U.S. patent application number 16/004753 was filed with the patent office on 2018-12-13 for capacitor for detecting viable microorganisms.
The applicant listed for this patent is American Sterilizer Company. Invention is credited to Michael A. Centanni, Kathleen A. Fix, Phillip P. Franciskovich.
Application Number | 20180355400 16/004753 |
Document ID | / |
Family ID | 59358912 |
Filed Date | 2018-12-13 |
United States Patent
Application |
20180355400 |
Kind Code |
A1 |
Centanni; Michael A. ; et
al. |
December 13, 2018 |
CAPACITOR FOR DETECTING VIABLE MICROORGANISMS
Abstract
This invention relates to a capacitor comprising two electrical
conductors separated by a dielectric, the dielectric comprising
microorganisms. The dielectric may comprise a biological indicator.
This invention relates to a process for determining whether the
microorganisms are alive or dead. The number of microorganisms can
be determined. This invention relates to a process for testing the
efficacy of a sterilization process using the capacitor.
Inventors: |
Centanni; Michael A.;
(Parma, OH) ; Franciskovich; Phillip P.; (Concord,
OH) ; Fix; Kathleen A.; (Willoughby, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
American Sterilizer Company |
Mentor |
OH |
US |
|
|
Family ID: |
59358912 |
Appl. No.: |
16/004753 |
Filed: |
June 11, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15375256 |
Dec 12, 2016 |
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16004753 |
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62425745 |
Nov 23, 2016 |
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62286621 |
Jan 25, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 2/28 20130101; C12Q
1/22 20130101; G01N 27/221 20130101; C12M 41/46 20130101; C12M
1/3407 20130101 |
International
Class: |
C12Q 1/22 20060101
C12Q001/22; C12M 1/34 20060101 C12M001/34; G01N 27/22 20060101
G01N027/22; A61L 2/28 20060101 A61L002/28 |
Claims
1-116. (canceled)
117. A process for counting test microorganisms on a treated
biological indicator using a capacitance test system comprising a
capacitor and a capacitance bridge, the process comprising: (a)
calibrating the capacitance test system to establish (1) an all
dead capacitance control value using an all dead control biological
indicator containing test microorganisms where all of the test
microorganisms are dead, and (2) an all live capacitance control
value using a live control biological indicator containing test
microorganisms where all of the test microorganisms are alive, the
all dead control biological indicator and the all live control
biological indicator being the same except for the presence of dead
or live test microorganisms, the all dead and all live control
biological indicators having the same estimated number of test
microorganisms; (b) determining the difference between the all live
capacitance control value and the all dead capacitance control
value to obtain a net capacitance control value; (c) dividing the
net capacitance control value by the estimated number of test
microorganisms on the all live control biological indicator to
obtain a capacitance value for each test microorganism; (d)
determining the capacitance value for a treated biological
indicator; (e) determining the difference between the capacitance
value for the treated biological indicator in (d) and the all dead
capacitance control value in (a) to obtain a net capacitance
treated value; and (f) dividing the net capacitance treated value
in (e) by the capacitance value for each test microorganism in (c)
to obtain the number of live test microorganisms on the treated
biological indicator.
118. A process for counting spores on a treated biological
indicator using a capacitance test system comprising a capacitor
and a capacitance bridge, the process comprising: (a) calibrating
the capacitance test system to establish (1) an all dead
capacitance control value using an all dead control biological
indicator containing spores where all of the spores are dead, and
(2) an all live capacitance control value using a live control
biological indicator containing spores where all of the spores are
alive, the all dead control biological indicator and the all live
control biological indicator being the same except for the presence
of dead or live spores, the all dead and all live control
biological indicators having the same estimated number of spores;
(b) determining the difference between the all live capacitance
control value and the all dead capacitance control value to obtain
a net capacitance control value; (c) dividing the net capacitance
control value by the estimated number of spores on the all live
control biological indicator to obtain a capacitance value for each
spore; (d) determining the capacitance value for a treated
biological indicator; (e) determining the difference between the
capacitance value for the treated biological indicator in (d) and
the all dead capacitance control value in (a) to obtain a net
capacitance treated value; and (f) dividing the net capacitance
treated value in (e) by the capacitance value for each spore in (c)
to obtain the number of live spores on the treated biological
indicator.
119-141. (canceled)
142. A process for counting microorganisms on a carrier using a
capacitance test system comprising a capacitor and a capacitance
bridge, the process comprising: (a) establishing a capacitance
value for the carrier; (b) establishing a capacitance value for the
carrier with a control deposit on the carrier of a known quantity
of microorganisms; (c) determining the difference between the
capacitance value in (b) and the capacitance value in (a) to obtain
a net capacitance value for the known quantity of microorganisms in
(b); (d) dividing the net capacitance value for the known quantity
of microorganisms in (c) by the known quantity of microorganisms in
(b) to obtain a capacitance value for each microorganism; (e)
determining a capacitance value for the carrier with a test deposit
of microorganisms on the carrier; (f) determining the difference
between the capacitance value for the carrier with the test deposit
of microorganisms in (e) and the capacitance value for the carrier
in (a) to obtain a net capacitance test value; and (g) dividing the
net capacitance test value in (f) by the capacitance value for each
microorganism in (d) to obtain the number of microorganisms in the
test deposit of microorganisms in (e).
143-146. (canceled)
147. A process for counting spores on a carrier using a capacitance
test system comprising a capacitor and a capacitance bridge, the
process comprising: (a) establishing a capacitance value for the
carrier; (b) establishing a capacitance value for the carrier with
a control deposit on the carrier of a known quantity of spores; (c)
determining the difference between the capacitance value in (b) and
the capacitance value in (a) to obtain a net capacitance value for
the known quantity of spores in (b); (d) dividing the net
capacitance value for the known quantity of spores in (c) by the
known quantity of spores in (b) to obtain a capacitance value for
each spore; (e) determining a capacitance value for the carrier
with a test deposit of spores on the carrier; (f) determining the
difference between the capacitance value for the carrier with the
test deposit of spores in (e) and the capacitance value for the
carrier in (a) to obtain a net capacitance test value; and (g)
dividing the net capacitance test value in (f) by the capacitance
value for each spore in (d) to obtain the number of spores in the
test deposit of spores in (e).
148-164. (canceled)
165. A process for counting microorganisms in a liquid using a
capacitance test system comprising a capacitor and a capacitance
bridge, the process comprising: (a) establishing a capacitance
value for the liquid; (b) establishing a capacitance value for the
liquid in (a) with a control sample of a known quantity of
microorganisms in the liquid; (c) determining the difference
between the capacitance value in (b) and the capacitance value in
(a) to obtain a net capacitance value for the known quantity of
microorganisms in (b); (d) dividing the net capacitance value for
the known quantity of microorganisms in (c) by the known quantity
of microorganisms in (b) to obtain a capacitance value for each
microorganism; (e) determining a capacitance value for the liquid
in (a) with a test sample of microorganisms in the liquid; (f)
determining the difference between the capacitance value for the
liquid with the test sample of microorganisms in (e) and the
capacitance value for the liquid in (a) to obtain a net capacitance
test value; and (g) dividing the net capacitance test value in (f)
by the capacitance value for each microorganism in (d) to obtain
the number of microorganisms in the test sample of microorganisms
in (e).
166-184. (canceled)
185. The process of claim 117 wherein the capacitance bridge has an
accuracy level of about 1 .mu.F or less.
186. The process of claim 117 wherein the capacitor comprises a
dielectric, the capacitance of the dielectric being in the range
from about 0.1 nF to about 20 mF.
187. The process of claim 118 wherein the spores on the all dead
control biological indicator, the all live control biological
indicator, and the treated biological indicator comprise bacterial
spores.
188. The process of claim 118 wherein the spores on the all dead
control biological indicator, the all live control biological
indicator, and the treated biological indicator comprise spores of
the Bacillus or Clostridia genera.
189. The process of claim 118 wherein the spores on the all dead
control biological indicator, the all live control biological
indicator, and the treated biological indicator comprise spores of
Geobacillus stearothermophilus, Bacillus atrophaeus, Bacillus
sphaericus, Bacillus anthracis, Bacillus pumilus, Bacillus
coagulans, Clostridium sporogenes, Clostridium difficile,
Clostridium botulinum, Bacillus subtilis globigii, Bacillus cereus,
Bacillus circulans, or a mixture of two or more thereof.
190. The process of claim 118 wherein the spores on the all dead
control biological indicator, the all live control biological
indicator, and the treated biological indicator comprise
Geobacillus stearothermophilus spores, Bacillus atrophaeus spores,
or a mixture thereof.
191. The process of claim 118 wherein the all dead control
biological indicator, the all live control biological indicator,
and the treated biological indicator comprise spores on a carrier,
the spore population on the carrier for each biological indicator
being in the range from about 500,000 to about 4,000,000
spores.
192. The process of claim 118 wherein the capacitor comprises two
electrical conductors, and the all dead control biological
indicator, the all live control biological indicator and the
treated biological indicator comprise spores on a carrier, the
carrier for each biological indicator comprising paper, plastic,
glass, ceramics, metal foil, one or both conductors of the
capacitor, or a combination of two or more thereof.
193. The process of claim 118 wherein the all dead control
biological indicator, the all live control biological indicator and
the treated biological indicator comprise spores on a carrier, the
carrier for each biological indicator having a length in the range
from about 1 to about 5 cm, a width in the range from about 0.1 to
about 1 cm, and a thickness in the range from about 0.5 to about 3
mm.
194. The process of claim 117 wherein the capacitor comprises
electrical conductors, the electrical conductors comprise aluminum,
copper, silver, gold, platinum, or a combination of two or more
thereof.
195. The process of claim 117 wherein the capacitor comprises
electrical conductors, the electrical conductors comprising indium
tin oxide on glass.
196. The process of claim 117 wherein the capacitor comprises two
electrical conductors, each electrical conductor having a length in
the range from about 1 to about 5 cm, and a width in the range from
about 0.5 to about 3 cm.
197. The process of claim 117 wherein the capacitor comprises two
electrical conductors, the separation between the electrical
conductors being in the range from about 0.5 to about 5 mm.
198. The process of claim 118 wherein all of the spores on the
treated biological indicator are dead.
199. The process of claim 118 wherein some of the spores on the
treated biological indicator are alive, the number of live spores
being in the range from 1 to about 4,000,000.
200. The process of claim 117 wherein the all dead capacitance
control value is in the range from about 0.1 nF to about 20 mF.
201. The process of claim 117 wherein the all live capacitance
control value is in the range from about 0.1 nF to about 20 mF.
202. The process of claim 118 wherein the capacitance value for
each spore is in the range up to about 10 pF.
203. The process of claim 118 wherein live spores are detected
within a period of time of up to about 2000 seconds.
204. The process of claim 118 wherein it is determined that all
spores are dead within a period of time of up to about 2000
seconds.
Description
[0001] This application claims priority under 35 U.S.C. .sctn.
119(e) to U.S. Provisional Application Ser. No. 62/286,621, filed
Jan. 25, 2016, and to U.S. Provisional Application Ser. No.
62/425,745, filed Nov. 23, 2016. These applications are
incorporated herein by reference.
TECHNICAL FIELD
[0002] This invention relates to a capacitor for detecting viable
microorganisms. The capacitor may comprise electrical conductors
and a dielectric. The dielectric may comprise a biological
indicator and an assay medium. The capacitor may be used for
evaluating the efficacy of a sterilization process and for counting
microorganisms.
BACKGROUND
[0003] Biological indicators, which typically comprise test
microorganisms (e.g., spores), are used for evaluating the efficacy
of sterilization processes. The biological indicator is placed in a
sterilization chamber and subjected to a sterilization process
along with the load intended for sterilization (e.g., a medical
device). Following the sterilization process, the biological
indicator is exposed to a growth media and incubated for the
purpose of determining if any of the test microorganisms are
viable. A successful sterilization process is indicated by a
complete inactivation (no outgrowth) of the test microorganisms. An
unsuccessful sterilization process is indicated by an incomplete
inactivation (outgrowth detected) of the test microorganisms.
SUMMARY OF THE INVENTION
[0004] Primarily in the health care industry, but also in many
other commercial and industrial applications, it is often necessary
to monitor the effectiveness of the processes used to sterilize
equipment such as medical and non-medical devices, instruments and
other articles and materials. It is often standard practice in
these sterilization processes to include a biological indicator in
the batch of articles to be sterilized. This allows a direct
approach to assay the lethality of the sterilization process.
[0005] Methods of sterility assurance typically involve exposing a
biological indicator containing test microorganisms to the
sterilization process and then measuring the outgrowth of any
surviving test microorganisms. Sterility may be assured if there is
no outgrowth of the test microorganisms following exposure to the
sterilization process. Bacterial spores (e.g., Geobacillus
stearothermophilus, Bacillus atrophaeus, and the like) may be used
as the test microorganisms. Upon completion of the sterilization
process, the biological indicator is exposed to a growth medium
under conditions that would promote the growth of any surviving
test microorganisms. The growth medium often contains a chemical
dye which changes color in response to actively growing
(metabolizing) cells. Because of the requirement for growth and
metabolism, the processes employing these test microorganisms
typically require about 24 to 72 hours of incubation before the
effectiveness of the sterilization process can be determined. A
problem with this process relates to the fact that many users of
sterilized articles, such as health care facilities and the like,
have limited resources and may reuse the "sterilized" articles
within 24 to 72 hours and sometimes immediately. In such settings,
the 24 to 72 hour holding period for sterility verification may be
impractical, costly and inefficient.
