U.S. patent application number 09/812204 was filed with the patent office on 2002-04-18 for apparatus and method for growing anaerobic microorganisms.
This patent application is currently assigned to Oxyrase, Inc.. Invention is credited to Adler, Howard I., Copeland, James C., Spady, Gerald E..
Application Number | 20020045245 09/812204 |
Document ID | / |
Family ID | 26931027 |
Filed Date | 2002-04-18 |
United States Patent
Application |
20020045245 |
Kind Code |
A1 |
Copeland, James C. ; et
al. |
April 18, 2002 |
Apparatus and method for growing anaerobic microorganisms
Abstract
An apparatus for growing anaerobic microorganisms is provided
having a dish top that contains a sealing ring upon which the media
surface in the dish bottom rests when the apparatus is inverted.
The contact between the sealing ring and the media surface forms a
seal that traps the gas in the headspace between the media surface
and the inside of the dish top. A oxygen reducing agent can also be
incorporated into the media together, in some instances, with a
substrate which react with oxygen in the media and with oxygen in
the headspace thereby creating an environment suitable for growing
anaerobic, microaerophilic and facultative anaerobic
microorganisms.
Inventors: |
Copeland, James C.;
(Ashland, OH) ; Adler, Howard I.; (Oak Ridge,
TN) ; Spady, Gerald E.; (Oak Ridge, TN) |
Correspondence
Address: |
Richard M. Klein
FAY, SHARPE, FAGAN, MINNICH & McKEE, LLP
1100 Superior Avenue, Seventh Floor
Cleveland
OH
44114
US
|
Assignee: |
Oxyrase, Inc.
|
Family ID: |
26931027 |
Appl. No.: |
09/812204 |
Filed: |
March 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09812204 |
Mar 19, 2001 |
|
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|
09321812 |
May 28, 1999 |
|
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Current U.S.
Class: |
435/305.3 ;
435/303.2 |
Current CPC
Class: |
C12M 23/10 20130101;
Y10S 435/801 20130101 |
Class at
Publication: |
435/305.3 ;
435/303.2 |
International
Class: |
C12M 001/22 |
Claims
1. A culture dish assembly comprising: a first dish having a wall
and a side wall extending therefrom to define a cavity; a second
dish having a wall and a side wall extending therefrom that
cooperate with the first dish to form a non-sealed assembly in a
first orientation of the first and second dishes and a closed
headspace in a second, inverted orientation of the first and second
dishes.
2. The assembly of claim 1 wherein the wall of the first dish has a
substantially circular configuration.
3. The assembly of claim 2 wherein the side wall of the first dish
extends outwardly in a substantially perpendicular direction from a
periphery of the circular wall.
4. The assembly of claim 3 wherein the side wall of the first dish
extends in a first direction from the circular wall.
5. The assembly of claim 1 wherein the wall of the second dish has
a substantially circular configuration.
6. The assembly of claim 5 wherein the side wall of the second dish
has a generally U-shaped configuration in cross-section defining an
annular recess dimensioned to receive at least a portion of the
side wall of the first dish therein.
7. The assembly of claim 6 wherein the side wall of the second dish
includes first and second legs disposed in generally parallel
relation, and the first leg is longer than the second leg and
disposed radially outward thereof.
8. The assembly of claim 7 wherein the second dish further includes
an annular, circumferentially continuous seal disposed radially
inward of the side wall and is disposed in a plane intermediate a
terminal end of the first leg and the wall.
9. The assembly of claim 8 wherein the side wall of the first dish
has a height less than the first leg of the first dish so that the
annular seal is spaced from the wall of the first dish in both the
first and second orientations of the first and second dishes.
10. The assembly of claim 8 wherein the side wall of the second
dish is circumferentially continuous.
11. The assembly of claim 1 wherein the wall of the first dish is
substantially planar.
12. The assembly of claim 1 wherein the wall of the second dish is
substantially planar.
13. The assembly of claim 1 wherein the side wall of the first dish
tapers radially outward as it extends from the wall.
14. The assembly of claim 1 wherein the side wall of the second
dish includes first and second legs defining an annular recess
therebetween, the first leg tapering radially outward as it extends
substantially perpendicular from the wall.
