Exercise 10: STUDY OF BACTERIAL SPORE

MSValdez & MTALlorin

Endospore

German botanist Ferdinand Cohn (1828 1898) discovered the existence of heatresistant bacterial endospores a special resistant, dormant structure formed by most Gram-positive bacteria within vegetative bacterial cells of several genera: Bacillus and Clostridium, Sporosarcina (cocci), and others.

Endospore
extraordinarily resistant to environmental stresses oheat, uv radiation, gamma radiation, chemical disinfectants, and desiccation osome endospores = viable for around 100,000 years actinomycete spores (not true endospores) alive after burial in the mud for 7,500 years

Endospore
aid in survival when moisture or nutrients are scarce forms when DNA produces a thick internal wall that encloses the DNA and some part of the cytoplasm

Schaeffer-Fulton / Wirtz-Conklin
1. first stained by heating bacteria with malachite green, which is a very strong stain that can penetrate endospores 2. the rest of the cell is washed free of dye with water

Schaeffer-Fulton
3. Counterstained with safranin Result: endospore cell

Step

Color of Cell

Color of Endospore Colorless Green

Green

Smear
Malachite Green Cooling/Washi ng

Colorless Green
Colorless

Safranin

Red

Green

Staining
Endospores are not stained well by most dyes, but once stained, they strongly resist decolorization

 Covering with absorbent paper : holds the stain; prevents the accumulation of artifacts during staining Steaming/heating: allows the penetration of the malachite green and binding with the spore as it softens the hard and durable outer layer of the endospore

Properties of Bacterial Spore

Thick walled Contains 4 layers:
core Cortex Coat exosporium

Shape: - ellipsoidal - ovoid - round

Bacillus megaterium
(-) distention central ellipsoidal

Heat resistance
due mainly to its ability to maintain a very low water content in the central DNAcontaining protoplast ***spores with a higher water content have a lower heat resistance bacterial endospores are more resistant to hydrostatic pressure ~1200 megapascal

Heat resistance
susceptibility can be increased considerably by: increasing temp; (low pressure) = SPORE GERMINATION ***spores lose their resistance to heat and to elevated pressure

Heat resistance
due to several factors: calcium-dipicolinate protoplast dehydration spore coat DNA repair stability of cell proteins in bacteria adapted to growth at high temperatures

Dipicolinic Acid
@ core 15% of spores dry wt. Complexed w/ calcium ions

Calcium

*before, it was thought to be correlated w/ spore heat resistance

Aid in resistance to weight heat, ox agent & dry heat, sometimes

Calcium-dipicolinate
may be the one that stabilizes spore nucleic acids

Dehydration of the protoplast

Cortex removes water from protoplast via osmosis = protected from heat & radiation damage

DNA-binding proteins
- discovered in the endospore - they saturate spore DNA and protect it during harsh conditions

Spore coat
- seems to protect against enzymes and chemicals such as hydrogen peroxide

DNA Repair Enzymes (some)

- DNA is repaired during germination and outgrowth after the core has become active once again

Germination Medium
Glucose Dipotassium hydrogen phosphate Ammonium sulfate Yeast extract Manganese sulfate monohydrate Magnesium sulfate Zinc sulfate Ferrous sulfate heptahydrate L-alanine Adenosine Copper sulfate pentahydrate Calcium chloride

Germination Medium
L-alanine (amino acid), Adenosine (glycoside of ribose = riboside) & Glucose (sugar): initiate RAPID germination Dipotassium hydrogen phosphate: buffer Ammonium sulfate N source

Germination Medium
Yeast extract vit. Source & other nutrients Manganese sulfate monohydrate stimulatory effect on sporulation Magnesium sulfate, Zinc sulfate source of ions to grow bacteria

Germination Medium
Ferrous sulfate heptahydrate: supply ions (iron) for bacterial growth Copper sulfate pentahydrate: source of ions (copper) for bact. growth Calcium chloride: source of ions; facilitates growth

Transformation
endospore vegetative state Occurs in 3 stages: (see pre-lab h.o.) oActivation oGermination proper oOutgrowth

Germination
@ 1st plating: - ALL veg. cells + mature spores @ 2nd plating: - spores GERMINATED after heating @ 3rd plating: - UNGERMINATED spores

Data (B. megaterium)

Dilution 1st plating Dilution 2nd plating

10-5 10-6 10-7

TNTC, TNTC 76, TNTC 114, 58 Dilution 10-2 10-3 10-4

10-3 10-4 10-5 3rd plating 7, 98 0, 4 2, 2

32, 87 21, 17 3, 0

Formulas
%spores in the stock: CFU/ml of 2nd plating + 3rd plating x 100 CFU/ml of 1st plating %ungerminated spores: CFU/ml of 3rd plating x 100 CFU/ml of 2nd + 3rd %germinated spores after heating: CFU/ml of 2nd plating x 100 CFU/ml of 2nd + 3rd
spores/ml in suspn: CFU/ml of 2nd plating + CFU/ml of 3rd plating

Results
% spores in the stock % ungerminated spores % germinated spores after heating spores / ml in suspension 0.003% 14.04% 85.96% 7.0 x 104

Questions:
1. Other genera of bacteria producing spores? 2. Difference between actinomycetes spores from bacterial endospores

Endospore formers
Alkalibacillus Actinomycetes Brevibacillus Coxiella Desulfotomaculum Desulfosporomusa Geobacillus Gracilibacillus Heliobacterium Piscibacillus Seinonella Sporoanaerobacter Sporobacterium Sporobacter Thermoanaerobacter Thermobacillus Thermoflavimicrobium Tuberibacillus Etc.

Difference
Both are respond to nutrient limitations Endospores develop from vegetative cells and resistant to environmental stresses The spores of actinomycetes from septal formation at the aerial hyphae. Reproductive spores.

References
Bhatia, R. and R.L. Ichhpujani. 2003. Microbiology For Nurses. 2nd ed. New Delhi: Jaypee Brothers Medical Publishers. p.9. Cain, D. et. al. 2011. Microbology Laboratory Manual. Collin College Publishing. Harrigan, W.F. 1998. Laboratory Methods in Food Microbiology. 3rd ed. London: Academic Press Limited. p.336. Madigan, M. T. et.al. 2006. Brock Biology of Microorganisms. 13th ed. CA: Pearson Education, Inc. pp. 69-73 and p.226. Precott, L.M. 2002. Microbiology. 5th ed. USA: Mc-Graw Hill Companies. pp.68-70. Sumbali, G. and R.S. Mehrotra. 2009. Principles of Microbiology. New Delhi: Tata McGraw Hill. p.343. http://edoc.ub.uni-muenchen.de/9774/1/Asen_Iris.pdf http://www.journal.su.ac.th/index.php/suij/article/viewFile/23/22