[0006] Thus, a problem in the art relates to determining the
efficacy of a sterilization process within a short period of time.
This invention provides a solution to this problem. With this
invention, the efficacy of a sterilization process can be
determined instantaneously, or within a period of time of up to
about 2000 seconds, or up to about 1500 seconds, or up to about
1000 seconds, or up to about 500 seconds, or up to about 200
seconds, or up to about 100 seconds, or up to about 50 seconds, or
up to about 30 seconds, or in the range from about 5 to about 2000
seconds, or from about 10 to about 1800 seconds, or from about 20
to about 1500 seconds, or from about 30 to about 1200 seconds, or
from about 50 to about 1000 seconds, or from about 60 to about 800
seconds.
[0007] This invention relates to a capacitor comprising two
electrical conductors separated by a dielectric, the dielectric
comprising a biological indicator and an assay medium, the
biological indicator comprising test microorganisms. In an
embodiment, the test microorganisms comprise bacteria. In an
embodiment, the test microorganisms comprise spores. In an
embodiment, the test microorganisms comprise bacterial spores. In
an embodiment, the biological indicator comprises spores on a
carrier. In an embodiment, the biological indicator comprises
bacterial spores on a carrier. In an embodiment, the electrical
conductors comprise metal plates or metal sheets. In an embodiment,
the capacitor is rolled to form a cylinder with an insulating layer
positioned between the electrical conductors. In an embodiment,
electric leads are connected to the electrical conductors. In an
embodiment, the capacitor is connected to a capacitance bridge. The
capacitance bridge may have an accuracy level of about 1 .mu.F or
less. In an embodiment, the dielectric has a capacitance in the
range from about 0.1 nF to about 20 mF, or about 1 to about 5000
nF. In an embodiment, the dielectric comprises from about 500,000
to about 4,000,000 colony forming units of the test microorganisms.
In an embodiment, the dielectric comprises spores, the spores being
on a carrier, the spore population on the carrier being in the
range from about 500,000 to about 4,000,000 spores. In an
embodiment, the test microorganisms are on a carrier, the carrier
comprising paper, plastic, glass, ceramics, metal foil, one or both
conductors of the capacitor, or a combination of two or more
thereof. In an embodiment, each electrical conductor has a length
in the range from about 1 to about 5 cm, a width in the range from
about 0.5 to about 3 cm. In an embodiment, the electrical
conductors are separated by a gap, the separation provided by the
gap being in the range from about 0.5 to about 5 mm. In an
embodiment, the dielectric comprises a liquid.
[0008] This invention relates to a capacitance device, comprising:
a first compartment containing a biological indicator, the
biological indicator comprising test microorganisms, the first
compartment containing two electrical conductors separated by a
gap, the biological indicator being positioned in the gap between
the electrical conductors, the first compartment being adapted to
permit a sterilant to be brought into contact with the biological
indicator during a sterilization process; and a second compartment
containing an assay medium, the second compartment being adapted to
maintain the assay medium separate from the biological indicator
during the sterilization process, and the second compartment being
adapted to permit the assay medium to flow into contact with the
biological indicator after the biological indicator has been
exposed to the sterilant, the biological indicator and the assay
medium forming a dielectric between the electrical conductors. In
an embodiment, the capacitance device is connected to a sensing
apparatus for ascertaining the effectiveness of the sterilization
process. The sensing apparatus may comprise a control unit, an
indicator, and a sensor. In an embodiment, the capacitance device
is connected to a capacitance bridge. The capacitance bridge may
have an accuracy level of about 1 .mu.F or less. In an embodiment,
the capacitance of the dielectric is in the range from about 0.1 nF
to about 20 mF, or about 1 to about 5000 nF. In an embodiment, the
test microorganisms comprise bacteria. In an embodiment, the test
microorganisms comprise spores. In an embodiment, the test
microorganisms comprise bacterial spores. In an embodiment, the
test microorganisms comprise spores on a carrier. The test
microorganism population on the carrier may be in the range from
about 500,000 to about 4,000,000 colony forming units. In an
embodiment, the carrier comprises paper, plastic, glass, ceramics,
metal foil, one or both conductors of the capacitor, or a
combination of two or more thereof. In an embodiment, each
electrical conductor has a length in the range from about 1 to
about 5 cm, and a width in the range from about 0.5 to about 3 cm.
In an embodiment, the separation between the electrical conductors
is in the range from about 0.5 to about 5 mm.
[0009] This invention relates to a process for analyzing a
biological indicator, comprising: placing the biological indicator
and an assay medium in a capacitor, the biological indicator
comprising test microorganisms, the capacitor comprising two
electrical conductors, the biological indicator and the assay
medium being placed between the two conductors and forming a
dielectric for the capacitor; applying an electrical signal to the
conductors; measuring the capacitance of the capacitor; and
determining from the capacitance of the capacitor whether any of
the test microorganisms are alive. In an embodiment, the capacitor
is connected to a capacitance bridge. The capacitance bridge may
have an accuracy level of about 1 .mu.F or less. In an embodiment,
the capacitance of the dielectric is in the range from about 0.1 nF
to about 20 mF, or about 1 to about 5,000 nF. In an embodiment, the
test microorganisms comprise bacteria. In an embodiment, the test
microorganisms comprise spores. In an embodiment, the test
microorganisms comprise bacterial spores. In an embodiment, the
test microorganisms are on a carrier, the test microorganism
population on the carrier being in the range from about 500,000 to
about 4,000,000 colony forming units. In an embodiment, the test
microorganisms comprise spores and the spores are on a carrier. The
spore population on the carrier may be in the range from about
500,000 to about 4,000,000 spores. In an embodiment, the test
microorganisms are on a carrier, the carrier comprising paper,
plastic, glass, ceramics, metal foil, one or both conductors of the
capacitor, or a combination of two or more thereof. In an
embodiment, each electrical conductor has a length in the range
from about 1 to about 5 cm, and a width in the range from about 0.5
to about 3 cm. In an embodiment, the separation between the
electrical conductors provided by the gap is in the range from
about 0.5 to about 5 mm. In an embodiment, all of the test
microorganisms are dead. In an embodiment, some of the test
microorganisms are alive, the number of live test microorganisms
being in the range from 1 to about 4,000,000, or from 1 to about
2,000,000, or from 1 to about 1,000,000, or from 1 to about
100,000, or from 1 to about 50,000, or from 1 to about 10,000
colony forming units.
[0010] This invention relates to a process for determining the
efficacy of a sterilization process, comprising: exposing an
article to be sterilized and a biological indicator to a sterilant,
the biological indicator comprising test microorganisms; placing
the biological indicator and an assay medium in a capacitor, the
capacitor comprising two electrical conductors, the biological
indicator and the assay medium being positioned between the two
electrical conductors and comprising a dielectric for the
capacitor; applying an electrical signal to the conductors;
measuring the capacitance of the capacitor; and determining from
the capacitance of the capacitor whether any of the test
microorganisms are alive. In an embodiment, the sterilant comprises
vaporous hydrogen peroxide, steam, ethylene oxide, peracetic acid,
ozone, ultraviolet light, radiation, or a combination of two or
more thereof. In an embodiment, the capacitor is connected to a
capacitance bridge. The capacitance bridge may have an accuracy
level of about 1 .mu.F or less. In an embodiment, the capacitance
of the dielectric is in the range from about 0.1 nF to about 20 mF,
or about 1 to about 5000 nF. In an embodiment, the test
microorganisms comprise bacteria. In an embodiment, the test
microorganisms comprise spores. In an embodiment, the test
microorganisms comprise bacterial spores. In an embodiment, the
test microorganisms are on a carrier. The test microorganism
population on the carrier may be in the range from about 500,000 to
about 4,000,000 colony forming units. In an embodiment, the carrier
comprises paper, plastic, glass, ceramics, metal foil, one or both
conductors of the capacitor, or a combination of two or more
thereof. In an embodiment, each electrical conductor has a length
in the range from about 1 to about 5 cm, and a width in the range
from about 0.5 to about 3 cm. In an embodiment, the separation
between the electrical conductors is in the range from about 0.5 to
about 5 mm. In an embodiment, all of the test microorganisms are
dead. In an embodiment, some of the test microorganisms are alive,
the number of live test microorganisms being in the range from 1 to
about 4,000,000, or 1 to about 2,000,000, or 1 to about 1,000,000,
or 1 to about 100,000, or from 1 to about 50,000, or from 1 to
about 10,000 colony forming units.
[0011] This invention relates to a process for determining the
efficacy of a sterilization process, comprising: (a) exposing an
article to be sterilized and a biological indicator to a sterilant,
the biological indicator comprising test microorganisms and being
positioned in a capacitor, the capacitor comprising two electrical
conductors, the biological indicator being positioned between the
two electrical conductors and comprising a dielectric for the
capacitor; (b) positioning an assay medium between the electrical
conductors in contact with the biological indicator to form a
dielectric for the capacitor; (c) applying an electrical signal to
the conductors; (d) measuring the capacitance of the capacitor; and
(e) determining from the capacitance of the capacitor whether any
of the test microorganisms are alive. In an embodiment, the
sterilant comprises vaporous hydrogen peroxide, steam, ethylene
oxide, peracetic acid, ozone, ultraviolet light, radiation, or a
combination of two or more thereof. In an embodiment, the capacitor
is connected to a capacitance bridge. The capacitance bridge may
have an accuracy level of about 1 .mu.F or less. In an embodiment,
the capacitance of the dielectric is in the range from about 0.1 nF
to about 20 mF, or about 1 to about 5000 nF. In an embodiment, the
test microorganisms comprise bacteria. In an embodiment, the test
microorganisms comprise spores. In an embodiment, the test
microorganisms comprise spores and the spores comprise bacterial
spores. In an embodiment, the test microorganisms are on a carrier.
The test microorganism population on the carrier may be in the
range from about 500,000 to about 4,000,000 colony forming units.
In an embodiment, the carrier comprises paper, plastic, glass,
ceramics, metal foil, one or both conductors of the capacitor, or a
combination of two or more thereof. In an embodiment, each
electrical conductor has a length in the range from about 1 to
about 5 cm, and a width in the range from about 0.5 to about 3 cm.
In an embodiment, the separation between the electrical conductors
is in the range from about 0.5 to about 5 mm. In an embodiment, all
of the test microorganisms are dead. In an embodiment, some of the
test microorganisms are alive, the number of live test
microorganisms being in the range from 1 to about 4,000,000, or 1
to about 2,000,000, or 1 to about 1,000,000, or 1 to about 100,000,
or from 1 to about 50,000, or from 1 to about 10,000 colony forming
units. In an embodiment, during step (a) the article to be
sterilized and the biological indicator are positioned in an
enclosure while being exposed to the sterilant, and during steps
(b), (c), (d) and (e) the biological indicator is positioned within
the enclosure. In an embodiment, during step (a) the article to be
sterilized and the biological indicator are positioned in an
enclosure while being exposed to the sterilant, and during steps
(b), (c), (d) and (e) the biological indicator is removed from the
enclosure.
[0012] This invention relates to a process for counting test
microorganisms on a treated biological indicator using a
capacitance test system comprising a capacitor and a capacitance
bridge, the process comprising: (a) calibrating the capacitance
test system to establish (1) an all dead capacitance control value
using an all dead control biological indicator containing test
microorganisms where all of the test microorganisms are dead, and
(2) an all live capacitance control value using a live control
biological indicator containing test microorganisms where all of
the test microorganisms are alive, the all dead control biological
indicator and the all live control biological indicator being the
same except for the presence of dead or live test microorganisms,
the all dead and all live control biological indicators having the
same estimated number of test microorganisms; (b) determining the
difference between the all live capacitance control value and the
all dead capacitance control value to obtain a net capacitance
control value; (c) dividing the net capacitance control value by
the estimated number of test microorganisms on the all live control
biological indicator to obtain a capacitance value for each test
microorganism; (d) determining the capacitance value for a treated
biological indicator; (e) determining the difference between the
capacitance value for the treated biological indicator in (d) and
the all dead capacitance control value in (a) to obtain a net
capacitance treated value; and (f) dividing the net capacitance
treated value in (e) by the capacitance value for each test
microorganism in (c) to obtain the number of live test
microorganisms on the treated biological indicator. This invention
relates to a process for counting spores on a treated biological
indicator using a capacitance test system comprising a capacitor
and a capacitance bridge, the process comprising: (a) calibrating
the capacitance test system to establish (1) an all dead
capacitance control value using an all dead control biological
indicator containing spores where all of the spores are dead, and
(2) an all live capacitance control value using a live control
biological indicator containing spores where all of the spores are
alive, the all dead control biological indicator and the all live
control biological indicator being the same except for the presence
of dead and live spores, the all dead and all live control
biological indicators having the same estimated number of spores;
(b) determining the difference between the all live capacitance
control value and the all dead capacitance control value to obtain
a net capacitance control value; (c) dividing the net capacitance
control value by the estimated number of spores on the all live
control biological indicator to obtain a capacitance value for each
spore; (d) determining the capacitance value for a treated
biological indicator; (e) determining the difference between the
capacitance value for the treated biological indicator in (d) and
the all dead capacitance control value in (a) to obtain a net
capacitance treated value; and (f) dividing the net capacitance
treated value in (e) by the capacitance value for each spore in (c)
to obtain the number of live spores on the treated biological
indicator. In an embodiment, the all dead capacitance control value
is higher than the all live capacitance control value. In an
embodiment, the all dead capacitance control value is lower than
the all live capacitance control value. In an embodiment, the
capacitance bridge has an accuracy level of about 1 .mu.F or less.
