U.S. patent application number 10/090057 was filed with the patent office on 2002-09-26 for biological indicator for sterilization processes with double buffer system.
This patent application is currently assigned to Ethicon, Inc.. Invention is credited to Feldman, Leslie A., Gorham, Richard A., Hui, Henry K..
Application Number | 20020137123 10/090057 |
Document ID | / |
Family ID | 24810632 |
Filed Date | 2002-09-26 |
United States Patent
Application |
20020137123 |
Kind Code |
A1 |
Hui, Henry K. ; et
al. |
September 26, 2002 |
Biological indicator for sterilization processes with double buffer
system
Abstract
A method and an apparatus for preventing reversion of the color
of an indicator dye in a biological indicator is disclosed. The
indicator dye changes color if viable microorganisms are present
after sterilization, because acidic byproducts are formed when the
microorganisms metabolize the growth medium. It has been found that
the dye can change color back to the original color after the
completion of the sterilization due to leaching or diffusion of
basic impurities into the growth medium. The method and the
apparatus employ a dual buffer system with one buffer which
operates at high pH to moderate pH fluctuations at the start of the
sterilization and a second buffer which operates at low pH to
minimize pH fluctuations after the sterilization is complete. Less
high pH buffer than low pH buffer is used in order to maximize the
speed and sensitivity of the biological indicator.
Inventors: |
Hui, Henry K.; (Laguna
Niguel, CA) ; Feldman, Leslie A.; (Calabasas Hills,
CA) ; Gorham, Richard A.; (Laguna Beach, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
620 NEWPORT CENTER DRIVE
SIXTEENTH FLOOR
NEWPORT BEACH
CA
92660
US
|
Assignee: |
Ethicon, Inc.
|
Family ID: |
24810632 |
Appl. No.: |
10/090057 |
Filed: |
February 27, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10090057 |
Feb 27, 2002 |
|
|
|
09699728 |
Oct 27, 2000 |
|
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Current U.S.
Class: |
435/31 |
Current CPC
Class: |
G01N 31/226 20130101;
A61L 2/28 20130101; C12Q 1/22 20130101 |
Class at
Publication: |
435/31 |
International
Class: |
C12Q 001/22 |
Claims
What is claimed is:
1. A method for determining the effectiveness of a disinfection or
sterilization process, said method comprising; providing a carrier
with microorganisms on the carrier; exposing said carrier to the
disinfection or sterilization process; incubating said carrier in a
growth medium comprising a first buffer having a first pK.sub.a, a
second buffer having a second pK.sub.a, and a pH-sensitive dye,
wherein said incubating is after said exposing; and determining
whether said microorganisms grow in said growth medium during said
incubating, wherein the growing of said microorganisms in said
growth medium generates acid, thereby changing pH in said growth
medium from a first pH within a first pH range to a second pH
within a second pH range, wherein said first pK.sub.a and said
second pK.sub.a are within said first pH range and said second pH
range respectively.
2. The method of claim 1, wherein said determining whether said
microorganisms have grown comprises determining whether the pH
changes in said growth medium from said first pH to said second
pH.
3. The method of claim 1, wherein said pH-sensitive dye has a first
color in said first pH range and a second color in said second pH
range, wherein said determining whether said microorganisms have
grown comprises determining whether the dye changes color from said
first color to said second color.
4. The method of claim 1, wherein said system comprises a lower
concentration of buffer having a pK.sub.a in said first pH range
than said buffer having a pK.sub.a in said second pH range.
5. The method of claim 1, wherein said growth medium is contained
in an openable enclosure and wherein incubating said carrier in a
growth medium further comprises opening the enclosure and immersing
said carrier in said growth medium.
6. The method of claim 1, wherein said carrier and said growth
medium are located in a container covered with a gas or vapor
permeable but microorganism impermeable barrier and wherein
exposing said carrier further comprises diffusing a germicide gas
or vapor from outside said container into said container through
said barrier.
7. The method of claim 1, wherein said microorganism comprises a
biological indicating microorganism for said disinfection or
sterilization process.
8. The method of claim 1, wherein said disinfection or
sterilization process comprises a process with a disinfecting or
sterilizing agent selected from the group consisting of steam,
heat, ethylene oxide, hydrogen peroxide, ozone, chlorine dioxide,
peracetic acid, performic acid, formaldehyde, glutaraldehyde,
ortho-phthalaldehyde, and hypochorite salts.
9. A self-contained biological indicator comprising; a carrier with
viable microorganisms on the carrier; a container containing said
carrier therewithin, wherein at least a portion of said container
is transparent and wherein said container comprises an opening
which is covered with a gas or vapor permeable but microorganism
impermeable barrier; at least one openable enclosure inside said
container, wherein said enclosure contains a culture medium which
is capable of supporting growth of the viable microorganisms; a dye
which changes color with a change in pH from a first pH range to a
second pH range; and a dual buffer system, wherein said dual buffer
system comprises a first buffer having a first pK.sub.a and a
second buffer having a second pK.sub.a, wherein said first pK.sub.a
and said second pK.sub.a are within said first pH range and said
second pH range respectively.
10. The self-contained biological indicator of claim 9, wherein
said carrier is selected from the group consisting of a porous
substrate, a non-porous substrate, an absorbent substrate, and a
non-absorbent substrate.
11. The self-contained biological indicator of claim 9, wherein
said gas or vapor permeable but microorganism impermeable barrier
is a nonwoven polyolefin.
