PROCEDURE OF SPORICIDE
1. A 2 x SG-Schaeffer sporulation medium (stock solution) : 16 g LB broth/liter, 0,5 g MgSO4 . 7H2O, 2 g KCl/lt.
2. Additional stock solutions include %10 glucose, 1M Ca(NO3)2, 0,1 M MnSO4, 0,001 M FeSO4.
3. Compenents of stock solutions are dissolved one at a time in 1 lt of d-distilled H2O.
4. 100 ml aliquots are poured into media bottles.
5. The caps are loosened and autoclaved in a pan of water for 20 min. on slow exhaust.
6. The bottles are allowed to cool comletely, caps are tightened, solutions are scored in the dark until use.
- As prepared, the Schaeffer nutrient base lasts approximately 1 week.
7. Stock solutions are filter sterilized yusing a 0,22 um filter.
Fermantation (Producing Spores)
1. 10 ml of Schaeffer medium is added to a 50 ml falcon, inoculated with a 10 ul culture (or one colony from plate) of B. Subtilis.
2. 5 days are allowed for growth and sporulation. (30 C, moderate aeration)
3. The culture is heat shocked at 65 C for 15 min to kill vegetative cells.
4. The spores are harvested by centrifiguation (16,000 x g for 25 min), then washed with d-distilled water 10 times.
5. The spores are resuspend in 1.7 ml of sterile d-distilled water.
6. A dilution series is performed on the suspension to numerate the number of viable spores.
7. Growth on nutrient agar medium deposited on petri dishes to determine the population of spores.
Preperation of the modified Fenton Reagents
1. Cupric chloride and ascorbic acid (AA) are dissolved in deionized water, tap water, or salt water (2 M NaCl). The most effectıve reagent solution includes 2M NaCl in water
2. A 1% surfactant (3 M FC-170 or FC-100 fluorinated surfactant) is also added in to reduce the surface tension of the aqueous solution in order to get the highest killing level.
- The most effetice reagent solution includes FC-100 type of sulfactant
3. The exposure time of spores to the reagent istypically 30 min, and exposure is performed at 25°C.
Sporicide Procedure (Aqueous)
1. Prepared aliquots of 1 ml of spore culture are placed in test tubes.
2. The tubes are spun for 10 min at 16,000 xg to pelletize the cells. The supernatant is discarded.
3. Three tubes are resuspended in the Fenton reagent to be tested (the precise volume and tube size varied).
- A fourth sample tube is resuspended in sterile d-distilled water as a control.
4. The tubes are exposed to the Fenton reagents for the specified exposure times (30 min unless noted otherwise).
5. Dilution with cold (0°C) liquid (sodium thiosulfate) quenches the reaction.
6. The sample tubes and the control are then spun at 16,000 xg for 10 min to pelletize the cells. The supernatant is removed.
7. Spores are rinsed two times in an appropriate diluent.
8. Spores are then resuspended in 1 ml of fresh sterile phosphate buffer.
9. Serial dilutions (generally over 5 to 7 logs of dilution) on both samples and controls are performed using nutrient agar growth medium plated on petri dishes. The dishes are incubated for 24 h minimum, 48 h maximum at 30°C.
NOTE: As described above, serial dilutions are then prepared for each tube, using sterile phosphate buffer as the diluent. In all cases, a standard serial dilution of the stock spore
culture is carried out to verify that the culture is still viable and that the number of CFU (CFU per milliliter) is constant over time. All spore-handling procedures use aseptic bacteriological techniques in laminar flow biohazard hoods.
Sporicide Procedure (Solid Surface)
1. Sterile 2-cm2 glass coupons are scored and cut from frosted glass microscope slides
2. The glass coupons are then inoculated with B. subtilisstock solution, approximately 5 x 10^7 spores/coupon. The pipette is fitted with a new tip for each coupon.
3. The inoculated coupon(s) then is dried at room temperature overnight in a desiccator containing Drierite.
4. Following exposure to Fenton reagents, coupons are placed in a cold (_0°C) neutralization agent (sodium thiosulfate) and then sonicated for 60 min to remove spores from the substrate. Separate tests verifythat the sonication procedure is capable of recovery from the coupons and that sonication do not induce spore deactivation.
5. The spore population is then determined by performing triplicate serial dilutions of the resulting suspension. Serial dilutions are prepared for each sample, using sterile phosphate buffer as the diluent.
6. Serial dilutions (generally over 5 to 7 logs of dilution) on both samples and controls are performed using nutrient agar growth medium plated on petri dishes. The dishes are incubated for 24 h minimum, 48 h maximum at 30°C.
NOTE: The magnitude of kill is then determined by comparing the treated colony count with that of untreated colonies. It is noted that the preparation techniques resulted in clumped spores on the impervious glass coupons.(J. B. Cross, 2003)