In an embodiment, the capacitor comprises a dielectric, the
capacitance of the dielectric being in the range from about 0.1 nF
to about 20 mF, or about 1 to about 5,000 nF. In an embodiment, the
spores on the all dead control biological indicator, the all live
control biological indicator, and the treated biological indicator
comprise bacterial spores. In an embodiment, the spores on the all
dead control biological indicator, the all live control biological
indicator, and the treated biological indicator comprise spores of
the Bacillus or Clostridia genera. In an embodiment, the spores on
the all dead control biological indicator, the all live control
biological indicator, and the treated biological indicator comprise
spores of Geobacillus stearothermophilus, Bacillus atrophaeus,
Bacillus sphaericus, Bacillus anthracis, Bacillus pumilus, Bacillus
coagulans, Clostridium sporogenes, Clostridium difficile,
Clostridium botulinum, Bacillus subtilis globigii, Bacillus cereus,
Bacillus circulans, or a mixture of two or more thereof. In an
embodiment, the spores on the all dead control biological
indicator, the all live control biological indicator, and the
treated biological indicator comprise Geobacillus
stearothermophilus spores, Bacillus atrophaeus spores, or a
combination thereof. In an embodiment, the all dead control
biological indicator, the all live control biological indicator,
and the treated biological indicator comprise spores on a carrier,
the spore population on the carrier for each biological indicator
being in the range from about 500,000 to about 4,000,000 spores. In
an embodiment, the capacitor comprises two electrical conductors,
and the all dead control biological indicator, the all live control
biological indicator and the treated biological indicator comprise
spores on a carrier, the carrier for each biological indicator
comprising paper or plastic, glass, ceramics, metal foil, one or
both conductors of the capacitor, or a combination of two or more
thereof. In an embodiment, the all dead control biological
indicator, the all live control biological indicator and the
treated biological indicator comprise spores on a carrier, the
carrier for each biological indicator having a length in the range
from about 1 to about 5 cm, a width in the range from about 0.1 to
about 1 cm, and a thickness in the range from about 0.5 to about 3
mm. In an embodiment, the capacitor comprises electrical
conductors, the electrical conductors being made of aluminum,
copper, silver, gold, platinum, or a combination of two or more
thereof. In an embodiment, the electrical conductors comprise
indium tin oxide (ITO) plates where indium tin oxide is deposited
on glass plates. In an embodiment, the capacitor comprises two
electrical conductors, each electrical conductor having a length in
the range from about 1 to about 5 cm, and a width in the range from
about 0.5 to about 3 cm. In an embodiment, the capacitor comprises
two electrical conductors, the separation between the electrical
conductors being in the range from about 0.5 to about 5 mm. In an
embodiment, the test microorganisms are spores and all of the
spores on the treated biological indicator are dead. In an
embodiment, the test microorganisms are spores and some of the
spores on the treated biological indicator are alive, the number of
live spores being in the range from 1 to about 4,000,000, or 1 to
about 2,000,000, or 1 to about 1,000,000, or 1 to about 100,000, or
from 1 to about 50,000, or from 1 to about 10,000.
[0013] In an embodiment, the all dead capacitance control value is
in the range from about 0.1 nF to about 20 mF. In an embodiment,
the all live capacitance control value is in the range from about
0.1 nF to about 20 mF. In an embodiment, the capacitance value for
each test microorganism or spore is in the range up to about 10 pF.
In an embodiment, live test microorganisms or spores are detected
within a period of time of up to about 2000 seconds. In an
embodiment, it is determined that all test microorganisms or spores
are dead within a period of time of up to about 2000 seconds.
[0014] This invention relates to a method for determining the
efficacy of a sterilization process, said method comprising:
placing spores within a region containing at least one item to be
sterilized; exposing the at least one item and the spores to a
sterilant; after exposure to the sterilant, placing the spores in
an assay medium located between a pair of electrical conductors,
wherein the spores and assay medium serve as a dielectric of a
capacitor; measuring the capacitance of the capacitor; and
determining whether the measured capacitance falls within a first
range of capacitance values indicative of the presence of live
spores or falls within a second range of capacitance values
indicative of the presence of dead spores, wherein the first range
of capacitance values does not overlap with the second range of
capacitance values. In an embodiment, the spores are Geobacillus
stearothermophilus spores, Bacillus atrophaeus spores, or a
combination thereof. In an embodiment, the assay medium comprises
glycerol. In an embodiment, the assay medium comprises about 20% by
volume glycerol in water. In an embodiment, the biological
indicator is an instant read biological indicator. In an
embodiment, the capacitor is a parallel plate capacitor. In an
embodiment, the sterilant is steam.
[0015] This invention relates to a biological indicator comprising:
a capacitive sensor including a capacitor having a pair of
electrical conductors and a dielectric comprised of an assay medium
and a plurality of spores that have been exposed to a sterilant;
and a control unit having a memory pre-stored with data associated
with a first range of capacitance values indicative of the presence
of live spores and data associated with a second range of
capacitance values indicative of the presence of dead spores,
wherein the first range of capacitance values does not overlap with
the second range of capacitance values. In an embodiment, the
spores are Geobacillus stearothermophilus spores, Bacillus
atrophaeus spores, or a combination thereof. In an embodiment, the
assay medium comprises glycerol. In an embodiment, the assay medium
comprises about 20% by volume glycerol in water. In an embodiment,
the biological indicator is an instant read biological indicator.
In an embodiment, the capacitor is a parallel plate capacitor. In
an embodiment, the sterilant is steam.
[0016] This invention relates to a system for determining the
efficacy of a sterilization process, comprising: a plurality of
spores that have been exposed to a sterilant; an assay medium; a
capacitive sensor including a capacitor having a pair of electrical
conductors and a dielectric comprised of the assay medium and the
plurality of spores; and a control unit having a memory pre-stored
with data associated with a first range of capacitance values
indicative of the presence of live spores and data associated with
a second range of capacitance values indicative of the presence of
dead spores, wherein the first range of capacitance values does not
overlap with the second range of capacitance values. In an
embodiment, the spores are Geobacillus stearothermophilus spores,
Bacillus atrophaeus spores, or a combination thereof. In an
embodiment, the assay medium comprises glycerol. In an embodiment,
the assay medium comprises about 20% by volume glycerol in water.
In an embodiment, the biological indicator is an instant read
biological indicator. In an embodiment, the capacitor is a parallel
plate capacitor. In an embodiment, the sterilant is steam.
[0017] This invention relates to a process for counting
microorganisms on a carrier using a capacitance test system
comprising a capacitor and a capacitance bridge, the process
comprising: (a) establishing a capacitance value for the carrier;
(b) establishing a capacitance value for the carrier with a control
deposit on the carrier of a known quantity of microorganisms; (c)
determining the difference between the capacitance value in (b) and
the capacitance value in (a) to obtain a net capacitance value for
the known quantity of microorganisms in (b); (d) dividing the net
capacitance value for the known quantity of microorganisms in (c)
by the known quantity of microorganisms in (b) to obtain a
capacitance value for each microorganism; (e) determining a
capacitance value for the carrier with a test deposit of
microorganisms on the carrier; (f) determining the difference
between the capacitance value for the carrier with the test deposit
of microorganisms in (e) and the capacitance value for the carrier
in (a) to obtain a net capacitance test value; and (g) dividing the
net capacitance test value in (f) by the capacitance value for each
microorganism in (d) to obtain the number of microorganisms in the
test deposit of microorganisms in (e). Those skilled in the art
will recognize that the carrier referred to in steps (a), (b) and
(e) may not be the exact same carrier for each step, but each will
at least be identical or comparable samples of the same carrier. In
an embodiment, the known quantity of microorganisms in (b) is in
the range from about 500,000 to about 4,000,000 colony forming
units. In an embodiment, the number of microorganisms in the test
deposit of microorganisms in (g) is in the range from 1 to about
4,000,000 colony forming units. In an embodiment, the capacitance
value for the carrier with the control deposit of the known
quantity of microorganisms in (b) is in the range from about 0.1 nF
to about 20 mF. In an embodiment, the capacitance value for each
microorganism in (d) is up to about 10 pF, or in the range from
about 0.05 to about 2 pF.
[0018] This invention relates to a process for counting spores on a
carrier using a capacitance test system comprising a capacitor and
a capacitance bridge, the process comprising: (a) establishing a
capacitance value for the carrier; (b) establishing a capacitance
value for the carrier with a control deposit on the carrier of a
known quantity of spores; (c) determining the difference between
the capacitance value in (b) and the capacitance value in (a) to
obtain a net capacitance value for the known quantity of spores in
(b); (d) dividing the net capacitance value for the known quantity
of spores in (c) by the known quantity of spores in (b) to obtain a
capacitance value for each spore; (e) determining a capacitance
value for the carrier with a test deposit of spores on the carrier;
(f) determining the difference between the capacitance value for
the carrier with the test deposit of spores in (e) and the
capacitance value for the carrier in (a) to obtain a net
capacitance test value; and (g) dividing the net capacitance test
value in (f) by the capacitance value for each spore in (d) to
obtain the number of spores in the test deposit of spores in (e).
Those skilled in the art will recognize that the carrier referred
to in steps (a), (b) and (e) may not be the exact same carrier for
each step, but each will at least be identical or comparable
samples of the same carrier. In an embodiment, the capacitance
bridge has an accuracy level of about 1 .mu.F or less. In an
embodiment, the capacitor comprises a dielectric, the capacitance
of the dielectric being in the range from about 0.1 nF to about 20
mF. In an embodiment, the spores comprise bacterial spores. In an
embodiment, the spores comprise spores of the Bacillus or
Clostridia genera. In an embodiment, the spores comprise spores of
Geobacillus stearothermophilus, Bacillus atrophaeus, Bacillus
sphaericus, Bacillus anthracis, Bacillus pumilus, Bacillus
coagulans, Clostridium sporogenes, Clostridium difficile,
Clostridium botulinum, Bacillus subtilis globigii, Bacillus cereus,
Bacillus circulans, or a mixture of two or more thereof. In an
embodiment, the spores comprise Geobacillus stearothermophilus
spores, Bacillus atrophaeus spores, or a mixture thereof. In an
embodiment, the known quantity of spores in (b) is in the range
from about 500,000 to about 4,000,000 spores. In an embodiment, the
carrier comprises paper, plastic, glass, ceramics, metal foil, one
or both conductors of the capacitor, or a combination of two or
more thereof. In an embodiment, the carrier has a length in the
range from about 1 to about 5 cm, a width in the range from about
0.1 to about 1 cm, and a thickness in the range from about 0.5 to
about 3 mm. In an embodiment, the capacitor comprises electrical
conductors, the electrical conductors comprise aluminum, copper,
silver, gold, platinum, or a combination of two or more thereof. In
an embodiment, the capacitor comprises electrical conductors, the
electrical conductors comprising indium tin oxide on glass. In an
embodiment, the capacitor comprises two electrical conductors, each
electrical conductor having a length in the range from about 1 to
about 5 cm, and a width in the range from about 0.5 to about 3 cm.
In an embodiment, the capacitor comprises two electrical
conductors, the separation between the electrical conductors being
in the range from about 0.5 to about 5 mm. In an embodiment, the
number of spores in the test deposit of spores is in the range from
1 to about 4,000,000. In an embodiment, the capacitance value for
the carrier is in the range from about 0.1 nF to about 20 mF. In an
embodiment, the capacitance value for the carrier with the control
deposit of the known quantity of spores in (b) is in the range from
about 0.1 nF to about 20 mF. In an embodiment, the capacitance
value for each spore is in the range up to about 10 pF, or from
about 0.05 to about 2 pF.
[0019] With this invention, it is possible to determine whether
live test microorganisms (e.g., spores) are present on a biological
indicator that has been subjected to a sterilization, and if so,
how many. The determination of whether live test microorganisms or
spores are present can be determined instantaneously, or within a
period of time of up to about 2000 seconds, or up to about 1500
seconds, or up to about 1000 seconds, or up to about 500 seconds,
or up to about 200 seconds, or up to about 100 seconds, or up to
about 50 seconds, or up to about 30 seconds, or in the range from
about 5 to about 2000 seconds, or from about 10 to about 1800
seconds, or from about 20 to about 1500 seconds, or from about 30
to about 1200 seconds, or from about 50 to about 1000 seconds, or
from about 60 to about 800 seconds.