15. The assembly of claim 14 wherein the second leg tapers radially
outward as it extends substantially perpendicular from the
wall.
16. The assembly of claim 14 wherein the wall of the first dish
includes a rib extending outwardly therefrom and radially
dimensioned to seat on a terminal end of the second leg of the
second dish.
17. The assembly of claim 16 wherein a terminal end of the side
wall of the first dish is dimensioned for radial receipt between
the first and second legs of the second dish.
18. The assembly of claim 14 wherein the first and second legs of
the second dish are connected by an interconnecting portion having
a raised protrusion for locating the wall of the first dish when
the first and second dishes are disposed in the first
orientation.
19. The assembly of claim 14 wherein the first and second legs of
the second dish are connected by an interconnecting portion that
has a radial dimension adapted to support the wall of a first dish
and a terminal end of a second leg of an adjacent second dish in a
first orientation of the dishes.
20. The assembly of claim 19 wherein the interconnection portion
includes a raised protrusion separating regions on which the wall
of a first dish and a terminal end of a second leg of an adjacent
second dish are supported in a first orientation of the dishes.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
Ser. No. 08/963/664, filed Nov. 3, 1997, now U.S. Patent No., which
is a continuation application of Ser. No. 08/237,773, filed May 4,
1994, now U.S. Pat. No. 5,830,746, issued Nov. 3, 1998.
BACKGROUND OF THE INVENTION
[0002] 1. Field of Invention
[0003] The present invention relates to an apparatus and method for
growing anaerobic microorganisms. The apparatus is comprised of a
specially designed culture dish which can be reconfigured such as
by inverting the dish to produce an anaerobic environment. An
oxygen reducing agent such as a biocatalytic oxygen reducing agent
can also be incorporated into the media present in the apparatus
together, in some circumstances, with a substrate. The biocatalytic
oxygen reducing agent and the substrate present in the media react
with oxygen enclosed in the culture dish to create an environment
suitable for growing and maintaining anaerobic microorganisms.
[0004] 2. Description of Prior Art
[0005] Microorganisms are important to our well being. This is
evident in health care, agriculture and industry. To be able to
simply and quickly isolate and grow microbes is economically
important. For example, being able to quickly and specifically
isolate and identify a microbe responsible for infection is
important in the human health care field. This basic technique is
also important in the agriculture industry. Large scale processing
of food requires constant microbial monitoring. The speed and
efficiency at which this can be done determines the length of time
finished food products must be held in storage before they can be
distributed for sale.
[0006] Control of the environment is necessary for control of
microbial growth. In particular, control of oxygen content in the
immediate environment is crucial for microbial growth.
Microorganisms can be divided into groups based on their need for,
and tolerance of, oxygen. There are those that require oxygen to
grow. These are "aerobes". Some microorganisms are able to grow
with or without oxygen. These are "facultative anaerobes". Another
group of microorganisms can grow only in the presence of very low
levels of oxygen. These are the "microaerophiles". Finally, some
microorganisms can not tolerate oxygen. They are inhibited by it or
may be killed by it. These are the "anaerobes".
[0007] This fundamental property of microorganisms, that is their
ability to grow in or tolerate oxygen, is used daily to isolate,
grow, and manipulate them. One basic technique in microbiology, is
the plating method. This generally involves the use of a dish,
developed by Petri (i.e. "Petri dish") in 1880's, and solidified
(agar or gelatin-based) medium.
[0008] A Petri dish is usually a round, shallow, flat-bottomed,
glass or plastic dish (often e.g. 10 cm diameter) with a vertical
side, that cooperates with a similar, slightly larger structure
which forms a loosely-fitting lid. Petri dishes are used in
microbiology, e.g., for the preparation of plates.
[0009] The purpose of the Petri dish is to provide a controlled
environment for selectively growing microbes. The dish is
sterilized and designed to maintain a sterile environment inside
while freely exchanging gases, normally air, with the outside
environment.
[0010] The medium utilized in conjunction with the Petri dish can
be formulated to provide a necessary and selective environment for
a specific microorganism. Solid medium in a Petri dish can be
prepared using aseptic technique by pouring sterile molten or
liquid (agar- or gelatin-based) medium into a Petri dish to a depth
of 3-5 mm and allowing it to set. Generally, freshly poured plates
to be used for separation and/or generation of microbes should be
left for 30 minutes in a 45.degree. C. hot-air incubator with the
lid partly off so that the surface moisture can evaporate. Such
"drying" before inoculation prevents unwanted spreading of the
inoculum in the surface film of the moisture.
