Why is heat necessary in endospore staining

Microbiology Lab

Microbiology Lab

Directions:
Answer following questions after reading the information and watching the video from the link below. Use color RED or BLUE for your answers. Submit the completed document on eCampus for grading. Refer to

· the textbook chapter 3 (3.2) (Nester- McGraw Hill)

· Link: Bacterial Staining Techniques-III, Virtual Edge Experiment-11

Endospore Staining (11A on Virtual Edge)
Watch the video of Endospore Staining, from textbook from chapter 3 review sections 3.2 (page 55), 3.7 (pages 76-77) to answer the following questions.

Actively growing bacterial cultures are not used for this experiment. Instead, cultures more than a week old are used. After a week, most nutrients are used up by bacteria, growth creates accumulation of waste products and less space, overall creating “unfavorable” conditions. Under such conditions, bacteria capable of producing endospores produce them to save their species.

1. What are bacterial endospores?

These are dormant and tough non-reproductive structures that are generated by bacteria in the phylum firmicutes.

2. Do all bacteria produce endospores?

Not all bacteria produce endospores. Very few bacteria are able to produce endospores.

3. Under what conditions bacterial endospores are produced?

In conditions deficient of nutrients in the gram positive bacteria.

4. Is sporulation a way of reproduction for bacteria?

This is the formation of spores occurring from the products of an asexual cell division. This is the formation of nearly dormant forms of bacteria.

5. What is germination of endospores and how does it start?

This is the irreversible conversion of a dormant bacterial endospore into a metabolically active cell. The process begins only after activation.

6. Can bacterial endospores be destroyed? How?

Bacterial endospores can be destroyed by autoclaving or burning at temperatures exceeding 100 degrees Celsius.

7. Why bacteria that are capable of producing endospores important in canning and health industry?

Because they are resistant to processes that usually kill vegetative cells e.g. freezing, desiccation, radiation, use of chemicals, and heating.

8. Name 1 bacterium that could be used easily in lab for this experiment.

Bacillus

9. This process differentiates between vegetative cells and endospores. True or False

True

10. What is the primary stain used in the endospore staining experiment?

Malachite green

11. Why is it necessary to steam the slide with smear while adding the primary stain?

Steam loosens up the waxy layer to promote the entrance of the primary stain in the cell.

12. Which counterstain is used in endospore staining?

Safranin

13. Which stain is retained by the endospores in this procedure?

Green (malachite green).

14. What stain is retained by the bacterial cells in this procedure?

Red from the safranin

15. Observe the following picture and point out bacterial endospores.

Bacterial endospores are the red components in the image.

ASMscience | Spore Stain of Bacillus subtilis

Capsule Staining- (4B on Virtual Edge):
The main purpose of capsule stain is to distinguish chemical composition of capsule from the bacterial cell. A capsule is a gelatinous outer layer secreted by bacterial cells and that surrounds and adheres to the cell wall. Most capsules are composed of polysaccharides, but some are composed of polypeptides. The capsule differs from the slime layer that some bacterial cells produce in that it is a thick, detectable, discrete layer outside the cell wall. The capsule stain uses an acidic stain and a basic stain to detect capsule production.

Watch the video from the link Capsule staining, read from the textbook chapter 3- section 3.2 (page55) and section 3.6 (page 72) to answer the following questions.

Mneomonics to remember capsulated bacteria
Some Killers Have Pretty Nice Capsule

Streptococcus pneumonia (commonly called Pneumococcus known to cause pneumonia, ear infections, sinus infections, meningitis, bacteremia) Klebsiella pneumonia (known to cause pneumonia, wound infections- surgical site infections, meningitis, bacteremia) Haemophilus influenza (known to cause pneumonia, meningitis, bacteremia, ear-throat infections, cellulitis, infectious arthritis) Pseudomonas aeruginosa (known to cause pneumonia, wound infections- surgical site infections, bacteremia) Neisseria meningitides (known to cause menigicoccal disease) Cryptococcus neoformans (a fungus known to have a capsule)

16. What is the function of bacterial capsule?

Bacterial capsule comprises of polysaccharides used to cover the cell wall composed of peptidoglycan and teichoic acid.

17. Do all bacteria produce capsule?

Some bacteria lack capsules e.g. gordonii, streptococcus oralis

18. What is the biochemical component that makes the bacterial capsule?

Hydrated polysaccharide

19. How does human immune system treat un-encapsulated bacteria?

As foreign objects and phagocytosis occurs.

20. Why does this procedure not involve “heat fixing” step?

A lot of bacteria capsules are comprised of polysaccharide and other genera produce polypeptide capsules. Capsular material is slimy and any heating process with cause it to shrink, hence, heating is not advised.

21. Which stain was used in this process?

Crystal violet

22. Which stain was retained by the cells?

Purple (crystal violet)

23. Which stain was retained by the capsule?

Capsules remain colorless

24. From the following picture, point out or describe the capsule:

Science Source - Bacterial Capsules, LM

The capsules appear colorless with stained cells against a dark background. The best way to view the capsule is to stain the background using an acidic stain.