12. The self-contained biological indicator of claim 9, wherein
said viable microorganism comprises a biological indicating
microorganism for a disinfection or sterilization process.
13. The self-contained biological indicator of claim 9, wherein
said openable container comprises a breakable glass ampoule.
14. The self-contained biological indicator of claim 9, wherein
said dye comprises Bromcresol Purple.
15. The self-contained biological indicator of claim 9, wherein
said first buffer comprises at least one phosphate salt.
16. The self contained biological indicator of claim 9, wherein
said second buffer comprises at least one acetate salt.
17. The self contained biological indicator of claim 16, wherein
said at least one acetate salt is sodium acetate.
18. The self-contained biological indicator of claim 9, wherein
said dual buffer system comprises a lower concentration of buffer
having a pK.sub.a in said first pH range than buffer having a
pK.sub.a in said second pH range.
19. The self-contained biological indicator of claim 9, further
comprising a cap with at least one opening above said barrier,
whereby gas or vapor can diffuse into said container through said
hole and said barrier.
20. The self-contained biological indicator of claim 9, further
comprising a chemical indicator for indicating exposure of said
self-contained biological indicator to a disinfection or
sterilization process.
21. A culture medium which is capable of supporting growth of
viable microorganisms comprising: a nutrient broth; a dye which
changes color with a change in pH from a first pH range to a second
pH range; and a dual buffer system comprising a first buffer having
a first pK.sub.a and a second buffer having a second pK.sub.a,
wherein said first pK.sub.a and said second pK.sub.a are within
said first pH range and said second pH range respectively.
22. The culture medium of claim 21, wherein said dye comprises
Bromcresol Purple.
23. The culture medium of claim 21, wherein said first buffer
comprises at least one phosphate salt.
24. The culture medium of claim 21, wherein said second buffer
comprises at least one acetate salt.
25. The culture medium of claim 24, wherein said at least one
acetate salt is sodium acetate.
26. The culture medium of claim 21, wherein said dual buffer system
comprises a lower concentration of buffer having a pK.sub.a in said
first pH range than buffer having a pK.sub.a in said second pH
range.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to a double buffer system for
preventing color reversion of the pH indicator dye which is used to
detect bacterial growth in a biological indicator for monitoring
the efficacy of a sterilization process.
[0003] 2. Description of the Related Art
[0004] A variety of sterilization systems are used to sterilize
medical devices in hospitals and other medical facilities. Steam,
heat, ethylene oxide, and hydrogen peroxide are commonly used as
sterilants. The STERRAD.RTM. sterilization system, available from
Advanced Sterilization Products of Irvine, Calif., is an exemplary
sterilization system which utilizes a combination of hydrogen
peroxide and plasma to sterilize medical equipment.
[0005] In all of these sterilization processes, biological
indicators are commonly used to confirm the effectiveness of the
sterilization process. The biological indicators generally include
a carrier which has been inoculated with spores or other
microorganisms. The effectiveness of the sterilization is assessed
by determining whether subjecting the biological indicator to the
sterilization cycle destroys all of the microorganisms on the
carrier.
[0006] The biological indicator is placed into the sterilizer
together with the equipment to be sterilized. After the completion
of the sterilization process, the biological indicator is removed
from the sterilizer, and the carrier is immersed into a sterile
culture medium. The culture medium and carrier are incubated for a
predetermined time at an appropriate temperature. At the end of the
incubation period, an indicator is used to determine whether any
microorganisms have survived. If no growth of the microorganisms
occurs, it is assumed that the articles in the sterilizer have been
properly sterilized. If microorganism growth has occurred, the
articles in the sterilizer may not be sterile.
[0007] A pH indicating chemical which changes color with a change
in pH can be used as an indicator, because acidic byproducts are
formed when microorganisms metabolize the growth medium. An acidic
change in the pH of the growth medium in the biological indicator
therefore indicates bacterial growth. Alternatively, the indicator
can be, for example, turbidity in the culture medium which results
from cell colonies produced by bacterial growth.
[0008] In some biological indicators, the microorganisms, the
culture medium, and the indicator are packaged in a way which
permits the microorganisms, the culture medium, and the indicator
to be combined without exposing the biological indicator to the
non-sterile surroundings. This type of biological indicator is a
"self-contained biological indicator" or SCBI. Use of SCBIs
simplify the test process and minimize the chance that external
contamination could affect the test results.
[0009] McCormick et al. (U.S. Pat. No. 4,743,537), for example,
disclose a SCBI having a compartment with a permeable opening which
permits the transmission of sterilant gas or steam while preventing
the passage of microorganisms into or out of the compartment. The
compartment contains a breakable ampoule with a culture medium.
Indicating microorganisms are placed on one end of a wick, and the
wick is placed in the compartment, with the end of the wick
containing the microorganisms away from the media-containing
ampoule and adjacent the permeable opening. After the sterilization
process, the device is removed from the sterilizer, and the ampoule
is broken, releasing the culture medium into the compartment. The
device is incubated and examined to determine whether or not
microorganism growth has occurred.
[0010] The apparatus and the method of the present invention
address the previously unrecognized cause of a problem when using
biological indicators containing indicator dyes for detecting
microbial growth to determine the efficacy of a sterilization
process.