[0011] The solid medium surface inside the dish provides a place to
grow microorganisms. By inoculating (or "plating") the surface of
the agar in a controlled way (i.e. "streaking"), single colonies of
a microorganism can be obtained. With this technique the
microbiologist can separate microbes one from another. Isolation
and purification is mandatory to further characterization and
study. Using this dish design, a microbiologist can isolate and
grow the great majority of microorganisms known today.
[0012] Working with microbes that are microaerophiles or anaerobes
poses a problem. The culture dishes for these microbes must be
incubated in a controlled gaseous environment that lacks oxygen, or
at least most of the oxygen, found in air. This is done by placing
the culture or Petri dish containing medium inside a container that
is sealed from the outside atmosphere. For one or a few dishes, a
sealable bag or jar (i.e., "Brewer Jar") is used (Becton Dickinson
Microbiology Systems, 1994 Catalog, p 89 p 94). In this case,
chemicals and a catalyst (see U.S. Pat. No. 4,287,306 issued Sep.
1, 1982 to Brewer entitled "Apparatus for Generation of Anaerobic
Atmosphere") are placed inside the container that, when activated
chemically, reacts with the oxygen in the container, thus removing
it. The catalyst is necessary to bring about the reaction at low
temperatures in a short time.
[0013] In addition, for many culture dishes, a sealed table-top
chamber can be used (Anaerobe Systems, San Jose, Calif.). This
chamber is evacuated and flushed with inert gases, such as nitrogen
and/or carbon dioxide. Sometimes chemicals and a catalyst are used
to consume the oxygen inside the chamber and fresh, inert gas is
supplied as needed. The microbiologist works with the culture
dishes inside of this chamber through ports fitted with gloves. A
means is provided for introducing materials into and taking items
out of the chamber without breaching the anaerobic environment
inside.
[0014] Work with microaerophiles and anaerobes under these
conditions is labor intensive, difficult, expensive, and time
consuming. The microbiologist is often frustrated by having to wait
for the slowest growing microbe in order to retrieve all culture
dishes from a bag or jar since once the bag or jar is opened, the
microbes are exposed to oxygen. A failure in the system can be
catastrophic for all of the microbial isolates inside.
[0015] To overcome many of these problems (see U.S. Pat. No.
2,348,448 issued May 9, 1944 to Brewer entitled "Apparatus for the
Cultivation of Anaerobic and Microaerophilic Organisms") Brewer
developed a culture dish lid (i.e., "Brewer Lid") that formed a
seal between a ring inside the lid with the agar or gelatin-based
surface. Within the dish, a very small, defined headspace is formed
by the lid and the agar surface. An anaerobic environment is
created inside this trapped headspace by reacting oxygen with
chemical reducing agents, such as thioglycollate, incorporated in
the medium. The limited volume of the headspace is important to the
function of the Brewer Lid.
[0016] However, a number of drawbacks exist in the use of the
Brewer Lid. The capacity and the rate for oxygen removal is limited
by the sensitivity of the microorganism to the chemical reducing
agent in the medium (see "Mechanism of Growth Inhibitory Effect of
cysteine on Escherichia coli." of Kari, et al., J. Gen. Microbiol.,
68, 1971, p. 349 and "Methods for General and Molecular
Bacteriology", Editor: Gerherdt, American Society for Microbiology,
1994, p. 146.). Moreover, the lid is made of heavy glass and is
expensive. It is available today (Kimble Glass Company, Vineland,
N.J.), but is not widely used because of serious limitations that
include cost, handling difficulties, and poor response of anaerobic
microorganisms.
[0017] Another limitation is caused by the material of
construction. The glass Brewer Lid is made very heavy to insure a
good seal between the ring inside the Brewer Lid and the agar
surface. Cultures dish bottoms fitted with the heavy Brewer Lid are
not easy to handle or to move about. They can not be stacked inside
an incubator. Thus, precious incubator space is wasted. Stacked
dishes crush the agar medium of the lowest dishes in the stack,
because of the weight of the dishes above them. This causes the
headspace above the agar to collapse resulting in contact between
the inside of the Brewer Lid and the agar surface. When this
happens, the microbial growth on the surface is spread out and
separation of individual colonies is lost. Motile microbes will
migrate and further frustrate separation.