[0020] This invention relates to a process for counting
microorganisms in a liquid using a capacitance test system
comprising a capacitor and a capacitance bridge, the process
comprising: (a) establishing a capacitance value for the liquid;
(b) establishing a capacitance value for the liquid in (a) with a
control sample of a known quantity of microorganisms in the liquid;
(c) determining the difference between the capacitance value in (b)
and the capacitance value in (a) to obtain a net capacitance value
for the known quantity of microorganisms in (b); (d) dividing the
net capacitance value for the known quantity of microorganisms in
(c) by the known quantity of microorganisms in (b) to obtain a
capacitance value for each microorganism; (e) determining a
capacitance value for the liquid in (a) with a test sample of
microorganisms in the liquid; (f) determining the difference
between the capacitance value for the liquid with the test sample
of microorganisms in (e) and the capacitance value for the liquid
in (a) to obtain a net capacitance test value; and (g) dividing the
net capacitance test value in (f) by the capacitance value for each
microorganism in (d) to obtain the number of microorganisms in the
test sample of microorganisms in (e). In an embodiment, the
capacitor comprises two electrical conductors and during step (e)
the test sample of microorganism is positioned between the
conductors and forms a dielectric for the capacitor. In an
embodiment, the capacitor comprises two electrical conductors and
during step (e) the test sample of microorganism flows between the
conductors and forms a dielectric for the capacitor. In an
embodiment, the concentration of microorganisms in the test sample
of microorganism in the liquid in (e) is determined by dividing the
number of microorganisms in the test sample of microorganisms in
(e) by the volume of the liquid in (e). In an embodiment, the known
quantity of microorganisms in (b) is in the range from about
500,000 to about 4,000,000 colony forming units. In an embodiment,
the number of microorganisms in the test sample of microorganisms
in (g) is in the range from 1 to about 4,000,000 colony forming
units. In an embodiment, the capacitance value for the liquid with
the control sample of the known quantity of microorganisms in (b)
is in the range from about 0.1 nF to about 20 mF. In an embodiment,
the capacitance value for each microorganism in (d) is in the range
up to about 10 pF. In an embodiment, the capacitance bridge has an
accuracy level of about 1 .mu.F or less. In an embodiment, the
microorganisms comprise bacteria, archaea, protozoa, fungi, algae,
virus, hetminths, or a combination of two or more thereof. In an
embodiment, the microorganisms comprise bacteria. In an embodiment,
the microorganisms comprise bacterial spores. In an embodiment, the
spores comprise spores of the Bacillus or Clostridia genera. In an
embodiment, the spores comprise spores of Geobacillus
stearothermophilus, Bacillus atrophaeus, Bacillus sphaericus,
Bacillus anthracis, Bacillus pumilus, Bacillus coagulans,
Clostridium sporogenes, Clostridium difficile, Clostridium
botulinum, Bacillus subtilis globigii, Bacillus cereus, Bacillus
circulans, or a mixture of two or more thereof. In an embodiment,
the spores comprise Geobacillus stearothermophilus spores, Bacillus
atrophaeus spores, or a mixture thereof. In an embodiment, the
microorganisms comprise yeast or lactobacillus microorganisms. In
an embodiment, the capacitor comprises electrical conductors, the
electrical conductors comprise aluminum, copper, silver, gold,
platinum, or a combination of two or more thereof. In an
embodiment, the capacitor comprises electrical conductors, the
electrical conductors comprising indium tin oxide on glass. In an
embodiment, the capacitor comprises two electrical conductors, each
electrical conductor having a length in the range from about 1 to
about 5 cm, and a width in the range from about 0.5 to about 3 cm.
In an embodiment, the capacitor comprises two electrical
conductors, the separation between the electrical conductors being
in the range from about 0.5 to about 5 mm.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In the annexed drawings, like parts and features have like
designations.
[0022] FIG. 1 is a sectional view taken from the side of a
capacitor configured with a sensing apparatus according to an
embodiment of the present invention.
[0023] FIG. 2 is a schematic diagram of an exemplary capacitive
sensor for determining the efficacy of a sterilization process,
according to an embodiment.
[0024] FIG. 3 is a schematic diagram illustrating an exemplary
capacitive sensor for determining the efficacy of a sterilization
process, according to another embodiment.
[0025] FIG. 4 is a schematic diagram of an exemplary capacitive
sensor for determining the efficacy of a sterilization process,
according to another embodiment.
[0026] FIG. 5 is a bar graph showing capacitance levels obtained in
Example 1.
[0027] FIG. 6 is a perspective view of a capacitance device which
can be used in accordance with the present invention.
[0028] FIG. 7 is a cross-sectional view of the capacitance device
of FIG. 6 showing a cap mounted on the capacitor in a first
non-activated position;
[0029] FIG. 8 is a cross-sectional view of the capacitance device
of FIG. 6 showing the cap mounted on the capacitor in a second
activated position, the capacitor being configured with a sensing
apparatus according to an embodiment of the present invention.
DETAILED DESCRIPTION
[0030] All ranges and ratio limits disclosed in the specification
and claims may be combined in any manner. It is to be understood
that unless specifically stated otherwise, references to "a," "an,"
and/or "the" may include one or more than one, and that reference
to an item in the singular may also include the item in the
plural.
[0031] The phrase "and/or" should be understood to mean "either or
both" of the elements so conjoined, i.e., elements that are
conjunctively present in some cases and disjunctively present in
other cases. Other elements may optionally be present other than
the elements specifically identified by the "and/or" clause,
whether related or unrelated to those elements specifically
identified unless clearly indicated to the contrary. Thus, as a
non-limiting example, a reference to "A and/or B," when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A without B (optionally including
elements other than B); in another embodiment, to B without A
(optionally including elements other than A); in yet another
embodiment, to both A and B (optionally including other elements);
etc.
[0032] The word "or" should be understood to have the same meaning
as "and/or" as defined above. For example, when separating items in
a list, "or" or "and/or" shall be interpreted as being inclusive,
i.e., the inclusion of at least one, but also including more than
one, of a number or list of elements, and, optionally, additional
unlisted items. Only terms clearly indicated to the contrary, such
as "only one of" or "exactly one of," may refer to the inclusion of
exactly one element of a number or list of elements. In general,
the term "or" as used herein shall only be interpreted as
indicating exclusive alternatives (i.e. "one or the other but not
both") when preceded by terms of exclusivity, such as "either,"
"one of," "only one of," or "exactly one of."
[0033] The phrase "at least one," in reference to a list of one or
more elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0034] The transitional words or phrases, such as "comprising,"
"including," "carrying," "having," "containing," "involving,"
"holding," and the like, are to be understood to be open-ended,
i.e., to mean including but not limited to.
[0035] The term "capacitor" refers to a two-terminal electrical
component used to store electrical energy temporarily. The
capacitor provided by the present invention comprises two
electrical conductors separated by a dielectric.
[0036] The term "dielectric" refers to an electrical insulator that
can be polarized by an applied electrical field. When a dielectric
is placed on an electrical field, electric charges do not flow
through the material as they do a conductor, but only slightly
shift from their average equilibrium positions causing dielectric
polarization. The dielectric may comprise microorganisms. The
dielectric may comprise microorganisms in combination with an assay
fluid. The dielectric may comprise test microorganisms. The
dielectric may comprise bacteria. The dielectric may comprise
spores. The dielectric may comprise a biological indicator. The
dielectric may comprise a biological indicator in combination with
an assay medium.
[0037] The term "microorganism" refers to a microscopic living
organism. The microorganisms may be unicellular, multicellular, or
in the form of cell clusters. The microorganisms may comprise
bacteria, archaea, protozoa, fungi, algae, viruses, multicellular
animal parasites (helminths), or a combination of two or more
thereof. The microorganisms may comprise spores. The microorganisms
may comprise bacterial spores. The microorganisms may comprise
spores of the Bacillus or Clostridia genera. The microorganisms may
comprise spores of Geobacillus stearothermophilus, Bacillus
atrophaeus, Bacillus sphaericus, Bacillus anthracis, Bacillus
pumilus, Bacillus coagulans, Clostridium sporogenes, Clostridium
difficile, Clostridium botulinum, Bacillus subtilis globigii,
Bacillus cereus, Bacillus circulans, or a mixture of two or more
thereof. The microorganisms may comprise Geobacillus
stearothermophilus spores, Bacillus atrophaeus spores, or a mixture
thereof. The microorganisms may comprise yeast or lactobacillus
microorganisms. In an embodiment, the term "microorganism" does not
include red or white blood cells (e.g., bovine blood).
[0038] The term "bacteria" refers to a domain of prokaryotic
microorganisms. The bacteria may be unicellular microorganisms. The
cells may be described as prokaryotic because they lack a nucleus.
The bacteria cells may have one of four major shapes: bacillus (rod
shaped), coccus (spherical shape), spirilla (spiral shape), or
vibrio (curved shape). The bacteria may have a peptidoglycan wall.
The bacteria may divide by bacteria fission. The bacteria may
possess flagella for motility. The bacteria may be classified as
either Gram-positive or Gram-negative when using Gram staining. The
bacteria may be divided based on their response to gaseous oxygen
into the following groups: aerobic (living in the presence of
oxygen), anaerobic (living without oxygen), and facultative
anaerobic (can live in both environments). The bacteria may be
classified as heterotrophs or autotrophs. Autotrophs make their own
food by using the energy of sunlight or chemical reactions, in
which case they are called chemoautotrophs. Heterotrophs obtain
their energy by consuming other organisms. The bacteria that use
decaying life forms as a source of energy may be called
saprophytes.
[0039] The term "spore" refers to a unit of asexual reproduction
that may be adapted for dispersal and survival for extended periods
of time under unfavorable conditions. Spores are highly resistant,
dormant cell types. Endospores (or simply spores) form within the
vegetative mother cell in response to adverse changes in the
environment, most commonly nutrient depletion. The mother cell
undergoes an asymmetrical cell division, where it replicates its
genetic material, which is then surrounded by multiple concentric
and spore specific layers. The mother cell then disintegrates,
releasing the mature dormant spore which requires neither
nutrients, water nor air for survival and is protected against a
variety of trauma, including extremes of temperature, radiation,
and chemical assault.
[0040] The term "bacterial spore" refers to a spore produced by
bacteria.
[0041] The term "test microorganism" refers to a microorganism that
may be used to test the efficacy of a sterilization process. The
test microorganism may be more resistant to a sterilization process
than the organisms intended for destruction during the
sterilization process. In theory, if the test microorganisms were
to die during a sterilization process, then all organisms intended
for destruction during the sterilization process that are less
resistant to the sterilization than the test microorganisms would
also die. The test microorganisms may comprise bacteria. The test
microorganisms may comprise spores. The test microorganisms may
comprise bacterial spores. The test microorganisms may comprise
spores of the Bacillus or Clostridia genera. The test
microorganisms may comprise spores of Geobacillus
stearothermophilus, Bacillus atrophaeus, Bacillus sphaericus,
Bacillus anthracis, Bacillus pumilus, Bacillus coagulans,
Clostridium sporogenes, Clostridium difficile, Clostridium
botulinum, Bacillus subtilis globigii, Bacillus cereus, Bacillus
circulans, or a mixture of two or more thereof. The test
microorganisms may comprise Geobacillus stearothermophilus spores,
Bacillus atrophaeus spores, or a mixture thereof.
[0042] The term "biological indicator" refers to an article or a
material that can be used to determine the efficacy of a
sterilization process. The biological indicator may comprise test
microorganisms (e.g., bacteria, spores or bacterial spores). The
biological indicator may comprise test microorganisms on a carrier.
The biological indicator may comprise bacteria, the bacteria may be
present within a defined space or deposited on a carrier. The
biological indicator may comprise spores (e.g., bacterial spores),
the spores may be present within a defined space or on a carrier.
The biological indicator may comprise a spore strip.
[0043] The term "carrier" refers to a support onto which
microorganisms may be deposited.
[0044] The term "killing" microorganisms or spores refers to
rendering microorganisms or spores incapable of reproduction,
metabolism and/or growth. The term "dead" microorganisms or spores
refers to microorganisms or spores which have been rendered
incapable of reproduction, metabolism and/or growth. The
microorganisms or spores used with a biological indicator may be
selected from those that would be more resistant to a sterilization
process for which they are intended to monitor than the organisms
to be killed by the sterilization process. The killing of the
microorganisms or spores of the biological indicator during a
sterilization process is indicative of a successful sterilization
process.
[0045] The term "live" microorganisms or spores refers to
microorganisms or spores that are capable of reproduction,
metabolism and/or growth.
[0046] The term "Farad" (F) refers to a unit of electrical
capacitance. Electrical capacitance is a measure of the ability of
a body to store an electrical charge. One Farad is the capacitance
across which, when charged with one coulomb, there is a potential
difference of one volt. For many applications, the Farad is an
impractically large unit of capacitance. As such, for many
electrical and electronics applications, the following prefixes are
used: 1 mF (milli Farad)=10.sup.-3 Farad; 1 .mu.F (micro
Farad)=10.sup.-6 Farad; 1 nF (nano Farad)=10.sup.-9 Farad; 1 pF
(pico Farad)=10.sup.-12 Farad; 1 fF (femto Farad)=10.sup.15 Farad;
and 1 aF (atto Farad)=10.sup.-18 Farad.