SUMMARY OF THE INVENTION
[0011] One aspect of the invention involves a method for
determining the effectiveness of a disinfection or sterilization
process. The method includes providing a carrier with
microorganisms on the carrier, exposing the carrier to the
disinfection or sterilization process, and incubating the carrier
in a growth medium to determine whether the microorganisms grow in
the growth medium. The growth medium contains a first buffer with a
first pK.sub.a, a second buffer with a second pK.sub.a, and a
pH-sensitive dye. The carrier is incubated in the growth medium
after exposure to the disinfection or sterilization process. Growth
of the microorganisms in the growth medium generates acid, changing
the pH in the growth medium from first pH in a first pH range to a
second pH in a second pH range. The first pK.sub.a and said second
pK.sub.a are within the first pH range and the second pH range,
respectively.
[0012] Advantageously, determining whether the microorganisms have
grown involves determining whether the pH changes in the growth
medium. Preferably, the pH-sensitive dye has a first color in the
first pH range and a second color in the second pH range. The
determination of whether the microorganisms have grown includes
determining whether the dye changes color from the first color to
the second color.
[0013] Preferably, there is a lower concentration of buffer having
a pK.sub.a in the first pH range than buffer having a pK.sub.a in
the second pH range. Advantageously, the growth medium is contained
in an openable enclosure, and the carrier is incubated in the
growth medium by opening the enclosure and immersing the carrier in
the growth medium. In an embodiment, the carrier and the growth
medium are located in a container covered with a gas or vapor
permeable but microorganism impermeable barrier. During the
disinfection or sterilization process, a germicide gas or vapor
diffuses from outside the container into the container through the
barrier.
[0014] Advantageously, the microorganism is a biological indicating
microorganism for the disinfection or sterilization process.
Preferably, the disinfection or sterilization process uses steam,
heat, ethylene oxide, hydrogen peroxide, ozone, chlorine dioxide,
peracetic acid, performic acid, formaldehyde, glutaraldehyde,
ortho-phthalaldehyde, or hypochorite salts as the disinfecting or
sterilizing agent.
[0015] Another aspect of the invention involves a self-contained
biological indicator including a carrier inside a container. The
carrier includes viable microorganisms. At least part of the
container is transparent, and the container has an opening which is
covered with a gas or vapor permeable but microorganism impermeable
barrier. At least one openable enclosure is located inside the
container. The enclosure contains a culture medium which is capable
of supporting growth of the viable microorganisms and a dye which
changes color with a change in pH from a first pH range to a second
pH range. The enclosure also contains dual buffer system made up of
a first buffer having a first pK.sub.a and a second buffer having a
second pK.sub.a. The first pK.sub.a and said second pK.sub.a are
within the first pH range and the second pH range,
respectively.
[0016] The carrier may be a porous substrate, a non-porous
substrate, an absorbent substrate, or a non-absorbent substrate.
Advantageously, the gas or vapor permeable but microorganism
impermeable barrier is a nonwoven polyolefin. Preferably, the
viable microorganism is a biological indicating microorganism for a
disinfection or sterilization process. In an embodiment, the
openable container is a breakable glass ampoule. Advantageously,
the dye is Bromcresol Purple. Preferably, the first buffer includes
at least one phosphate salt. In an embodiment, the second buffer
includes at least one acetate salt. Preferably, the acetate salt is
sodium acetate.
[0017] Advantageously, the dual buffer system contains a lower
concentration of buffer having a pK.sub.a in the first pH range
than buffer having a pK.sub.a in the second pH range. Preferably,
the self-contained biological indicator also has a cap with at
least one opening above the barrier, so that gas or vapor can
diffuse into said container through the hole and the barrier. In an
embodiment, the self-contained biological indicator also has a
chemical indicator for indicating exposure of the self-contained
biological indicator to a disinfection or sterilization
process.
[0018] Another aspect of the invention involves a culture medium
which is capable of supporting growth of viable microorganisms. The
culture medium includes a nutrient broth, a dye which changes color
with a change in pH from a first pH range to a second pH range, and
a dual buffer system. The dual buffer system contains a first
buffer having a first pK.sub.a and a second buffer having a second
pK.sub.a, where the first pK.sub.a and the second pK.sub.a are
within the first pH range and the second pH range,
respectively.
[0019] Advantageously the dye in the culture medium is Bromcresol
Purple. Preferably, the first buffer includes at least one
phosphate salt. In an embodiment, the second buffer includes at
least one acetate salt. In an exemplary embodiment, the acetate
salt is sodium acetate. Advantageously, the dual buffer system
includes a lower concentration of buffer having a pK.sub.a in the
first pH range than buffer having a pK.sub.a in the second pH
range.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a cross-sectional view of a self-contained
biological indicator suitable for use with the embodiments of the
method of the present invention;
[0021] FIG. 2 is a perspective view of the self-contained
biological indicator of FIG. 1; and
[0022] FIG. 3 is a graph showing bacterial growth (left hand axis)
and pH (right hand axis) versus time in a culture medium in a
biological indicator with a dual buffer system according to
embodiments of the method of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0023] The presence of viable microorganisms in a biological
indicator after completion of a sterilization cycle is often
determined with a pH indicator, because acidic byproducts are
formed when the microorganisms metabolize the growth medium. An
acidic shift in the pH in the growth medium is therefore an
indication of incomplete sterilization. However, it has been found
that pH-raising (basic) contaminants can enter the growth medium
after the pH sensitive dye has changed color due to acid generation
from bacterial growth. The basic contaminants can unintentionally
shift the pH of the growth medium, changing the color of the
indicator dye back to its original color so that the indicator
color is no longer stable. It has been found that the basic
contaminants can sometimes enter the growth medium, for example, by
leaching or diffusion out of the plastic material from which many
self-contained biological indicators are made.