[0018] Because of their weight and material of construction, Brewer
Lids do not lend themselves to commercial production of pre-made
agar or gelatin-based plates. The commercial process requires
assembly line filling of the dishes, packaging the filled dishes in
stacks, and handling and storing these dishes. Pre-made agar plates
are widely used in clinical microbiological laboratories. This
limitation of the Brewer Lid is economically significant.
[0019] The headspace inside the Brewer Lid formed by the lid and
agar surface is very small. This limited headspace is determined by
the ability of the chemical reducing agent (H.sub.2S, cysteine,
thioglycollate, etc.) to reduce oxygen in the headspace. The amount
of chemical reducing agent used in the medium in turn is
constrained by anaerobic microorganism's sensitivity to it. The sum
of these limitations is a very small head space that imparts severe
problems to the function of the Brewer Lid for its intended
purpose, i.e. to grow anaerobic and microaerophilic
microorganisms.
[0020] Another limitation of the Brewer Lid is that the very
limited head space can not hold much moisture. Fresh agar medium is
generally greater than 98 percent water. Upon incubation, water in
the medium evaporates and condenses upon the upper surface of the
inside of the lid. This condensate can become sufficient to fall to
the agar surface and to flood it. Under such conditions, the plate
is ruined and can not be used for isolation and purification of the
microbe.
[0021] The very limited headspace imposes still more limitations on
the Brewer Lid. No provision is made to incorporate CO.sub.2 into
the headspace above the agar surface. This is important for the
rapid growth of some microorganisms and may be required by others.
Yet this feature should be made optional for the microbiologist,
because for some uses of the culture dish the microbiologist may
not want to include CO.sub.2 in the headspace. Reports show that
CO.sub.2 can change the pH of the medium it contacts. This in turn
can interfere with the determination of susceptibility to some
antibiotics (see "Effect of CO.sub.2 on Susceptibilities of
Anaerobes to Erythromycin, Azithromycin, Clarithromycin, and
Roxithromycin", Spangler, et al., Antimicrob. Agents Chemotherapy,
38, p. 20, 1994). Since CO.sub.2 is generated in anaerobic jars and
bags by commercial catalysts products, this problem is commonly
encountered. CO.sub.2 is a component of the gas used to flush
anaerobic chambers and incubators too.
[0022] Another desired feature for a self contained culture dish is
an indicator to show that the headspace is anaerobic. These
features are difficult to impossible to include in the Brewer Lid
because of the very small space between inside the lid top and the
agar surface.
[0023] Several attempts have been made to design a culture dish
that provides a self-contained environment for growing anaerobic
microorganisms (see U.S. Pat. No. 2,701,229 issued Feb. 1, 1955 to
Scherr entitled "Apparatus for the Cultivation of Microorganisms";
U.S. Pat. No. 3,165,450 issued Jan. 12, 1965 to Scheidt entitled
"Anaerobic Culturing Device"; U.S. Pat. No. 4,294,924 issued Oct.
13, 1981 to Pepicelli, et al. entitled "Method and Container for
Growth of Anaerobic Microorganisms"; U.S. Pat. No. 4,299,921 issued
Nov. 10, 1981 to Youssef entitled "Prolonged Incubation
Microbiological Apparatus and Filter Gaskets Thereof"; and U.S.
Pat. No. 4,859,586 issued Aug. 8, 1989 to Eisenberg entitled
"Device for Cultivating Bacteria"). The fact that the Brewer Lid
and none of these inventions are commonly or commercially available
or used widely by microbiologists today, attest to their
limitations and shortcomings. The need to simplify and reduce the
cost for isolating and growing anaerobic and microaerophilic
microorganisms still exists today.
[0024] It is therefore an object of the present invention to
provide an improved apparatus and method for cultivating and/or
enumerating anaerobic microorganisms which obviate the
above-mentioned disadvantages of the prior art.