[0047] The term "log reduction" is a mathematical term to show the
number of live microorganisms or spores killed by contacting the
microorganisms or spores with a sterilant during a sterilization
process. A "4 log reduction" means that the number of live
microorganisms or spores at the end of the sterilization process is
reduced by 10,000-fold. A "5 log reduction" means that the number
of live microorganisms or spores is reduced by 100,000-fold. A "6
log reduction" means that the number of live microorganisms or
spores is reduced by 1,000,000-fold. Thus, for example, if a
carrier has 1,000,000 live microorganisms or spores on it, a 6-log
reduction would reduce the number of live microorganisms or spores
to 1.
[0048] The term "sterilization" may be used to refer to a process
wherein there is a total absence of living test microorganisms
remaining after the sterilization process has been completed.
However, processes that are less rigorous than sterilization
processes including, for example, disinfection, sanitization,
decontamination, cleaning processes, and the like, may be of value
and are taken into account with this invention. Unless otherwise
indicated, the term "sterilization" is used herein to refer to
sterilization processes as well as less rigorous processes such as
disinfection, sanitation, decontamination, cleaning, and the
like.
[0049] The term "sterilant" refers to any medium or energy that can
be used to sterilize a substrate (e.g., a medical device, the
interior of a room, etc.). The sterilant may comprise a liquid or a
gas. The sterilant may comprise vaporous hydrogen peroxide, steam,
ethylene oxide, peracetic acid, ozone, or a combination of two or
more thereof. The sterilant may comprise ultraviolet light or
radiation. The radiation may comprise x-ray radiation, gamma
radiation, or electron beam radiation.
[0050] The sterilization process provided for herein may employ any
sterilant. The sterilization process may be conducted for an
effective period of time to achieve at least a 4 log reduction, or
at least a 5 log reduction, or at least a 6 log reduction in the
number of test microorganisms capable of reproduction, metabolism
and/or growth. When at least a 6 log reduction is achieved, the
process may be referred to as a sterilization process. When a 4 log
reduction or a 5 log reduction is achieved, the process may be
considered to be less rigorous than a sterilization process, but
nevertheless useful for various disinfection, sanitization,
decontamination and/or cleaning applications.
[0051] The biological indicator may comprise test microorganisms
(e.g., spores) deposited on a carrier. The test microorganism
population for the biological indicator may be in the range from
about 500,000 to about 4,000,000 colony forming units (cfu), or
from about 500,000 to about 2,500,000 cfu, or from about 500,000 to
about 1,500,000 cfu, or from about 750,000 to about 1,200,000 cfu,
or about 10.sup.6 cfu. If the test microorganisms are spores, the
spore population for the biological indicator may be in the range
from about 500,000 to about 4,000,000 spores, or from about 500,000
to about 2,500,000 spores, or from about 500,000 to about 1,500,000
spores, or from about 750,000 to about 1,200,000 spores. The spore
population may be about 10.sup.6 spores. The biological indicator
may be referred to as a spore test strip.
[0052] The spores may comprise bacterial spores. These may include
spores of the Bacillus or Clostridia genera. The spores may be
spores of Geobacillus stearothermophilus, Bacillus atrophaeus,
Bacillus sphaericus, Bacillus anthracis, Bacillus pumilus, Bacillus
coagulans, Clostridium sporogenes, Clostridium difficile,
Clostridium botulinum, Bacillus subtilis globigii, Bacillus cereus,
Bacillus circulans, or a combination of two or more thereof. The
spores may comprise spores of Geobacillus stearothermophilus,
Bacillus atrophaeus, or a combination thereof.
[0053] The carrier may comprise a strip, sheet or film of any
material that does not dissolve or deteriorate during the
sterilization processes. The carrier may comprise a paper strip,
e.g., a cellulose strip, or a plastic sheet or film. The plastic
may comprise a polyolefin, polystyrene, polycarbonate,
polymethacrylate, polyacrylamide, polyimide, polyester, or a
combination of two or more thereof. The carrier may comprise glass,
ceramics, metal foil, or a combination of two or more thereof. The
carrier may comprise one or both conductors of the capacitor. The
carrier may have a length in the range of about 1 to about 5 cm, or
about 2 to about 4 cm; a width in the range from about 0.1 to about
1 cm, or about 0.4 to about 0.7 cm; and a thickness in the range
from about 0.2 to about 3 mm, or from about 0.5 to about 1.5
mm.
[0054] The biological indicator may comprise a spore test strip.
These may include Geobacillus stearothermophilus test strips for
use in monitoring steam sterilizations; Bacillus atrophaeus test
strips for monitoring ethylene oxide and dry heat sterilizations;
Bacillus pumilus test strips for irradiation sterilizations;
combined species spore test strips, G. stearothermophilus and B.
atrophaeus, for monitoring steam, ethylene oxide and dry heat
sterilizations; and the like. These test strips may be
characterized by spore populations in the range from about 500,000
to about 4,000,000 spores, or from about 500,000 to about 2,500,000
spores, or from about 500,000 to about 1,500,000 spores, or from
about 750,000 to about 1,200,000 spores per test strip, or about
10.sup.6 spores per test strip.
[0055] The biological indicator may comprise a VERIFY.RTM. Spore
Test Strip for 540.RTM. Sterilant Concentrate supplied by STERIS
Corporation. This test strip may be used for monitoring liquid
chemical sterilizations, e.g., peracetic acid sterilizations. These
test strips are characterized by spore populations of at least
about 10.sup.5 Geobacillus stearothermophilus spores per test
strip.
[0056] The capacitor may comprise a passive two-terminal electrical
component that has two electrical conductors (plates) separated by
a dielectric. The plate area of the capacitor may be in the range
from about 0.5 to about 15 cm.sup.2, or about 1 to about 10
cm.sup.2. The gap between the plates, or the plate separation, may
be in the range from about 0.5 to about 5 mm, or from about 1 to
about 3 mm. The plates may comprise aluminum, copper, silver, gold,
platinum, indium tin oxide deposited on glass, or a combination of
two or more thereof. The dielectric may comprise the biological
indicator in combination with an assay medium. The biological
indicator may comprise a spore test strip.
[0057] The assay medium may comprise any fluid (e.g., gas or
liquid) that can be combined with the microorganisms, test
microorganisms, spores, or biological indicator to form a
dielectric for the capacitor. The assay medium may comprise any
liquid or gas having a dielectric constant in the range from 1 to
about 90, or from about 5 to about 85, or from about 10 to about
80, measured at a temperature in the range from about -10.degree.
C. to about 60.degree. C., or about 0.degree. C. to about
50.degree. C., or about 0.degree. C. to about 40.degree. C. The
assay medium may comprise air, one or more solvents (e.g., water,
dimethyl sulfoxide, deuterium oxide), one or more alcohols or
polyols (e.g., methyl alcohol, ethyl alcohol, isopropyl alcohol,
butyl alcohol, isoamyl alcohol, hexyl alcohol, octyl alcohol,
phenol, biphenyl, benzyl alcohol, creosol, glycol, pentandiol,
glycerol), aldehydes (e.g., acetaldehyde, benzaldehyde,
butaldehyde, butraldehyde, saliylaldehyde), ketones (e.g., acetone,
methylethyl ketone, diethyl ketone, heptone, benzophenone, benzoyl
acetone, chloroacetone, cyclohexanone, hexanone), hydrocarbons and
halogen substituted hydrocarbons (e.g., chloromethane,
bromomethane, benzyl chloride, cyclohexane, cyclohexene,
cyclopentane) nitrogenous compounds (e.g., acetonitrile,
nitrotoluene, butronitrile, lactonitrile, ammonia, formamide,
hydrazine, nitrobenzene, pyridine, proprionitrile, nitrobenzene),
anhydrides (e.g., maleic anhydride, butyric anhydride, acetic
anhydride), oils (e.g., castor oil), acetates and cyanoacetates
(e.g., methylscyanoacetate, methylchloroacetate, ethyl
acetoacetate, cyanoethylacetate), thiocyanates (e.g.,
ethylthiocyanate, amylthiocyanate), hydrocyanic acid, hydrogen
peroxide, trifluoroacetic acid, lactic acid, dichloracetic acid, or
a mixture of two or more thereof. The assay medium may comprise a
glycerol in water solution (e.g., 20% by volume glycerol in water).
The assay medium, when combined with the microorganisms or
biological indicator may be used in an effective amount to fill the
gap between electrical conductors of the capacitor.
[0058] In an embodiment, the biological indicator (after it has
been exposed to a sterilization process) may be combined with an
additional sheet of carrier material (e.g., capacitor paper), two
sheets of metal, and an insulating layer, to form a capacitor. The
biological indicator may comprise test microorganisms on a carrier,
e.g., a spore test strip. The biological indicator may have a
thickness of about 0.2 to about 3 mm, or about 0.5 to about 1.5 mm,
or about 1 mm. The additional sheet of carrier material may be
placed over the biological indicator to cover the test
microorganisms. The thickness of the additional sheet of carrier
material may be from about 0.0001 to about 0.01 mm, or about 0.001
to about 0.008 mm, or about 0.005 mm. The combined thickness of the
biological indicator and the additional sheet of carrier material
may be in the range from about 0.21 to about 3.1 mm, or about 0.5
to about 1.5 mm, or about 1 mm. The biological indicator and the
additional sheet of carrier material may be square or rectangular
in shape with lengths in the range from about 1 to about 5 cm, and
widths in the range from about 0.1 to about 1 cm. The biological
indicator and additional sheet of carrier material may be placed
between the two sheets of metal (e.g., aluminum, copper, gold,
silver, platinum, or a combination of two or more thereof) which
may be used as electrical conductors. The two sheets of metal may
each comprise a metal foil. The two metal sheets may be square or
rectangular in shape with lengths of about 1 to about 5 cm, and
widths of about 0.5 to about 3 cm. The metal sheets may have
thicknesses in the range from about 0.001 to about 0.02 mm, or
about 0.003 to about 0.006 mm. The insulating layer may be
constructed of paper, a polymer, an elastomer, or a combination of
two or more thereof. The insulating layer may have a thickness in
the range from about 0.1 to about 5 mm, or about 0.5 to about 1.5
mm. The insulating layer may be square or rectangular in shape with
lengths in the range from about 1 to about 5 cm, and widths of
about 0.1 to about 1 cm. The biological indicator and the
additional sheet of carrier material may be placed between the two
sheets of metal, and the resulting construction may then be rolled
with the insulating layer positioned between the metal sheets to
form a capacitor. The insulating material may be used to avoid
shorting. Electrical leads may be placed in contact with the metal
sheets.
[0059] The capacitor may be connected to a capacitance bridge to
detect capacitance levels for the biological indicator. The
capacitance bridge may be any capacitance bridge that may detect
capacitance levels of about 0.1 nF to about 20 mF, or about 1 to
about 5,000 nF, or about 10 to about 2,000 nF, or about 1,500 nF or
less. An example of capacitance bridge that may be used is
available from Andeen-Hagerling under the trade designation
AH2700A. The AH2700A bridge is identified as a 50 Hz-20 kHz
capacitance/loss bridge. The AH2700A bridge has the following
precision specifications:
TABLE-US-00001 Temperature Frequency Accuracy Stability Coefficient
Resolution kHz ppm ppm/year ppm/.degree. C. aF ppm 0.1 .+-.9
.+-.<1.9 .+-.0.07 16 0.8 1 .+-.5 .+-.<1.0 .+-.0.035 0.8 0.16
10 .+-.11 .+-.<1.9 .+-.0.07 2.4 0.5
[0060] The capacitance of the biological indicator can be measured
after a sterilization process to determine whether any spores
survive the sterilization process and, if so, how many spores
survived. The capacitance level readings may be used to determine
if all spores are killed, or if 1, 2, 3, etc., spores survived the
sterilization process.
[0061] For some applications, it may be sufficient to use the
capacitor to determine whether all test microorganisms (e.g.,
spores) of the biological indicator have been killed, or whether
any test microorganisms remain alive following a sterilization
process. For other applications, it may be of value to count the
number of test microorganisms, if any, that survive a sterilization
process. With this invention it is possible not to only determine
whether or not all test microorganisms of the biological indicator
have been killed, but also count the number of test microorganisms
that survive a sterilization process and thereby determine what
level of sterilization (or disinfection, sanitation,
decontamination and/or cleaning) is achieved.
[0062] Since results may vary depending on the particular
biological indicator and capacitor being used, a "control" can be
programmed into the software used in the control unit (discussed
below) where the results for the specific biological indicator
(e.g., a known commercial spore strip) being used, where all test
microorganisms are dead, and results for the specific capacitor
being used, are stored. By comparing the results for the tested
biological indicator and capacitor being used to the control, a
capacitance reading can be obtained that can be translated into a
reading of the number of live test microorganisms, if any, on the
biological indicator being tested.
[0063] The number of live test microorganisms, if any, on a treated
biological indicator can be determined by the process indicated
below. With this process a capacitance test system comprising a
capacitor and a capacitance bridge is used. The capacitance test
system is initially calibrated using all dead and all live control
biological indicators which contain either all dead or all live
test microorganisms. The system is then used to evaluate a treated
biological indicator which has been subjected to a sterilization.
