http://www.slideshare.net/Ashraf05/bacterial-spores-ashraf-24915787

2. Definition, history
List of medically important spore forming bacteria
Spore formation/ Sporulation
Structure
Classification based on shape and position
Properties
Resistance and destruction
Germination
Demonstration of spores
Uses of spores

3. Bacterial spores are dormant forms of bacteria which are
thick walled, highly refractile and resistant.
– Nutritional deprivement(depletion of N2 or C source
or both) is the triggering factor
– Exposure to suboptimal temperature

4. • Endospores were reported by numerous scientists
Perty 1852;
Pasteur 1869;
Koch, 1876 and
Cohn, 1872.

5. Spore-forming bacteria
Bacillus:
– Bacillus anthracis,
– Bacillus subtilis,
– Bacillus cereus,
– Bacillus clausii,
– Bacillus halodenitrificans
Clostridium:
– Clostridium botulinum,
– Clostridium difficile,
– Clostridium perfringens,
– Clostridium tetani and Clostridium sordellii.
– Clostridium thermocellum (ethanol)
– Clostridium acetobutylicum (acetone)
– Clostridium diolis (propane-di-ol and to convert fatty acids to yeasts)
–
Thermophilic actinomycetes:
– Thermoactinomyces vulgaris

6. Spore formation
Stages
Stage I Pre-sporulation phase
DNA assembles as an axial filament
Stage II Spore septum formation
Stage III Engulfment of the forespore
Stage IV Cortex formation
Stage V Synthesis of spore coats, dipicolinic acid, calcium uptake
Stage VI Spore maturation (coats more thick)
Stage VII Lysis of cell and liberation of mature spore

8. 5.Exosporium
made of glycoprotein and
Lipoproteins.
4. Outer Membranes
(Spore Coats)
• keratin-like protein (consists
sulphur containing aminoacids
with disulphide bonds
• impermeable
• protects spore from chemicals
3. Cortex
• thickest spore layer,
• made of modified peptidoglycan
• susceptible to lysozyme
2.Core Membrane/germ cell wall
structure similar to that in the
vegetative cell wall
1. Spore protoplast/Core
• contains DNA,RNA
• protein
• dipicolinic acid
• Divalent ions (calcium)
1. Core
2. Core Membrane
3. Cortex
4. Spore Coats
5. Exosporium
STRUCTURE

10. SHAPES
AND
POSITION

11. • Shapes
– Spherical, oval or elongated in shape
– May be narrower or broader(bulge) than parent cell
spore
Bacterial cell
spore
Bacterial cell
Bulged
Ex- Clostridium sp.
Not bulged
Ex- Bacillus sp.

12. Position
• Terminal (located at one of the poles)
• Sub terminal or sub central(between center & one of the poles)
• Equatorial (central)
Courtesy-microrao.com/anatomyofbacteria

13. Bacteria can be classified based on the shape
and position of spores.
TERMINAL SUBTERMINAL CENTRAL
OVAL Clostridium tetani
( drumstick appearance)
Clostridium welchii,
Clostridium septicum
Clostridium sporogenes
Bacillus anthracis
SPHERICAL Clostridium tertium ------- -------
spore spore spore
spore

14. PROPERTIES
• Endospores
• Extremely resistant
• Non-reproducing
mechanism of survival rather than a mechanism of
reproduction(unlike fungal spores)
• Not stained readily with dyes (spore coats)

15. • Metabolically inactive (dormant forms)
– No DNA replication (transcription)
– mRNA not produced (translation)
– Spore protein synthesis is defective
• DNA of the endospores is protected and stabilized by
calcium dipiclonate
• Specialized DNA binding proteins saturate it and
protects it from heat, radiation, chemicals.
• Has DNA repair enzymes

16. PROPERTY
VEGETATIVE
CELLS
ENDOSPORES
Surface coats
murein cell wall
polymer
Thick cortex with
spore coats
Microscopic appearance Non-refractile Refractile
Calcium dipicolinic acid Absent Present in core
Cytoplasmic water activity High Very low
Metabolic activity Present Absent
RESISTANCE
Heat resistance Low High
Resistance to chemicals Low High
Radiation resistance Low High
SENSITIVE
Sensitivity to lysozyme Sensitive Resistant

17. RESISTANCE
• Bacterial spores are the toughest forms of life
known.
“ They are so resistant to destruction that some
scientists have proposed that life arrived on
earth when bacterial spores drifting through
space fell to earth.”

18. RESISTANCE
Extremely resistant due to
• low water content (removal by osmosis)
• thick wall and impermeability of spore coat
• high content of dipicolinic acid
• ability to concentrate calcium

19. • Resists boiling for prolonged periods.
• Can survive
–drying,
–radiation, and
–many damaging chemicals.
• Capacity to germinate many years after
formation, may be centuries or ages.

