Staining of bacterial endospore by Dorner’s Method

1. Define an endospore?

Answer :-

Endospores are thick-walled refractile, heat, desiccation, chemical, and radiation-resistant, a dormant spore that is formed within a parent cell.

2. What is a spore?

Answer :-

A spore is an asexual reproductive or dormant body formed by the differentiation of vegetative cells or structures under unfavorable environmental conditions particularly with the exhaustion of nutritional carbon. source.

A spore is resistant to the deleterious effects of excessive heat freezing, radiation, desiccation: and chemical agents including commonly used stains. 

A spore is metabolically inactive and may revert to active forms when favorable spore environmental conditions are available. They are usually unicellular and may develop into vegetative organisms or gametes.

3. What is a cyst ?

Answer :-

A cyst is a dromant form enclosed in a wall and are formed by protozoa and a few bacteria for withstanding adverse conditions, such as low nutrient concentration, dessication, low pH, and lack of oxygen.

They may be reproductive cysts that are normal stage in the life cycle of protozoa. Cysts are comparatively less resistant to heat and chemical agents as compared to endospores.

4. Name a few microorganisms which produce cysts.

Answer :-

MicroorganismGenus
EubacteriaAzotobacter
Methylomonas
Methylococcus
ProtozoaEntamoeba
Giardia
Toxoplasma

5. Name an actinomycetes which produces true endospores.

Answer :-

Thermoactinomyces produce true endospores, which are very heat resistant (survive at 90° C for 30 min.)

6. Name a few methods for endospore staining.

Answer :-

  • (a) Domer’s method. 
  • (b) Bartholomew and Mittwer’s method. 
  • (c) Schaeffer – Fulton method.

7. Give the principle of spore staining.

Answer :-

The endospore outer layers show a very low rate of penetration to ordinary dye in aqueous solution at room temperature. A strong basic dye such as carbol fuschin is allowed to react with the spore with heating.

Spores once stained are resistant to decolourization by the nigrosine stain, which also stains the background. The vegetative cells appear colourless, with pink endospore and dark background.

8. What is the full form of Z.N.C.F.?

Answer :-

Z.N.C.F. stands for Ziehl – Neelsen Carbol Fuschin.

9. Name a few Gram positive endspore forming eubacteria.

Answer :-

GenusMorphologyPhysiology
AmphibacillusRodsFacultative
Bacillus
B. cereus
B. megaterium
B. subtilis
B. thuringiensis
B. stereothermophilus
RodsAerobic or Facultative anaerobic
Clostridium
C. botulinum
C. difficle
C. pasteurianum
C. perfringens
C. tetani
RodsAnaerobic
DesulfotomaculumRodsAnaerobic
OscillospiraCurved rodsAnaerobic (requires 3% to 12% NaCl)
SporohalobacterRodsAerobic
Sporolactobacillus
Sporosarcina
Sulfobacillus
Rods
Rods
Facultative
Aerobic
Aerobic  

10. Which endospore forming eubacteria are employed for validation of autoclaves or canning equipment and procedures ?

Answer :-

Bacillus stearothermophilus spores are commonly used in validation of autoclaves and canning equipment i.e. as a biological indicator for checking autoclaving.

Clostridium sporogenes PA 3679 is also used. Exposure to 115° C for 15 minutes is generally considered necessary in canning.

11. Give the mechanism for development of heat resistance by endospore?

Answer :-

Endospore heat resistance is due to several factors. Calcium – dipicolinate, stabilization of components like DNA, protoplast dehydration due to the removal of wäter osmótically by the cortex, the greater stability of cell proteins in bacteria adapted to growth at high temperatures, etc.

12. What is a sporangium or spore mother cell?

Answer :-

The sporangium or spore mother cell is the vegetative cell which produces endospore.

13. Which are the parts of a spore?

Answer :-

Endospore structure is very complex and it can be seen by electron microscopy.

Its parts are :

ExosporiumOutermost delicate covering (present in some)
Spore coatA thick layer, below the exosporium, which consists of several protein layers.
CortexA structure below the spore coat, which forms 2/3 of the spore and consists of peptidoglycan
Spore cell wall or core wallA layer below cortex, which surrounds the protoplast or core
CoreCentral part of spore with RNA, Ca and dipicolinic acid nucleoid.

14. How is spore formation useful in identification?

Answer :-

Spore position in the mother cell or sporangium is of considerable value in identification of bacteria. Spores may bė located centrally, close to one end (sub-terminal) or at one end (terminal). 

15. How are the spores of fungi and actinomycetes different from bacterial endospore?

Answer :- 

Fungi and actinomycetes produce spores that increase their survival. However, these spores are not heat resistant but can withstand other adverse conditions.

16. What is cryptobiosis? 

Answer :-

The state of dormancy (inactive state) of a spore is known as cryptobiosis.

17. What are conidia?

Answer :-

Some filamentous becteria and fungi such as micromonospora and streptomyces, form asexual; aerial spores with thick outer walls, called conidia (sing conidium). Conidia are temporarily dormant, but are not especially resistänt to heat and drying.

18. What is germination?

Answer :-

The process by which a spore returns to its vegetative state, is called germination.

19. What are the stages of germination?

Answer :-

Germination ocurs in three stages; activation (by low pH or heat), germination proper and outgrowth.

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