The process involves the following steps: (a) calibrating the
capacitance test system to establish (1) an all dead capacitance
control value using an all dead control biological indicator
containing test organisms or spores where all of the test organisms
or spores are dead, and (2) an all live capacitance control value
using a live control biological indicator containing test
microorganisms or spores where all of the test microorganisms or
spores are alive, the all dead control biological indicator and the
all live control biological indicator being the same except for the
presence of dead or live test microorganisms or spores, the all
dead and all live control biological indicators having the same
estimated number of test microorganisms or spores; (b) determining
the difference between the all live capacitance control value and
the all dead capacitance control value to obtain a net capacitance
control value; (c) dividing the net capacitance control value by
the estimated number of test microorganisms or spores on the all
live control biological indicator to obtain a capacitance value for
each test microorganism or spore; (d) determining the capacitance
value for a treated biological indicator; (e) determining the
difference between the capacitance value for the treated biological
indicator in (d) and the all dead capacitance control value in (a)
to obtain a net capacitance treated value; (f) dividing the net
capacitance treated value in (e) by the capacitance value for each
test microorganisms or spore in (c) to obtain the number of live
test microorganisms or spores on the treated biological
indicator.
[0064] In performing the above-indicated test procedure, the same
biological indicator (e.g., spore strip) type used to calibrate the
capacitance test system (i.e., the dead and live control biological
indicators) is also used as the treated biological indicator (e.g.,
treated spore strip), the treated biological indicator having been
subjected to a sterilization. Thus, for example, if VERIFY.RTM.
Spore Test Strip for 540.RTM. Sterilant Concentrate supplied by
STERIS Corporation are used as the dead and live control biological
indicators, then a VERIFY.RTM. Spore Test Strip for 540.RTM.
Sterilant Concentrate supplied by STERIS Corporation will also be
used as the treated biological indicator.
[0065] The all dead capacitance control value may be from about 0.1
nF to about 20 mF, or from about 1 to about 5,000 nF, or from about
100 to about 2,000 nF, or about 1000 nF. The all live capacitance
control value may be from about 0.1 nF to about 20 mF, or about 1
to about 5000 nF, or from about 100 to about 1,000 nf, or about 600
nF. The capacitance value for each test microorganism or spore may
be up to about 10 pF, or from about 0.05 pF to about 2 pF, or from
about 0.1 to about 1 pF, or about 0.3 pF.
[0066] For many sterilizations, the ideal is that no test
microorganisms or spores survive the sterilization process.
However, if any test microorganisms survive, this process can be
used to detect the number that survive. Even if the test
microorganisms have not been subjected to a sterilization process,
they can nevertheless be counted using the inventive method. This
may be applicable to other processes, for example, counting
microorganisms in a liquid (e.g., milk, beer, etc.). The number of
microorganisms that may be detected and counted may be, for
example, from 1 to about 4,000,000 colony forming units (cfu), or
from 1 to about 3,000,000 cfu, or from 1 to about 2,000,000 cfu, or
from 1 to about 1,000,000 cfu, or from 1 to about 500,000 cfu, or
from 1 to about 200,000 cfu, or from 1 to about 100,000 cfu, or
from 1 to about 50,000 cfu, or from 1 to about 10000 cfu, or from 1
to about 5000 cfu, or from 1 to about 2000 cfu, or from 1 to about
1000 cfu, or from 1 to about 500 cfu, or from 1 to about 200 cfu,
or from 1 to about 100 cfu, or from 1 to about 50 cfu, or from 1 to
about 20 cfu, or from 1 to about 10 cfu, or from 1 to about 5
cfu.
[0067] The number of spores that may be detected and counted may
be, for example, from 1 to about 4,000,000 spores, or from 1 to
about 3,000,000 spores, or from 1 to about 2,000,000 spores, or
from 1 to about 1,000,000 spores, or from 1 to about 500,000
spores, or from 1 to about 200,000 spores, or from 1 to about
100,000 spores, or from 1 to about 50,000 spores, or from 1 to
about 10000 spores, or from 1 to about 5000 spores, or from 1 to
about 2000 spores, or from 1 to about 1000 spores, or from 1 to
about 500 spores, or from 1 to about 200 spores, or from 1 to about
100 spores, or from 1 to about 50 spores, or from 1 to about 20
spores, or from 1 to about 10 spores, or from 1 to about 5 spores,
or from about 5 to about 10000 spores, or from about 5 to about
5000 spores, or from 5 to about 1000 spores, or from 5 to about 500
spores, or from 5 to about 200 spores, or from 5 to about 100
spores, or from 5 to about 50 spores, or from 5 to about 20 spores,
or from about 10 to about 10000 spores, or from about 10 to about
5000 spores, or from 10 to about 1000 spores, or from 10 to about
500 spores, or from about 10 to about 200 spores, or from about 10
to about 100 spores, or from about 10 to about 50 spores, or from
about 10 to about 30 spores, or from about 15 to about 10000
spores, or from about 15 to about 5000 spores, or from about 15 to
about 2000 spores, or from about 15 to about 1000 spores, or from
about 15 to about 500 spores, or from about 15 to about 200 spores,
or from about 15 to about 100 spores, or from about 15 to about 50
spores, or from about 15 to about 30 spores, or from about 20 to
about 10000 spores, or from about 20 to about 5000 spores, or from
about 20 to about 1000 spores, or from about 20 to about 500
spores, or from about 20 to about 200 spores, or from about 20 to
about 100 spores, or from about 20 to about 50 spores, or from
about 20 to about 40 spores, or from about 25 to about 10000
spores, or from about 25 to about 5000 spores, or from about 25 to
about 1000 spores, or from about 25 to about 500 spores, or from
about 25 to about 200 spores, or from about 25 to about 100 spores,
or from about 25 to about 50 spores, or from about 25 to about 40
spores. It is possible with this invention to detect the fact that
1 spore or no spores survive a sterilization process.
[0068] The number of test microorganisms, if any, that survive a
sterilization process can be determined instantaneously, or within
a time period of up to about 2000 seconds, or up to about 1500
seconds, or up to about 1000 seconds, or up to about 500 seconds,
or up to about 200 seconds, or up to about 100 seconds, or up to
about 50 seconds, or up to about 30 seconds, or from about 5
seconds to about 2000 seconds, or from about 10 to about 1800
seconds, or from about 20 to about 1500 seconds, or from about 30
to about 1200 seconds, or from about 50 to about 1000 seconds, or
from about 60 to about 800 seconds, or from about 100 to about 600
seconds, or from about 200 to about 600 seconds, or from about 300
to about 600 seconds. This is also applicable to other systems or
processes (e.g., liquids such as milk or beer) wherein
microorganisms are detected and/or counted.
[0069] The biological indicator may be used to release loads or
validate sterilization chamber functionality in healthcare
settings. In the scientific setting, the biological indicator may
be used to validate the functionality of sterilization chambers,
release loads of goods, or validate that a process meets required
functionality.
[0070] The biological indicator may be used by subjecting it to the
same sterilant and sterilization conductions as the articles for
which sterilization is desired. Following sterilization, the
capacitance of the biological indicator may be tested to determine
if live test microorganisms or spores survived the sterilization
process. If desired, the number of live test microorganisms or
spores that survived the sterilization may be determined.
[0071] Referring to the drawings, FIG. 1 shows a system 10
comprised of a capacitance device A that includes a biological
indicator comprising test microorganisms (e.g., spores) (not shown
in FIG. 1), and a sensing apparatus 50 to ascertain the efficacy of
a sterilization process. The sensing apparatus 50 may comprise a
capacitance bridge. Sensitive capacitance bridges can be
inexpensive, making the instant read biological indicator of the
present invention a sensitive and inexpensive device.
[0072] Capacitance device A includes a biological indicator housing
assembly B, a cap C, and an assay medium housing D. A biological
indicator comprising test microorganisms (not shown in FIG. 1) is
positioned in the housing assembly B. Cap C substantially envelopes
the biological indicator housing assembly B. A tortuous path E is
defined by the cap C and the housing B between test microorganisms
in the biological indicator housing assembly B, and the environment
around the capacitance device A. Cap C is movable with respect to
assay medium housing D to open and block the tortuous path E. The
cap C further provides indirect access for the sterilant to the
biological indicator housing B assembly via the tortuous path E.
The sterilant may comprise a gaseous sterilant, vaporous sterilant,
or a combination thereof. Examples include steam, vaporous hydrogen
peroxide, peracetic acid, ozone, ethylene oxide, and the like.
[0073] The assay medium housing D defines a holding compartment or
reservoir for holding assay medium F. The combination of the
biological indicator housing assembly B, the cap C, and the media
housing D forms a mechanism that, after a sterilization cycle, is
sealed. The test microorganisms are then transferred from the
housing assembly B to the assay medium housing D wherein they are
immersed into the assay medium F. A pair of electrical conductors
(e.g., conducting plates) 301 and 302 are located inside assay
medium housing D. The combination of the test microorganisms and
the assay medium F forms a dielectric positioned between the
conductors 301 and 302.
[0074] The tortuous path E discourages external contamination after
the internal surfaces and the test microorganisms have been
microbially decontaminated. At the same time, the tortuous path E
permits efficient entrance and exit of sterilant between the test
microorganisms and the surrounding environment.
[0075] A microporous, preferably hydrophilic, membrane G is
positioned within the cap C in the tortuous path E between the
environment and the biological indicator. The microporous membrane
covers and encloses a cavity (not shown) within the biological
indicator housing assembly B.
[0076] Membrane G performs at least two functions. The first
function is to prohibit any of the test microorganisms from moving
out of the biological indicator housing assembly B. The second
function is to allow entrance of the sterilant into the housing
assembly B in contact with the test microorganisms, and removal of
the sterilant from the housing assembly B. This allows a secure
storage of the test microorganisms within the biological indicator
housing assembly B while testing the effectiveness of a
sterilization process.
[0077] The effectiveness of the sterilization process may be tested
by contacting the test microorganisms with the sterilant in the
same manner as the load being sterilized. The sterilant flows along
the tortuous path E to biological indicator housing assembly B
where the sterilant flows over and among the test microorganisms.
After completion of a sterilization process, assay medium housing D
is compressed into the cap C. This compression simultaneously
introduces the test microorganisms into the assay medium F and
closes off the tortuous path E. This closing off of the tortuous
path seals off the test microorganisms from the environment.
[0078] In an embodiment, the capacitance device illustrated in
FIGS. 6-8 may be used. Referring to FIGS. 6-8, capacitance device
100 includes cap 110, first compartment 120 and second compartment
130. First compartment 120 holds biological indicator 160, and
contains electrical conductors 301 and 302. The biological
indicator 160 comprises test microorganisms (e.g., spores) on a
carrier. Second compartment 130 holds frangible ampoule 140 which
contains assay medium 150. The frangible ampoule 140 may be a glass
ampoule.
[0079] When used in a sterilization process, the cap 110 is held in
an open position as illustrated in FIG. 7. The capacitance device
100 and items to be sterilized are then subjected to the
sterilization process. During the sterilization process, the
sterilant flows through openings between the cap 110 and the second
compartment 130, as indicated by arrows 121, and then into the
first compartment 120, as indicated by arrows 131, where it
contacts and acts upon the test microorganisms on the biological
indicator 160.
[0080] After the sterilization process is complete, the capacitance
device 100 is activated by screwing the cap 110 downward into a
closed position as shown in FIG. 8. This results in the frangible
ampoule 140 being broken. Assay medium 150 then flows from the
second compartment 130 into the first compartment 120 and contacts
the biological indicator 160. The combination of the biological
indicator 160 and the assay medium 150 forms a dielectric
positioned between the conductors 301 and 302. The capacitance
device 100 is then placed in dock 306 which contains electrical
contacts 307 and 308. The electrical contacts 307 and 308 contact
electrical conductors 301 and 302, respectively.
[0081] Sensing apparatus 50 is comprised of control unit 60,
indicator 70, and sensor 300. A power source (e.g., a battery),
which is not shown, provides power to control unit 60, indicator 70
and sensor 300. Control unit 60 may be a microprocessor or a
microcontroller. Control unit 60 may also include (or is connected
with) a data storage device for storing data. Indicator 70 may take
the form of a visual and/or an audible indicator. These may include
one or more LEDs, LCDs, speakers, and/or alarms.
[0082] Referring to FIG. 2, sensor 300 includes a capacitor 305
that acts as a sensing element. Capacitor 305 is comprised of a
pair of electrical conductors 301 and 302 located within assay
medium housing D of capacitance device A, or within first
compartment 120 of capacitance device 100. In capacitance device A,
the assay medium F is located between electrical conductors 301 and
302. Assay medium F in combination with test microorganisms
introduced into assay medium housing D act as a dielectric for
capacitor 305. In capacitance device 100, the assay medium 150
flows into first compartment 120, and the combination of the assay
medium 150 and the biological indicator 160 function as a
dielectric between the electrical conductors 301 and 302.
[0083] Electrical properties of the capacitor are responsive to the
physical condition of the test microorganisms (i.e., live vs. dead)
that contact the assay medium after the sterilization process in
completed. In this respect, live spores, for example, tend to be
spheroidal in nature, whereas dead spores tend to be similar in
morphology to deflated balloons. The electrical properties of the
capacitor are measurably different with the presence of live test
microorganisms than with the presence of dead test microorganisms,
since the dielectric constant of the assay medium combined with
live test microorganisms differs from the dielectric constant of
the assay medium combined with dead test microorganisms. As a
result of these different dielectric constants, the capacitance of
the capacitor is measurably different with assay medium combined
with live test microorganisms, as compared to assay medium combined
with dead test microorganisms. By observing these differences in
capacitance, it can be determined whether a sterilization process
has been effective.