[0024] The color reversion of the dye would falsely indicate that
no microorganism growth had occurred. If an observer simply looked
at the biological indicator at the end of the incubation period,
the observer would believe that there had been no bacterial growth,
because the dye would have reverted to its original color. Further,
if the observer monitored the color of the biological indicator on
a regular basis, he would see the color of the dye change from the
original color to the color of the dye in a basic solution and then
back to the original color. The observer would likely be confused
by the multiple color changes and might believe that the biological
indicator is defective. The recognition of the previously
unrecognized cause of the problem of dye reversion from leaching or
diffusion of basic contaminants into the growth medium is one
aspect of the method and the apparatus of the present
invention.
[0025] The embodiments of the apparatus and the method of the
present invention address the previously unrecognized cause of the
problem of dye reversion through the use of a dual buffer system.
Although single buffer systems have been previously used in
biological indicators, the need for a dual buffer system was not
previously recognized. The present invention therefore includes
both the recognition of a cause of the problem as well as the
solution for the problem.
[0026] Although the dual buffer system of the present invention is
described in the context of a dual buffer system for biological
indicators where the dual buffer system includes a small amount of
high pH buffer and a large amount of low pH buffer, it is to be
understood that the dual buffer system has broad application, and
the concept of the dual buffer system is not meant to be limited to
the embodiment of biological indicators. The broader application of
dual buffer systems will be described in more detail after the
description of the more specific application to biological
indicators.
[0027] The single buffer system previously used provided some
stability to the biological indicator to minimize the number of
false positives due to small amounts of acid impurities. For
example, if a small amount of acid were present in the atmosphere
in the sterilization system at the beginning of the sterilization
process, the acid would be retained by the buffer system in the
biological indicator with only a minimal pH change. The pH would
not be shifted sufficiently to cause a change of color in the
indicator dye in the biological indicator system.
[0028] The single buffer system stabilizes the pH in the system
from both acidic and basic conditions. Choosing a single buffer
with a pK.sub.a about 7 stabilizes the system at a pH of
approximately 7. A pH of about 7 is suitable for microorganism
growth.
[0029] With the dual buffer system according to embodiments of the
method and the apparatus of the present invention, one buffer
system has a pH range sufficient to allow the indicator dye to
change color if acid is generated from metabolism of the growth
medium by bacteria. The second buffer controls the pH after the dye
has changed color so that the pH of the system is not significantly
shifted by the basic impurities, causing the indicator dye to
revert to its original color. The dual buffer system overcomes the
previously unrecognized cause of the problem of color reversion of
the indicator dye.
[0030] FIG. 1 depicts a cross-sectional view of a sterility
indicator 10 suitable for use with the dual buffer system according
to the apparatus and method of the present invention. The structure
of the sterility indicator 10 of FIG. 1 is somewhat similar to the
SCBI described in U.S. Pat. No. 5,552,320, hereby incorporated by
reference, except that the sterility indicator of FIG. 1 has only
one inner container rather than the two inner containers of the
SCBI of U.S. Pat. No. 5,552,320. A wide variety of both biological
indicators and self-contained biological indicators (SCBIs) are
suitable for use in the method and the apparatus of the embodiments
of the invention, and the example of the sterility indicator 10 of
FIG. 1 is not meant to be limiting on the embodiments of either the
method or the apparatus of the present invention.
[0031] Returning to FIG. 1, transparent vial 12 has liquid
impermeable walls 14 and open end 16. A medium ampoule 20 inside
the transparent vial 12 contains a liquid culture medium 22. The
medium ampoule 20 is sealed, and the interior of the medium ampoule
20 is not in fluid communication with the interior of the
transparent vial 12 until the medium ampoule 20 is opened. A
carrier 24 is located at a bottom of the transparent vial 12 at an
end of the transparent vial 12 opposite the open end 16. The
carrier 24 is made of a material such as TYVEK.TM., stainless
steel, fiberglass, and the like. The carrier 24 may be a porous
substrate, a non-porous substrate, an absorbent substrate, or a
non-absorbent substrate. The carrier can be a disk or a strip or
have another suitable shape. If the carrier 24 retains or absorbs
disinfectant or sterilant, another chemical may be needed in the
sterility indicator 10 to neutralize or decompose the sterilant or
disinfectant.
[0032] The carrier 24 is inoculated with a predetermined
concentration of viable spores or other microorganisms. One
suitable microorganism is Bacillus stearothermophilus spores,
although other suitable microorganisms may also be used with the
apparatus and method of embodiments of the invention. Bacillus
stearothermophilus is normally used as a biological indicating
microorganism with steam sterilization processes. Bacillus subtilis
var. niger is used as a biological indicating microorganism with
ethylene oxide and dry heat sterilization processes. Either
Bacillus stearothermophilus or Bacillus subtilis var. niger may be
used as biological indicating microorganisms in the STERRAD.RTM.
process depending on the carrier and how easily the sterilant can
diffuse into the biological indicator. The STERRAD.RTM. process
utilizes a combination of hydrogen peroxide and plasma. Other
microorganisms are suitable for use with the method and the
apparatus of the embodiments of the invention, and the
microorganisms described above are not meant to be limiting.
