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) 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. 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
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
CFU (as observed on
(includes dilution factor)
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
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.