[0084] While not wishing to be bound by theory, it is believed that
as the test microorganisms die, ions are emitted and the emission
of these ions is, in part, what is producing the difference in the
observed capacitance measurements. Live and dead test
microorganisms express significantly different capacitances that do
not require signal accumulation time or growth promotion incubation
in order to be detected. As such, with the present invention it is
possible to obtain an instantaneous read on whether a sterilization
process has been successful by measuring the capacitance of the
biological indicator at the conclusion of the sterilization
process.
[0085] Sensor 300 is in the form of a "bridge circuit." The bridge
circuit may be used to determine the value of an unknown impedance
in terms of other impedances of known value. Highly accurate
measurements are possible because a null condition is used to
determine the unknown impedance. The bridge circuit is used to
determine the presence of live or dead spores inside media housing
D (FIG. 1) or in first compartment 120 (FIGS. 6-8) between
electrical conductors 301 and 302.
[0086] Sensor 300 is comprised of a voltage source 322, a null
detector 330, an electronic potentiometer 340, a capacitor 315 of a
known capacitance C.sub.1, and capacitor 305 having a capacitance
C.sub.x. Capacitance C.sub.x of capacitor 305 will vary in response
to the presence of live or dead test microorganisms (e.g., spores)
inside assay media housing D (FIG. 1) or in first compartment 120
(FIGS. 6-8).
[0087] In an embodiment, the inventive capacitor may be a parallel
plate capacitor. However, it should be appreciated that the
capacitor may be constructed in a different form, including, but
not limited to, a cylindrical or spherical-shaped capacitor. If a
spherical capacitor is used as the capacitor, holes may be placed
in the outer shell of the capacitor such that the test
microorganisms may enter and exit the capacitor. The electrical
conductors may be made of copper, aluminum, silver, gold, platinum,
or a combination of two or more thereof. The electrical conductors
may comprise indium tin oxide (ITO) on glass.
[0088] Electronic potentiometer 340 functions in the same manner as
a mechanical potentiometer. In this regard, electronic
potentiometer 340 may be a three terminal device. Between two of
the terminals is a resistive element. The third terminal known as
the "wiper" may be connected to various points along the resistive
element. In the illustrated embodiment, the wiper is digitally
controlled by control unit 60. The wiper divides the resistive
element into two resistors R.sub.BC and R.sub.AC. Electronic
potentiometer 340 may take the form of a digitally programmable
potentiometer (DPPTM) available from Catalyst Semiconductor, Inc.
of Sunnyvale, Calif.
[0089] In an embodiment, voltage source 322 provides an AC voltage
signal, such as a sinusoidal or pulse waveform. Null detector 330
is a device for detecting a null condition (i.e., a short circuit),
such as a galvanometer, a voltmeter, a frequency-selective
amplifier, and the like.
[0090] Operation of sensor 300 will now be described with reference
to FIG. 2. The elements of the bridge circuit are connected between
junctions AC, BC, AD, and BD. Electronic potentiometer 340 is
operated by control unit 60 to vary the resistances R.sub.BC and
R.sub.AC until the potential difference between junctions A and B
(V.sub.AB) is zero. When this situation exists, the bridge is said
to be balanced or is "nulled." The following relationships then
hold for voltages in the main branches:
V.sub.AC=V.sub.BC, and V.sub.AD=V.sub.BD,
where V.sub.AC is the voltage between junctions A and C, V.sub.BC
is the voltage between junctions B and C, V.sub.AD is the voltage
between junctions A and D, and V.sub.BD is the voltage between
junctions B and D. Accordingly,
V.sub.AD/V.sub.AC=V.sub.BD/V.sub.BC
V.sub.AD=V.sub.BD/(V.sub.AC/V.sub.BC)
[0091] Capacitor 305 of capacitance C.sub.x is connected between
junctions A and D, and capacitor 315 of known capacitance C.sub.1
is connected between junctions B and D. Electronic potentiometer
340, connected from junction A to junction C to junction B, is
adjusted by control unit 60 to vary the voltages V.sub.AC and
V.sub.BC.
[0092] When a null is detected by null detector 330, current
I.sub.1 flows from junction C to junction A to junction D, and a
current I.sub.2 flows from junction C to junction B to junction D.
The voltage V.sub.AC across junctions A to C, and the voltage
V.sub.BC across junctions B to C are:
V.sub.AC=I.sub.1R.sub.AC, and V.sub.BC=I.sub.2R.sub.BC.
[0093] The voltage across a capacitor with capacitance C, current
I, and frequency f is:
V = I 2 .pi. fC ##EQU00001##
Therefore, the voltages V.sub.AD and V.sub.BD may be expressed
as:
V AD = I 1 2 .pi. fC x V BD = I 2 2 .pi. fC 1 ##EQU00002##
[0094] As discussed above, V.sub.AD=V.sub.BD/(V.sub.AC/V.sub.BC),
V.sub.AC=I.sub.1R.sub.AC, and V.sub.BC=I.sub.2R.sub.BC.
Therefore,
C x = C 1 ( R BC R A C ) . ##EQU00003##
[0095] In view of the forgoing relationship, when a null condition
is detected, the resistance values for R.sub.BC and R.sub.AC, along
with the known capacitance C.sub.1 of capacitor 315, can be used to
determine the unknown value of capacitance C.sub.x of capacitor
305.
[0096] By configuring capacitor 305 as an element of a bridge
circuit, a measure of resistance values R.sub.AC and R.sub.BC, when
the bridge is balanced or nulled, can be used to determine the
capacitance C.sub.x of capacitor 305. Changes to the capacitance
C.sub.x of capacitor 305 is indicative of the presence of live or
dead spores in assay media F.
[0097] For a parallel plate capacitor,
C=(k.epsilon..sub.0)(A/d)=(.epsilon.)(A/d), where C is capacitance,
k is the dielectric constant, .epsilon..sub.0 is the permittivity
of free space (8.85.times.10.sup.-12 F/m), .epsilon. is the
permittivity (Farads/meter) of the capacitor dielectric, A is the
area of the capacitor plates (m.sup.2), and d is the separation in
meters between the capacitor plates. As .epsilon. increases, the
capacitance C will increase. Where the capacitor is a parallel
plate capacitor with circular plates of diameter D,
C=(.pi.D.sup.2.epsilon.)/(4d).
[0098] The dielectric constant k of the capacitor can be determined
according to the following expression:
k = 4 dC .pi. D 2 0 , ##EQU00004##
where the value of capacitance, C, is determined as discussed
above. The dielectric constant of the capacitor can also be
determined by determining the capacitance with the dielectric in
place between the conducting plates (C.sub.d), and then determining
the capacitance without the dielectric in place (C.sub.o). The
ratio of the two capacitances equals the dielectric constant,
k = C d C 0 . ##EQU00005##
[0099] The response of a capacitor is influenced by the
characteristics (e.g., frequency) of the AC waveform applied
thereto. In this regard, capacitive reactance (X.sub.c) is a
function of frequency. Capacitive reactance is the opposition
offered to the flow of alternating current by pure capacitance, and
is expressed in ohms (X.sub.c=1/(2.pi.fC)). Accordingly, frequency
of the waveform generated by voltage source 322 influences the
response of capacitors.
[0100] While sensor 300 is shown as being in the form of a bridge
circuit, other types of circuits and techniques (including other
types of bridge circuits, and capacitance meters) may be used to
measure capacitance. For example, FIG. 3 illustrates an alternative
sensor 300A. Sensor 300A is an LC resonant circuit, including a
variable capacitor 325 (having a capacitance C.sub.A), and
capacitor 305 (having a capacitance C.sub.x) that acts as the
sensing element, as described above. Since the resonance frequency
.omega..sub.0=[L(C.sub.A+C.sub.x)].sup.-1/2, the unknown
capacitance C.sub.x of capacitor 305 can be determined.
[0101] FIG. 4 illustrates yet another alternative sensor 300B
suitable for use in connection with the present invention. Sensor
300B is a "charge transfer" sensor circuit. Charge transfer sensor
circuits are recognized to provide resolutions of fractions of a
femtoFarad. In a charge transfer sensor circuit the unknown
capacitance C.sub.x of a sense electrode is determined by charging
the sense electrode to a fixed potential, and then transferring
that charge to a charge detector comprising a capacitor 335 of
known capacitance C.sub.s. In sensor 300B, capacitor 305 of unknown
capacitance C.sub.x acts as a sensing element, as described above.
In this regard, an assay medium and spores fill the gap between the
conducting plates of capacitor 305, thereby acting as an insulator
or "dielectric" of capacitor 305. Capacitor 305 is first connected
to a DC reference voltage (V.sub.r) via a switch S.sub.1. Switch
S.sub.1 is reopened after capacitor 305 is satisfactorily charged
to the potential of V.sub.r. Then, after as brief as possible a
delay so as to minimize leakage effects caused by conductance,
switch S.sub.2 is closed and the charge (Q) present on capacitor
305 is transferred to capacitor 335 (i.e., the charge detector).
Once the charge Q is satisfactorily transferred to capacitor 335,
switch S.sub.2 is reopened. By reading voltage V.sub.s, the
capacitance C.sub.x of capacitor 305 can be determined. V.sub.s may
be input to an amplifier to provide the scaling necessary to
present an analog-to-digital converter (ADC) with a useful range of
voltage for digital processing. Switch S.sub.3 acts as a reset
means to reset the charge between charge transfer cycles, so that
each charge transfer cycle has a consistent initial condition.
Switches S.sub.1, S.sub.2 and S.sub.3 may be electromechanical
switches or transistors. Preferably, digital control logic is used
to control switches S.sub.1, S.sub.2 and S.sub.3. Capacitor 335 may
be significantly larger than capacitor 305.
[0102] The equations governing sensor 300B are as follows:
V.sub.s=V.sub.r[C.sub.y/(C.sub.y+C.sub.s)], therefore
C.sub.y=V.sub.sC.sub.s/[V.sub.r-V.sub.s].
[0103] The charge-transfer sensor has been applied in a
self-contained capacitance-to-digital-converter (CDC) integrated
circuit (IC). For example, Quantum Research Group produces a
QProx.TM. CDC sensor IC (e.g., QT300 and QT301 CDC sensor ICs) for
detecting femtofarad level changes in capacitance. The CDC sensor
IC outputs a digital value corresponding to the detected input
capacitance. The value of an external sampling capacitor controls
the gain of the sensor.
[0104] Other high sensitivity circuitry is provided by such devices
that may be used include the PTL 110 capacitance transducer from
Process Tomography Limited of Cheshire, United Kingdom. The PTL 110
measures small values of capacitance (up to 10 pF) with a
resolution of 1 fF. A 7600 Plus Precision LCR Meter Capacitance
Bridge from IET Labs, Inc. of Westbury, N.Y., allows for
measurement of capacitances in the range from 0.01 fF to 10 F.
Tektronix produces the Tektronix 130 LC Meter that measures
capacitance from 0.3 pF to 3 pF. It has also been acknowledged in
the prior art literature that capacitance sensor circuits using
modern operational amplifiers and analog-to-digital converters
(ADCs) can easily obtain resolutions to 0.01 pF. In an embodiment,
a dielectric cell may be used to provide a more accurate
capacitance reading by screening out extraneous electrical signals;
see, ASTM D150.
[0105] Operation of the present invention, as illustrated in FIG.
1, will now be summarized. The capacitance device A is located
within an enclosure containing at least one item to be sterilized.
The test microorganisms in the hosing assembly B along with the
item to be sterilized are then exposed to a sterilant for an
effective period of time to provide for sterilization. During the
sterilization process the test microorganisms are maintained within
the housing assembly B as illustrated in FIG. 1. After the
sterilization process is completed, the test microorganisms are
combined with the assay medium F. The assay medium, combined with
the test microorganisms, is placed between electrical conductors
301 and 302 to form a dielectric. Sensing apparatus 50 determines a
measured capacitance of the capacitor to ascertain whether the test
microorganisms are alive or dead.
[0106] In accordance with an embodiment of the present invention, a
method for determining the efficacy of a sterilization process,
includes the steps of: (a) placing a biological indicator
comprising test microorganisms within a region containing at least
one item to be sterilized; (b) exposing the at least one item and
the biological indicator to a sterilant; (c) after exposure to the
sterilant, placing the biological indicator and an assay medium
between a pair of electrical conductors of a capacitor, wherein the
biological indicator and the assay medium serve as a dielectric for
the capacitor; (d) measuring the capacitance of the capacitor; and
(e) determining whether the measured capacitance values indicate
the presence of live test microorganisms. The capacitance values
may be used to count the live test microorganisms, if any, that
survive the sterilization process. The determination of whether
live test microorganisms are present, and if so, how many, can be
accomplished instantaneously, or within a period of time of up to
about 2,000 seconds, or up to about 1500 seconds, or up to about
1000 seconds, or up to about 500 seconds, or up to about 200
seconds, or up to about 100 seconds, or up to about 50 seconds, or
up to about 30 seconds, or in the range from about 5 to about 2000
seconds, or from about 10 to about 1800 seconds, or from about 20
to about 1500 seconds, or from about 30 to about 1200 seconds, or
from about 50 to about 1000 seconds, or from about 60 to about 800
seconds, or from about 100 to about 600 seconds, or from about 200
to about 600 seconds, or from about 300 to about 600 seconds.