[0033] The open end 16 of the transparent vial 12 is covered by a
gas or vapor-permeable, microorganism-impermeable closure sheet 26.
The closure sheet 26 can be made of a nonwoven fabric such as
TYVEK.TM., CSR wrap, or other suitable material. CSR wrap is
nonwoven polypropylene. TYVEK.TM. is a tradename for spun-bonded
polyethylene. In an exemplary embodiment, the closure sheet 26 is
made of a nonwoven polyolefin. Alternatively, the closure sheet 26
can be any kind of hydrophobic filter which is gas or vapor
permeable but impermeable to microorganisms. The closure sheet 26
is held in place by a cap 28. The cap 28 snaps over the open end 16
of the transparent vial 12 and is held in place by friction. A
chemical indicator 30 is preferably located at a top of the cap 28
and is held in place on the end of the cap 28 by a plurality of
flexible tabs 32. At least one hole 34 is located in the cap 28.
The hole 34 allows germicide to enter the cap 28.
[0034] The culture medium 22 in the medium ampoule 20 contains dye
which undergoes a visible change with a change of pH in the culture
medium 22. The visible change in the dye with a change in pH can be
detected through the walls 14 of the transparent vial 12. As used
in the present specification and claims, "transparent" is the
quality of the walls 14 that permits visible changes in the color
of the dye in the culture medium 22 to be detected from the
outside. The walls 14 of the transparent vial 12 may therefore be
translucent rather than transparent. Further, in some embodiments,
the walls 14 of the transparent vial 12 are opaque, and there is a
transparent or translucent window in the transparent vial 12 to
allow for observation of the color change of the dye.
Alternatively, all or a portion of the cap 28 may be transparent or
translucent.
[0035] The transparent vial 12 is preferably made of a flexible
plastic which is resistant to hydrogen peroxide. Polypropylene is
an exemplary plastic for use in fabricating the transparent vial
12. Although it is not necessary that the cap 28 be transparent or
translucent, in an exemplary embodiment, the cap 28 is transparent
or translucent and is made of the same plastic as the transparent
vial 12. In other embodiments, the cap 28 is made from a material
which is different than the material from which the transparent
vial 12 is made.
[0036] The medium ampoule 20 is made of a frangible material such
as glass. In an embodiment, the walls 14 of the transparent vial 12
are deformable so that the medium ampoule 20 can be opened (by
crushing, for example) without breaking the walls 14 of the
transparent vial 12.
[0037] The chemical indicator 30 is any indicator which indicates
exposure to a sterilant. One suitable chemical indicator 30 for
oxidative sterilants such as hydrogen peroxide is a chromate coated
aluminum alloy disk coated with Bordeaux Red dye, as described in
detail in U.S. Pat. No. 5,942,438, hereby incorporated by
reference. Bordeaux Red dye has the tradename Aluminum Bordeaux RL.
Other chemical indicators 30 are suitable for use with the
apparatus and the method of the embodiments of the present
invention. The chemical indicator 30 of U.S. Pat. No. 5,942,438
changes color from red to a yellow/gold color when exposed to an
oxidation-type sterilant. The chemical indicator 30 of U.S. Pat.
No. 5,942,438 can therefore be used as a means of determining
whether or not the sterility indicator 10 has been exposed to an
oxidative sterilant. Other forms of chemical indicators 30 can be
used to indicate exposure to sterilants such as steam, heat, or
ethylene oxide. The chemical indicator 30 is optional and is not
necessary for the embodiments of the apparatus or the method of the
present invention.
[0038] FIG. 2 shows a perspective view of the sterility indicator
10 of FIG. 1, showing in more detail how the cap 28 fits onto the
transparent vial 12. FIG. 2 also shows more clearly the tabs 32
which hold the chemical indicator 30 in place on the top of the cap
28. FIG. 2 also shows the hole 34 in the cap 28. The hole 34 allows
sterilant or germicide to enter the interior of the sterility
indicator 10.
[0039] The medium ampoule 20 of FIG. 1 contains a culture medium 22
and a dye. One suitable culture medium 22 is Tryptic Soy Broth
(available from SGM Biotech, Bozeman, Mont.). The dye has a first
color at high pH and a second color at low pH, so that the color in
the culture medium 22 changes if the pH changes from a high pH
range to a low pH range during incubation of the sterility
indicator 10 due to generation of acidic byproducts from the
metabolism of the culture medium 22 by the surviving
microorganisms. In other embodiments, the dye is in the transparent
vial 12 outside of the medium ampoule 20. The dye is then brought
into contact with the culture medium 22 when the medium ampoule 20
is opened.
[0040] The sterility indicator 10 is placed in the sterilizer with
the equipment to be sterilized. The equipment to be sterilized and
the sterility indicator 10 are then subjected to a sterilization
cycle. The sterilization cycle may be any suitable sterilization
cycle, including, but not limited to, sterilization cycles with a
disinfecting or sterilizing agent such as heat, steam, ethylene
oxide, plasma, hydrogen peroxide, a combination of hydrogen
peroxide and plasma, chlorine dioxide, ozone, peracetic acid,
formaldehyde, or any other suitable sterilization process. During
the sterilization cycle, the steam or germicide gas or vapor may
enter the sterility indicator 10 through the hole 34 in the cap 28.
