Method •The primary stain used is crystal violet. • Some of the other requirements include: A sample (Organism grown for 36-48 hours) Glass slide Inoculating loop 12
- 13. Anthony's Stain MethodCont. Procedure • Introduce a drop of crystal violet on to a clean glass slide • Using a sterile loop, place the sample on to the glass slide • Obtain another glass slide and at an angle, spread the drop and sample to form a thin film • Allow the film to dry (air dry) for about 6 minutes • Title the slide and rinse with 20% copper sulfate solution • Allow the slide to air dry for about 3 minutes • Place the slide on to the microscope stage and observe using oil immersion 13
- 14. Anthony's Stain MethodCont. • This is a positive staining method used in capsule staining • The smear/sample is treated with a hypertonic solution (copper sulfate) for the purposes of creating an ionic difference that ultimately results in the diffusion of a stain towards the outer surface of the cell • After the slide is air dried, it becomes possible to observe the stain that remained on the capsular layer • One will see a dark violet color of the cell and a light violet color of the capsule 14
- 15. Additional cares • Don’tHeat • Don’t rinse with water • Add drop of serum • Sterile technique/practice 15
- 16. REFERENCES • https://www.microscopemaster.com/capsule- stain.html • https://microbenotes.com/capsule-stain- principle-procedure-and-result-interpretation/ •https://microbiologyinfo.com/capsule- staining-principle-reagents-procedure-and- result/ • https://microbeonline.com/capsule-stain- principle-procedure-results/ 16
- 17.
Editor's Notes
- #4 they protect the bacteria from phagocytic actions of such cells as neutrophils allowing the bacteria to thrive. This is achieved due to the fact that the capsule is very smooth and has a negative charge that prevents attachments/adherence. Desiccation is the state of extreme dryness, or the process of extreme drying. A desiccant is a hygroscopic (attracts and holds water) substance that induces or sustains such a state in its local vicinity in a moderately sealed container.