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Geobacillus Stearothermophilus and Bacillus Subtilis
Time-Kill Procedure for Ulta-Calm Medical Industrial Cleaner


Manufacturer: Biobased USA, Solution: Ulta-Calm Industrial Cleaner Organisms: Geobacillus Stearothermophilus and Bacillus Subtilis Geobacillus stearothermophilus (formally Bacillus stearothermophilus)[1][2] is a rod-shaped, Gram-positive bacterium and a member of the division Firmicutes. The bacteria is a thermophile and is widely distributed in soil, hot springs, ocean sediment, and is a cause of spoilage in food products. It will grow within a temperature range of 30-75 degrees Celsius. Some strains are capable of oxidizing carbon monoxide aerobically. It is commonly used as a challenge organism for sterilization validation studies and periodic check of sterilization cycles. The biological indicator contains spores of the organism on filter paper inside a vial. After sterilizing, the cap is closed, an ampule of growth medium inside of the vial is crushed and the whole vial is incubated. A color and/or turbidity change indicates the results of the sterilization process; no change indicates that the sterilization conditions were achieved, otherwise the growth of the spores indicates that the sterilization process has not been met. Bacillus subtilis, known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium.[3] A member of the genus Bacillus, B. subtilis is rod-shaped, and has the ability to form a tough, protective endospore, allowing the organism to tolerate extreme environmental conditions. B. subtilis has historically been classified as an obligate aerobe, though recent research has demonstrated this is not strictly correct. Although this species is commonly found in soil, more evidence suggests B. subtilis is a normal gut commensal in humans. A 2009 study compared the density of spores found in soil (about 106 spores per gram) to that found in human feces (about 104 spores per gram). The number of spores found in the human gut is too high to be attributed solely to consumption through food contamination. Soil simply serves as a reservoir, suggesting B. subtilis inhabits the gut and should be considered as a normal gut commensal. Biological indicators are used in conjunction with chemical indicators and process indicators to validate sterilization processes. Overview: Time Kill procedure as routinely conducted by Steris Corporation for disinfectants was performed for Ulta-Calm Industrial cleaner at recommended dilution for use (1/2 oz. Ulta-Calm Cleaner to 1 gallon of water). Solution was then challenged with microbial populations. Samples were taken at specific time intervals and evaluated for microbial inactivation. At each time interval, duplicate, one-milliliter samples were taken and plated for incubation. An additional 1:1000 dilution was made for each organism and time interval and plated (resulting dilution 1:1000). After appropriate incubation time at the appropriate temperature, plates were removed and counted for organism survival. Organisms were purchased to have known populations per volume. Populations were verified at time of testing to ensure that labeled populations were correct Test Results:

 

Time Interval

Organism

Dilution

CFU (as observed on plate)

Corrected Pop (includes dilution factor)

Starting Population

Geobacillus Stearothermophilus

 

Labeled: 106

 

216, 500

358,000,000

Actual: 108

5 minutes

None

0,0

0

1:1000

349, 346

348,000

7 minutes

None

0, 0

0

1:1000

211, 221

216,000

10 minutes

None

0, 0

0

1:1000

191, 274

232,500

15 minutes

None

0, 0

0

1:1000

226, 717

471,500

Starting Population

Bacillus Subtilis

 

Labeled: 104

 

18, 21

195,000

Actual: 105

5 minutes

None

395, 458

427

1:1000

5, 14

9500

7 minutes

None

482, 501

492

1:1000

7, 7

7000

10 minutes

None

462, 215

339

1:1000

2, 10

6000

15 minutes

None

345, 383

364

1:1000

4, 4

4000

Each time point was diluted 1:1000 at time of sampling in anticipation of the remaining population being too much to count without dilution. Conclusions: Bacillus Subtilis and Geobacillus Stearothermophilus were chosen for evaluation since both organisms are spore-forming organisms. This type of organism poses a challenge in cleaning, as they are more resistant to cleaning solutions. Cleaning solutions, especially sporocides, used to eliminate these in the cleanroom environment are usually corrosive and uncomfortable for the operator to use due to the highly concentrated fumes. Previous studies conducted by manufacturer for this Ulta-Calm cleaner did not evaluate cleaner effectiveness on spore-forming organisms. Geobacillus Stearothermophilus was inactivated at all time intervals from the starting population and overall exhibited a 3-log population reduction in 15 minutes. This organism is incubated at 55-60░C for growth. Growth of organisms at higher dilutions (103) when growth is not observed at no dilution for same time interval are unusual and were observed with this organism. This could be explained by the fact that the Ulta-Calm cleaner may have been keeping the organism from growing when undiluted, exhibiting a microbiostasis quality. When the sample was diluted, this could have caused a dilution of the Ulta-Calm cleaner also present in the sample. Reduction in concentration of Ulta-Calm cleaner could have allowed the organism to grow. Also, increased incubation temperature could also attribute to unusual results. Additionally, this organism may be more susceptible to the Ulta-Calm cleaner than Bacillus subtilis. Bacillus subitilis was inactivated at all time intervals and overall exhibited a 2-log population reduction in 15 minutes. Results from the non-diluted and diluted sample demonstrate a slight microbiostasis in the undiluted sample, similar to the results seen for Geobacillus Stearothermophilus. In conclusion, Ulta-Calm cleaner was effective in reducing spore populations. For use in cleanrooms, the FDA recommends that a 10 6ápopulation be reduced by three logs at minimum in a defined contact time. Additional requirements for cleaners are based on the surface limits for the specific cleanroom manufacturing environment. For example, ISO 7 (Class 10,000) surfaces are only allowed 5 CFU per contact plate (25 cm2) for all surfaces. ISO 8 (Class 100,000) surfaces are allowed 5 CFU for walls and ceilings and only 10 CFU for floors. Both rooms would require a more effective cleaner. It is possible that the Ulta-Calm cleaner would completely inactivate a 106ápopulation; however, it would require a longer contact time than evaluated in this study. Current sporocides used in TSS manufacturing cleanrooms completely inactivate a 106ápopulation in 15 minutes. Additional studies for Ulta-Calm cleaner would need to be conducted to determine complete inactivation time for spore-forming organisms. This Ulta-Calm cleaner would not be an effective replacement for current sporocides, but may be an effective cleaner to use for routine daily cleaning.