The germicide gas or vapor can be a disinfectant or a sterilant. In
the context of this application, disinfectant is to be understood
to be a germicide which kills microorganisms in the vegetative
stage but does not necessarily kill spores. A sterilant is to be
understood to be a germicide which kills all microorganisms,
including spores. Because the closure sheet 26 on the open end 16
of the transparent vial 12 is vapor-permeable or gas-permeable, the
germicide gas or vapor can penetrate the closure sheet 26. The
microorganisms on the carrier 24 are therefore exposed to the
sterilizing atmosphere during the sterilization cycle. If heat is
used as the sterilization cycle, the sterility indicator 10 is
subjected to the same heat conditions as the equipment to be
sterilized.
[0041] After completion of the sterilization cycle, the sterility
indicator 10 is removed from the sterilizer, and the medium ampoule
20 is opened to expose the microorganisms on the carrier 24 to the
culture medium 22. The medium ampoule 20 may be opened in any
suitable manner. One method of opening the medium ampoule 20 is to
deform the walls 14 of the transparent vial 12 to crush the medium
ampoule 20, though any suitable method of opening the medium
ampoule 20 may be used.
[0042] The sterility indicator 10 is then placed in a conventional
incubator at a temperature and for a time suitable for growing the
microorganism in the culture medium. For example, with Bacillus
stearothermophilus spores, incubation of 48 hours or more at a
temperature of 58.degree. C. is suitable for growth of the
microorganism.
[0043] If the microorganism grows during the incubation cycle, the
metabolism of the culture medium 22 by the microorganism produces
acidic byproducts, lowering the pH of the culture medium 22. The
change in pH causes a change in color of the dye in the culture
medium 22. A change in the color of the dye during incubation of
the sterility indicator 10 indicates that sterilization was not
complete. The absence of a color change confirms that the
sterilization cycle was effective. In alternative embodiments, the
change in pH may be detected with a pH meter, pH paper, or by any
suitable pH measuring method.
[0044] FIG. 3 shows graphs of the growth curve of the bacteria
versus time (left hand scale) and the curve of pH versus time
(right hand scale) for a typical sterility indicator 10. As shown
in FIG. 3, the pH in the growth medium 22 changes from a high pH
range to a low pH range during the course of the incubation due to
generation of acid byproducts from the metabolism of the culture
medium 22 by the bacteria. If Bromcresol Purple dye is used as the
indicator dye, the Bromcresol Purple dye changes from purple above
a pH of about 6.8 to yellow below a pH of about 5.2. A change of
color from purple to yellow with a sterility indicator 10
containing Bromcresol Purple dye in the culture medium 22 therefore
indicates bacterial growth. The example of Bromcresol Purple is
used to illustrate the method, and the embodiments of the apparatus
and the method of the present invention are not limited to
Bromcresol Purple dye. (Bromcresol Purple dye is also known as
Bromocresol Purple dye.)
[0045] At the start of the incubation period, even a small amount
of acid contamination due to the presence of carbon dioxide or
other acidic impurities could shift the pH enough to change the
color of the dye. For example, in a system with Bromcresol Purple,
a shift in pH from 7 to about 6.0 would be sufficient to cause the
Bromcresol Purple indicator to start to change from the purple
color. A buffer is therefore commonly added to the culture medium
22 to reduce the sensitivity to false positives as a result of
small amounts of acidic contaminants. In order to make the system
as sensitive as possible and to make the response of the system as
fast as possible, only a small amount of buffer is used.
[0046] If base were to be introduced into the culture medium 22
after the pH in the culture medium 22 had decreased sufficiently
for the dye to change color, the pH in the culture medium 22 could
increase sufficiently to change the color of the dye back to the
original color. In the example of Bromcresol Purple, a shift in pH
from about 5.2 to above about 6 would change the color of the
Bromcresol Purple dye from yellow at low pH to purple at high pH,
falsely indicating that no bacterial growth had occurred.
[0047] Although the dye reversion would not be an issue if the
sterility indicator 10 were monitored regularly, the sterility
indicators 10 are sometimes not monitored until the end of the
seven day incubation period, long enough for dye reversion to take
place. In such a case of dye reversion, even though a positive
readout has occurred sometime during the seven day incubation
period, a subsequent readout would show that the original color had
returned, indicating a shift in pH toward the original pH value.
The reading is then unreliable, because it can lead to confusion
and uncertainty, in some cases leading to bacterial growth being
seen as no growth.
[0048] It has been found that a basic contaminant leaches or
diffuses out of certain batches of transparent vial 12, causing a
change in pH to above 6.0, with a resulting color change of the
Bromcresol Purple dye back to purple during the seven day
incubation period, a color reversion. The color reversion would
falsely indicate no bacterial growth. Color reversion is, of
course, not limited to Bromcresol Purple dye, and the example of
Bromcresol Purple dye is illustrative only. The recognition of the
reasons for the color reversion from leaching or diffusion of basic
contaminants is a recognition of a previously unrecognized cause of
a problem and is a part of the present invention.
[0049] Dual Buffer System
[0050] A dual buffer system for sterility indicators 10 has been
developed which eliminates the problem of color reversion while
retaining the speed and sensitivity of the determination of the
effectiveness of the sterilization process. Returning to FIG. 3,
the growth medium 22 contains a small amount of buffering agent.