20. Sporostatic Sporicidal

21. GERMINATION
• Germination is the process whereby spores differentiate
into vegetative cells that grow, reproduce & metabolize.
• ONE spore--ONE vegetative cell.
• complex process which happens in 3 stages:
1. Endospores activation
2. germination
3. outgrowth

22. 1. Endospore activation
• Activation- breaks the dormancy in spores,
• most spore properties retained
• Reversible
• Activators(triggering agents)
– Heating at 60°C for more than 1 hour
– Decreasing the pH
– Treatment with mercaptoethanol
• Conformational change in spore macromolecules
• No morphological changes

23. 2. Germination:
• Germination of activated spores occurs when exposed
to certain substances(nutrients)
– aminoacids
– sugars
– nucleosides
• Irreversible
• Removal of cortex (enzyme Ƀ-N acetyl glucosaminidase)- renders
it susceptible to chemical agents
• Loss of refractility
• In vitro germination
incubated at 37°C, 5% CO2, FBS (fetal bovine serum)
– Dulbecco’s modified Eagle’s medium(DMEM)+10% FBS
– Roswell Park Memorial Institute(RPMI)1640 medium +10%FBS,

24. (b) development of
phase dark forms
c) Germination completes
with full conversion to
phase dark cells
(a) mature, phase-bright
(REFRACTILE) spores

25. 3. Outgrowth
• The core of the endospores leave the old spore coat to
develop into a fully functional vegetative cell.
• First visible change is swelling of cell--water uptake
• Molecular biosynthetic process starts
– RNA synthesis– first
– Protein
– DNA synthesis
• Cell wall synthesis coincides with swelling

27. ACTIVATION GERMINATION OUTGROWTH
Morphology No change Swelling followed by
elongation
Reversibility
(back to spore state)
reversible Not reversible --
Metabolism Inert Active RNA, protein and
DNA synthesis starts
Resistance retained Loss of resistance,
Susceptible to most of
chemical agents
Not resistant
Phase contrast
microscopy
Refractile Loss of refractility
staining Not readily
stained
Stained Stained readily with
simple aniline dyes

28. DEMONSTRATION OF SPORES

29. Microscopy:
Unstained preparation
– Using Phase contrast microscope
– Morphology best observed
– large, refractile, oval or spherical bodies within
mother cells
Stained preparation

30. Stained preparation
•Gram stain
•ZN method (modified)
•Dorner’s method
•Malachite green stain
1. Schaeffer and fulton method
2. Ashby modification

31. Gram stain
Reveals
– deep blue bacilli,
– colorless spore within the bacilli

32. Modified Ziehl-Neelsen technique
• 0.25% sulphuric acid used as decolorizer
• Spores- pink
Bacteria - blue

34. Dorner’s method
• First spore staining described (1922)
• Differential staining method
– Primary stain – Carbol fuchsin for 5min
– Decolorizer – acid alcohol for 1min
– Counterstain- Nigrosin 10%

35. Dorner’s Method
Nigrosin (negative stain)
red endospores in colorless
vegetative cells.

36. Schaeffer-Fulton Stain Procedure
1. Make a smear. Air Dry. Heat fix
2. Flood the smear with Malachite Green stain
3. flooded smear covered with a square of filter paper
4. Steam slide for 10 minutes (every minute, add a few
more drops of Malachite Green stain)
5. Allow slide to cool (after the 10 min. steam process)
Malachite green staining

37. Schaeffer-Fulton Stain Procedure (continued)
6. Drain slide and rinse for 30 seconds with DI water
(discard filter paper)
7. Put slide on steam rack
8. Flood smear with Safranin (counter stain). This stains
the vegetative cell. (Leave for 1 minute)
9. Drain the slide and rinse with DI water
10. Blot Dry
11. Use oil immersion objective to view

38. Schaeffer-fulton Method
Spore: green Vegetative cell: red
http://www.arches.uga.edu/~howie/MVC-052endoS.JPG
http://homepages.wmich.edu/~rossbach/bios312/LabProcedures/endospore.jpg

39. Summary of all staining methods
Primary stain Decoloriser Counter stain
Dorner’s Carbol fuchsin
5%
10min Acid- alcohol
1min
Nigrosin 10%
Schaeffer &
Fulton's
Malachite green
0.5%
10 min Tap water Safranin
ASHBY’s
modification
Malachite green
5%
1min Tap water Dilute carbol fuchsin
Or
0.5% safranin
Modified ZN Strong carbol
fuchsin
5-7min 0.25% H2SO4 Methylene blue

40. Uses of spores
• Sterilization control
Bacillus steothermophilus
• Nanobiotechnology
– substrate for DELIVERY of biomolecules
– vaccine vehicles spores
– source for understanding unknown self-assembling
molecules.

41. Anthrax bioterrorism
• Concentrated anthrax spores were used for
bioterrorism in the 2001, in U.S.
• Spores mailed in the form of powder
• led to 11 cases of cutaneous anthrax and 11 cases of
inhalational anthrax, with five deaths in the latter.
• Used as agent for biological warfare by germans.

43. References
• Topley & Wilson’s principles of bacteriology, virology and immunity, 8th
edition. Vol-1
• Mackie & McCartney Practical Medical Microbiology, 14th edition
• A. D. RUSSELL “Bacterial Spores and Chemical Sporicidal Agents”
Clinical microbiology reviews, ASM, Apr. 1990, p. 99-119.
• en.wikipedia.org/wiki/2001_anthrax_attacks
• Emerging Applications of Bacterial Spores in Nanobiotechnology, Journal
of Nanobiotechnology http://www.jnanobiotechnology.com/content/1/1/6