[0107] A control capacitance value may be determined in advance and
pre-stored in a memory of control unit 60. The control capacitance
value may be dependent upon several factors, including the type of
assay medium, number of test microorganisms, physical configuration
of the capacitor (e.g., dimensions and shape of the capacitor
plates), etc.
[0108] Indicator 70 may be used to provide a visual and/or audible
indication of whether viable test microorganisms are detected. For
instance, a green LED may be illuminated to indicate the absence of
viable test microorganisms (i.e., a successful sterilization
cycle), while a red LED may be illuminated to indicate the presence
of viable test microorganisms (i.e., an unsuccessful sterilization
cycle). Alternatively, an audible alarm may be activated when it is
determined that viable test microorganisms are present.
Example 1
[0109] A Keysight Technologies model U1701B Handheld Capacitance
Meter with measurement range from 1000 pF to 199.99 mF is used to
test two sets of spore test strips. One set of spore test strips
(treated) is subjected to a steam sterilization process, and the
other set (untreated) is not subjected to the sterilization
process. The Keysight capacitance meter is electrically connected
to a Bio-Rad Shock Pod. The Bio-Rad Shock Pod provides electrical
communication with a Mirus Ingenio 0.2 cm cuvette. The cuvette is a
disposable plastic container that is 4.5 cm tall (without its cap)
and 1.2 cm on each side. The sidewalls of each of two opposing
sides of the cuvette are constructed of aluminum plates (each being
2 cm tall and 1 cm wide). The aluminum plates extend through the
walls and communicate with the interior of the cuvette. The
aluminum plates function as electrical conductors. The gap between
the plates inside the cuvette is 0.2 cm.
[0110] The test strips are VERIFY.RTM. Spore Test Strips for
540.RTM. Sterilant Concentrate supplied by STERIS Corporation.
These test strips are cellulose strips that are 0.6 cm wide, 3.8 cm
long, and less than 0.1 cm thick. These test strips are
characterized by a population of Geobacillus stearothermophilus
spores of approximately 10.sup.6 spores. One of each of these test
strips is folded over so that it is 1.9 cm long. The test strips
are inserted into the cuvette, and positioned between the aluminum
plates. A 20% glycerol (v/v in water) solution is added to the
cuvette to cover the tops of the plates, thus filling all voids.
The test strip in combination with the glycerol solution form a
dielectric. The aluminum plates function as electrical conductors.
The capacitance of each test strip is determined by applying an
electrical signal to the conductors and measuring capacitance using
the Keysight capacitance meter.
[0111] An additional sample of each test strip (treated and
untreated) is transferred to a growth medium to confirm the state
of the spores strips. The spores on the untreated test strip grow
overnight, while the spores on the treated test strip do not grow.
This indicates a complete spore kill for the treated test strip,
i.e., a successful sterilization.
[0112] The treated test strip shows a capacitance level of 1004
nanofarads (nF), with a standard deviation of 33 nF. The untreated
test strip shows a capacitance level of 626 nF, with a standard
deviation of 23 nF. These results are shown in FIG. 5.
Example 2
[0113] The capacitance value at one standard deviation for the
untreated test strip (live spores) in Example 1 is: 626 nF+23
nF=649 nF to 626 nF-23 nF=603 nF. The capacitance value at one
standard deviation for the treated test strip (dead spores) in
Example 1 is: 1004-33=971 nF to 1004+33 nF=1037 nF. For purposes of
this example, it is assumed that the spore population for the test
strip is 10.sup.6 spores. To determine the smallest accuracy level
needed for the capacitor to detect one live spore, the following
difference in capacitance levels is determined: 971 nF-649 nF (the
smallest value for 10.sup.6 dead spores minus the largest value for
10.sup.6 live spores)=322 nF. This number is then divided by
10.sup.6 spores to yield 0.32 pF/spore (or about 0.3 pF/spore).
This indicates that in using a capacitance bridge with an accuracy
of less than about 0.3 pF, it is possible to detect one live spore
out of 10.sup.6 spores. If no live spores can be detected, then all
of the spores are dead and the sterilization process is
successful.
Example 3
[0114] The software for an Andeen-Hagerling AH 2700A capacitance
bridge is used to calculate capacitance levels for counting sores.
The resolution of the AH 2700A bridge is 0.8 aF. Since as indicated
in Example 2 a change of 0.3 pF/spore is required to detect the
presence of live spores, the level of resolution of 0.8 aF is 375
times better than needed: 0.3 pF/0.8 aF=375. The accuracy of the AH
2700A bridge is 5 ppm or 5E (-6). If it is assumed that this
accuracy is based on the largest value the AH 2700A bridge can
measure (1.5 microfarads), the accuracy is
5E(-6).times.1.5E(-6)=7.5E(-12)=7.5 pF. However, as indicated
above, an accuracy of 0.3 pF is needed to indicate the presence of
one live spore. At an accuracy of 7.5 pF, it is possible to show a
capacitance change that translates to 25 live sores ((7.5 pF)(0.3
pF/spore)=25 spores).
[0115] Using software provided for the AH2700A bridge, the
capacitance level that would be required to provide an accuracy
level of less than 0.3 pF is determined to be 15 nF or less. This
indicates that it is possible to provide a capacitor that can
detect one live spore on a treated test strip that initially
contains 10.sup.6 live spores prior to sterilization. The values
from the AH2700A software that demonstrate this are as follows:
TABLE-US-00002 Capacitance Accuracy in PPM Accuracy in Farads
Maximum Number of Spores 1004 nf 68.1 (1004 nf)(68.1E-6) = 68.4 pF
(68.4 pF)/(0.3 pF/spore) = 228 spores 500 nF 46.5 (500 nF)(46.5E-6)
= 23.25 pF (23.25 pF)/0.3 pF/spore) = 77.5 spores 60 nF 35.2 (60
nF)(35.2E-6) = 2.1 pF (2.1 pF)/(0.3 pF/spore) = 7 spores 40 nF 25.2
1 pF 4 spores 20 nF 25.1 0.5 pF 2 spores 15 nF 15.1 0.23 pF 1 spore
10 nF 15.1 0.15 pF 0 spores
Example 4
[0116] The procedure used in Example 1 is repeated except that the
test strip is an untreated test strip that contains Bacillus
atrophaeus spores. This is compared to a blank cellulose strip. The
untreated test strip shows a capacitance level of 609.2 nF. The
blank cellulose strip shows a capacitance of 1042.7 nF.
Example 5
[0117] A statistical analysis is conducted using the test strips
shown in Example 1 to determine whether there is a statistical
difference in capacitance levels for the treated and the untreated
test strips. Two cuvettes (one for a treated test strip, and one
for an untreated test strip) are separately placed in the Bio-Rad
Shock Pod and the Keysight capacitance meter is activated. With
each test strip, an initial capacitance reading is taken, and then
readings are taken every 5 seconds for 15 minutes (180 readings).
At the conclusion of the first 15 minute trial (180 data points),
the data are collected. The results for this first trial are
reported in the tables below as "live 1" (first trial, untreated
spore strip) and "dead 1" (first trial, treated spore strip). The
capacitance meter is then activated for a second trial, and then a
third trial, with each trial consisting of readings taken every 5
seconds for 15 minutes (180 readings). The results for the second
and third trials are reported in the tables below as follows: "live
2" (second trial, untreated spore strip); "dead 2" (second trial,
treated spore strip); "live 3" (third trial, untreated spore
strip); and "dead 3" (third trial, treated spore strip). The data
for the three trials are also combined (540 readings), and reported
below as "all live" (first, second and third trials combined,
untreated spore strip), and "all dead" (first, second and third
trials, treated spore strip). The numerical values shown below are
capacitance levels measured in nanofarads (nF).
[0118] The analysis that is used can be referred to as a Two-Sample
T-Test which determines whether the means of two independent
populations are equal to a target value. In the data provided
below, "N" is the number of readings or data points (180 for each
analysis, except for the last analysis where all data points from
the treated and the untreated spores strips are shown and N is
540). The term "Mean" refers to the sum of the capacitance levels
measured in nF divided by the number (N) of data points. The term
"St Dev" refers to standard deviation, which is a measure of the
variability of the capacitance levels within a single sample. The
term "SE Mean" refers to the standard error of the mean which is a
measure of the variability of the capacitance levels between
samples. The term "Difference=.mu. (live_)-.mu. (dead_)" refers to
the difference between the means of the live test with the dead
test. The term "Estimate for difference" refers to an estimated
difference between the two means based upon spore population
statistics. The term "95% CI for difference" refers to the bounds
of the 95% confidence interval, if the set includes 0 the sample
set means would be considered equivalent. The term "T-Test of
difference" refers to a hypothesis for test that both means are
equivalent (T=0). The term "T-Value" refers to the calculated
t-test value for comparison to T=0. The term "P-Value" refers to
the value normally used for determination of equivalence. The
confidence interval used here is 95%, therefore, any value above
0.05 for the P-value indicates the spore population means are
equivalent. The term "DF" refers to degrees of freedom in the
test.
[0119] The results are indicated below:
TABLE-US-00003 N Mean StDev SE Mean (A) live 1, dead 1 live 1 180
579 129 9.6 dead 1 180 931 206 15 Difference = .mu. (live 1) - .mu.
(dead 1) Estimate for difference: -351.9 95% CI for difference:
(-387.7, -316.2) T-Test of difference = 0 (vs .noteq.): T-Value =
-19.38 P-Value = 0.000 DF = 301 (B) live 1, dead 2 live 1 180 579
129 9.6 dead 2 180 876 185 14 Difference = .mu. (live 1) - .mu.
(dead 2) Estimate for difference: -296.3 95% CI for difference:
(-329.5, -263.2) T-Test of difference = 0 (vs .noteq.): T-Value =
-17.59 P-Value = 0.000 DF = 320 (C) live 1, dead 3 live 1 180 579
129 9.6 dead 3 180 911 176 13 Difference = .mu. (live 1) - .mu.
(dead 3) Estimate for difference: -331.3 95% CI for difference:
(-363.4, -299.2) T-Test of difference = 0 (vs .noteq.): T-Value =
-20.32 P-Value = 0.000 DF = 328 (D) live 2, dead 3 live 2 180 577
110 8.2 dead 3 180 911 176 13 Difference = .mu. (live 2) - .mu.
(dead 3) Estimate for difference: -333.5 95% CI for difference:
(-363.9, -303.0) T-Test of difference = 0 (vs .noteq.): T-Value =
-21.54 P-Value = 0.000 DF = 299 (E) live 2, dead 2 live 2 180 577
110 8.2 dead 2 180 876 185 14 Difference = .mu. (live 2) - .mu.
(dead 2) Estimate for difference: -298.5 95% CI for difference:
(-330.1, -266.9) T-Test of difference = 0 (vs .noteq.): T-Value =
-18.59 P-Value = 0.000 DF = 291 (F) live 2, dead 1 live 2 180 577
110 8.2 dead 1 180 931 206 15 Difference = .mu. (live 2) - .mu.
(dead 1) Estimate for difference: -354.1 95% CI for difference:
(-388.4, -319.8) T-Test of difference = 0 (vs .noteq.): T-Value =
-20.32 P-Value = 0.000 DF = 272 (G) live 3, dead 1 live 3 180 72
104 7.8 dead 1 180 31 206 15 Difference = .mu. (live 3) - .mu.
(dead 1) Estimate for difference: -359.5 95% CI for difference:
(-393.5, -325.6) T-Test of difference = 0 (vs .noteq.): T-Value =
-20.86 P-Value = 0.000 DF = 265 (H) live 3, dead 2 live 3 180 572
104 7.8 dead 2 180 876 185 14 Difference = .mu. (live 3) - .mu.
(dead 2) Estimate for difference: -303.9 95% CI for difference:
(-335.2, -272.7) T-Test of difference = 0 (vs .noteq.): T-Value =
-19.17 P-Value = 0.000 DF = 282 (I) live 3, dead 3 live 3 180 572
104 7.8 dead 3 180 911 176 13 Difference = .mu. (live 3) - .mu.
(dead 3) Estimate for difference: -338.9 95% CI for difference:
(-369.0, -308.9) T-Test of difference = 0 (vs .noteq.): T-Value =
-22.19 P-Value = 0.000 DF = 290 (J) all live, all dead All live 540
576 115 4.9 All dead 540 906 191 8.2 Difference = .mu. (all live) -
.mu. (all dead) Estimate for difference: -329.78 95% CI for
difference: (-348.59, -310.97) T-Test of difference = 0 (vs
.noteq.): T-Value = -34.41 P-Value = 0.000 DF = 884
[0120] These test results indicate that, with a 95% confidence
level, there is a statistical difference between the capacitance
levels for the spore strips with live spores (untreated) compared
to the spore strips with dead spores (treated).
[0121] While the invention has been explained in relation to
various embodiments, it is to be understood that various
modifications thereof will become apparent to those skilled in the
art upon reading the specification. Therefore, it is to be
understood that the invention disclosed herein includes any such
modifications that may fall within the scope of the appended
claims.
* * * * *