The buffer stabilizes the pH somewhat against small additions of
ions that can change the pH of the solution, which could change the
color change of the indicator. Potassium phosphate, dibasic
(K.sub.2HPO.sub.4) is an exemplary buffer. A potassium phosphate,
dibasic buffer maintains the starting pH level at about 7.0, a pH
which is suitable for bacterial growth.
[0051] During bacterial growth, the metabolic acids and by-products
act to reduce the pH. When the buffering capacity of small amount
of buffer in the growth medium 22 is overcome, the pH value drops.
If Bromcresol Purple dye is used as the indicator dye, the
indicator dye changes color at a pH level around 5.2 or below,
giving visible indication of growth. The incubation and readout
period is 2-7 days. If the color change is observed during
incubation at any time before the end of the second day, the result
is recorded as positive growth of the biological indicator. If the
user is unable to read the sterility indicator 10 after the second
day, the sterility indicators may be kept up until seven days until
read.
[0052] In order to prevent reversion, one approach would be to
ensure that the contaminant level in the polypropylene transparent
vials 12 is kept very low. However, the amount of basic
contaminants necessary to shift the pH and cause reversion in the
present system is small, because of the small amount of buffer in
the present system. The one buffer system is not effective to
buffer the medium at the lower pH range. If an occasional lot of
polypropylene vials contained an basic contaminant which could
leach out of the polypropylene, dye reversion could occur, and the
contaminated lot of vials would have to be discarded.
[0053] However, if basic contamination is introduced, such as by
leaching or diffusion of ions from the material of the transparent
vial 12, the pH may shift back again toward neutral. If the pH
rises sufficiently, the indicator dye begins to return to the
original color, causing confusion or even giving a false negative
reading. Although the amount of buffer in the culture medium 22
could be increased to lessen the possibility of this color
reversion, increasing the amount of buffer would decrease the
sensitivity and speed of the method.
[0054] According to an embodiment of the method of the invention,
adding a second buffer with a buffering range near the end point of
the indicator acts to stabilize the pH in the region of the end
point. If Bromcresol Purple is used as the indicator dye, the end
point of the system is in the pH 4-5 range, where the dye is
yellow. Rather than adding more buffer having a pK.sub.a of about
7, which would decrease the sensitivity of the system, a second
buffer with a pK.sub.a of about 4-5, near the end point, is added
to the system. The second buffer with the pK.sub.a of about 4-5
tends to hold the pH steady in the pH 4-5 range. The second buffer
having a pK.sub.a in the 4-5 range stabilizes the system in the pH
range of 4-5 when the bacterial acid lowers the pH into this range.
Because the second buffer stabilizes the system in the pH 4-5
range, basic contaminants which leach or diffuse into the system
are neutralized by the second buffer, and the pH remains in the pH
4-5 range. Because only a small amount of the first buffer in the
pH 7 range is used, the system retains the speed and sensitivity of
the indicator readout. The larger quantity of buffer having a pKa
near the pH of the end point stabilizes the system after the end
point is reached, minimizing the possibility of dye reversion while
not decreasing the sensitivity of the system.
[0055] The advantages of the dual buffer system are that it:
[0056] 1. preserves the speed and sensitivity of the readout
while
[0057] 2. increasing the readout stability and reliability.
[0058] The dual buffer system thus eliminates the trade-off between
sensitivity and reliability as in the single buffer system.
[0059] If Bromcresol Purple is used as the indicator dye, the dye
changes color from purple to yellow in the pH range of about 6.8 to
about 5.2. A suitable dual buffer system for the system with
Bromcresol Purple includes a relatively small amount of buffer with
a pK.sub.a in the range of about 7 and a relatively large amount of
buffer with a pK.sub.a in the range of about 4-5.
[0060] The larger amount of buffer with a pK.sub.a in the range of
4-5 does not affect the sensitivity of the system, because the
color change indicating bacterial activity occurs in the pH range
of about 5-6, outside the buffering range of the buffer with a
pK.sub.a of 4-5. The small amount of buffer with a pK.sub.a of
about 7 therefore protects against false positives, while the
larger amount of buffer with a pK.sub.a of about 4-5 protects
against shifts in pH due to leaching or diffusion of base,
eliminating color reversion of the dye.
[0061] There are a variety of dual buffer systems and dyes which
are suitable for the sterility indicator 10 operating in the pH
range of 7 at the start and about 4-5 at the end. Phosphate buffer
(KH.sub.2PO.sub.4 and K.sub.2HPO.sub.4) with a pKa of 7.0 is a
suitable buffer for controlling the pH at the start of the
incubation period at the pH of approximately 7.0. The pH of about 7
is suitable for bacterial growth. The phosphate buffer is
preferably at a concentration of approximately 0.2 grams/liter
(about 1 mmolar).
[0062] Table 1 shows some suitable buffer systems with pK.sub.a's
in the range of 4-5. The buffer systems with pK.sub.a's in the 4-5
range are suitable for preventing dye reversion.
1TABLE 1 Buffer Systems Having pK.sub.a's in the 4-5 Range Chemical
Concentration pK.sub.a Citric Acid 0.01-0.1 M 4.76 Sodium Acetate
0.01-0.1 M 4.76
[0063] As seen in Table 1, the buffer having a pK.sub.a in the 4-5
range is present in a concentration of about 0.01-0.1 molar,
compared to approximately 1 millimolar for the buffer with a
PK.sub.a of about 7. The buffer with a pK.sub.a of 7 is therefore
present in a concentration of about 1/10 to 1/100 the concentration
of the buffer with a pK.sub.a in the 4-5 range. By using a small
amount of buffer with a pK.sub.a of 7, the sensitivity of the
method is retained.
[0064] Table 2 shows a series of dyes which are suitable for use in
the system shown in FIG. 3. All of the dyes have a pH range of
about 4-7.
2TABLE 2 Suitable Dyes Dye Concentration Color Change pH Range
Bromcresol Purple 8-12 mg/l Yellow-Purple 5.2-6.8 Chlorophenol Red
8-12 mg/l Yellow-Purple 4.8-6.6 Chrysoidin 8-12 mg/l Orange-Yellow
4.0-7.0 Alizarin 8-12 mg/l Yellow-Red 5.6-7.2
[0065] The dual buffer system is not limited to the embodiment of
having a pH change from 7 to a range of 4-5, as previously
described. The dual buffer system can be applied to any system in
which a pH change occurs. In the broader concept of the dual buffer
system, the dual buffer system is applicable to any system in which
a change in pH from a first pH range to a second pH range occurs,
where the pH change is indicative of a result. In the present case,
the pH change indicates bacterial growth and the ineffectiveness of
the sterilization cycle.
[0066] In the broad concept of the dual buffer system and method,
the dual buffer system contains a first buffer which has a pK.sub.a
in the first pH range and a second buffer which has a pK.sub.a in
the second pH range. The first buffer stabilizes the pH in the
region of the first pH range, and the second buffer stabilizes the
pH in the second pH range. The relative amounts of the two buffers
are chosen depending on whether it is important for the system to
be sensitive in the region of the first pH or the second pH. If it
is important to have sensitivity in the region of the first pH, a
small amount of the first buffer which has a pK.sub.a close to the
first pH is used. Similarly, if it is important to have sensitivity
in the region of the second pH, a small amount of the second buffer
which has a pK.sub.a close to the second pH is used. The relative
amounts of the first buffer and the second buffer are therefore
chosen to optimize the relative sensitivity of the system in the
region of the first pH range and the second pH range.
[0067] The two buffers are chosen depending on the pH range of the
pH-sensitive dye. One buffer is chosen so that the pK.sub.a of the
buffer is around or higher than the high end of the pH range of the
dye. The second buffer is chosen so that the pKa of the buffer is
around or lower than the low end of the pH range of the
pH-sensitive dye. The pKa's of the two buffers therefore bracket
the pH range of the dye. The relative quantities of the two buffers
are selected so that a lower concentration of buffer is used at the
end of the pH range of the dye where more sensitivity is desired.
The dye changes color from a first pH range to a second pH range.
The first pH range is the pH around or higher than the high end of
the pH range of the dye. The second pH range is the pH of the
buffer around or lower than the pH range of the dye.
[0068] The embodiments of the method of the present invention for
minimizing or eliminating dye reversion by using a dual buffer
system are not limited to self-contained biological indicators as
shown in FIG. 1. For example, the embodiments of the method are
applicable to a wide range of biological indicators. In particular,
the dual buffer system can be used with conventional biological
indicator carriers. It can also be applied to the biological
indicator carriers used for liquid disinfectant or sterilant, such
as glutaraldehyde, performic acid, peracetic acid, hydrogen
peroxide, formaldehyde, ortho-phthalaldehyde, or hypochlorite salts
such as sodium hypochlorite. When the method is applied to these
biological indicators, the carrier, after treatment with liquid,
gas, or vapor disinfectant or sterilant, is placed into a culture
medium 22 which contains a dual buffer system. The dual buffer
system according to embodiments of the method of the present
invention will eliminate or at least minimize the possibility of
dye reversion, when conventional biological indicator strips are
incubated in the culture medium which contains the dual buffer
system according to embodiments of the present invention.
[0069] The dual buffer system is also applicable to a wide variety
of self-contained biological indicators (SCBIs) such as the SCBI of
FIGS. 1 and 2, as well as a wide variety of other SCBIs. In
embodiments of the method of the present invention, the
conventional culture medium 22 which is utilized in many SCBIs is
augmented or supplemented with a dual buffer system according to
embodiments of the present invention.
[0070] A culture medium 22 according to embodiments of the
invention contains a nutrient broth, the dual buffer system, and a
pH-sensitive dye. In some embodiments, the dual buffer system
and/or the pH-sensitive dye are separate from the nutrient broth.
The nutrient broth, the dual buffer system, and the pH-sensitive
dye are brought into contact with each other when the medium
ampoule 20 is opened.
[0071] Although the dual buffer system has been described with
respect to a hydrogen peroxide/plasma sterilization system, the
dual buffer system can be applied to any system where a pH change
occurs. In particular, the dual buffer system can be applied to
indicators for sterilization with ethylene oxide, heat, steam,
hydrogen peroxide, plasma, a combination of hydrogen peroxide and
plasma, glutaraldehyde, peracetic acid, performic acid,
formaldehyde, ortho-phthaldehyde, hypochlorite salts such as sodium
hypochlorite, ozone, chlorine dioxide, or a variety of
germicides.
[0072] Various modifications and alterations of this invention will
be apparent to those skilled in the art without departing from the
scope and spirit of this invention. It is to be understood that the
invention is not limited to the embodiments disclosed therein, and
that the claims should be interpreted as broadly as the prior art
allows.
* * * * *