[0025] Another object of the present invention is to provide an
improved anaerobic culturing apparatus which is extremely simple,
inexpensive and easy to use and wherein the proper anaerobic
environment is produced and maintained in an extremely efficient
manner.
[0026] These and other additional objects and advantages of the
present invention will become apparent from the following
description of the invention.
SUMMARY OF THE INVENTION
[0027] The present inventors have designed a novel culture
apparatus or dish in order to eliminate many of the difficulties
observed in the prior art. It has been found that the use of the
new culture dish (i.e., "OxyDish.TM.") together with an oxygen
reducing agent (preferably a biocatalytic oxygen reducing agent)
and, in some instances, a substrate, produces a controlled,
self-contained environment for isolating, enumerating, identifying
and growing facultative aerobes, microaerophiles and anaerobes. The
use of the specially designed culture dish along with an oxygen
reducing agent makes possible the design and function of a culture
dish that utilizes some features of the Brewer Lid, but overcomes
its limitations and makes possible novel and improved
characteristics.
[0028] In this regard, the present invention is directed to a
specifically designed culture dish with a dish top or cover that
contains a sealing Sing on the inside upon which the solid media
surface in the bottom dish rests when the dish is inverted to form
a media-ring seal. The seal so formed traps the gas in the
headspace between the media surface and the inside of the dish top
or cover. In addition, an oxygen reducing agent, such as a
biocatalytic oxygen reducing agent, can be incorporated into the
media present in the culture dish together, in some instances, with
a substrate which reacts with oxygen in the media and the headspace
to create an environment suitable for growing anaerobic
microorganisms.
[0029] The preferred biocatalytic oxygen reducing agent (see "A
Novel Approach to the Growth of Anaerobic Microorganisms" of Adler,
et al., Biotechnol. Bioegn. Symp. 11, J. Wiley & Sons, New
York, 1981, p. 533 and U.S. Pat. No. 4,476,224 issued Oct. 9, 1984
to Adler entitled "Material and Method for Promoting the Growth of
Anaerobic Bacteria") utilized in the invention is comprised of
oxygen scavenging membrane fragments which contain an electron
transport system which reduces oxygen to water in the presence of a
hydrogen donor. These oxygen scavenging membrane fragments can be
derived from the cytoplasmic membranes of bacteria (U.S. Pat. No.
4,476,224) and/or from the membranes of mitochondrial organelles of
a large number of higher non-bacterial organisms. Other known
biocatalytic oxygen reducing agents such as glucose oxidase,
alcohol oxidase, etc. can also be utilized.
[0030] The biocatalytic oxygen reducing agents suitable for use in
the invention are non-toxic to microorganisms. Being catalysts,
they are dynamic and highly efficient at reducing the oxygen in the
trapped headspace in the specially designed culture dish. The
biocatalytic oxygen
[0031] In this regard, the present invention is directed to a
specifically designed culture dish with a dish top or cover that
contains a sealing ring on the inside upon which the solid media
surface in the bottom dish rests when the dish is inverted to form
a media-ring seal. The seal so formed traps the gas in the
headspace between the media surface and the inside of the dish top
or cover. In addition, an oxygen reducing agent, such as a
biocatalytic oxygen reducing agent, can be incorporated into the
media present in the culture dish together, in some instances, with
a substrate which reacts with oxygen in the media and the headspace
to create an environment suitable for growing anaerobic
microorganisms.
[0032] The preferred biocatalytic oxygen reducing agent (see "A
Novel Approach to the Growth of Anaerobic Microorganisms" of Adler,
et al., Biotechnol. Bioegn. Symp. 11 J. Wiley & Sons, New York,
1981, p. 533 and U.S. Pat. No. 4,476,224 issued Oct. 9, 1984 to
Adler entitled "Material and Method for Promoting the Growth of
Anaerobic Bacteria") utilized in the invention is comprised of
oxygen scavenging membrane fragments which contain an electron
transport system which reduces oxygen to water in the presence of a
hydrogen donor. These oxygen scavenging membrane fragments can be
derived from the cytoplasmic membranes of bacteria (U.S. Pat. No.
4,476,224) and/or from the membranes of mitochondrial organelles of
a large number of higher non-bacterial organisms. Other known
biocatalytic oxygen reducing agents such as glucose oxidase,
alcohol oxidase, etc. can also be utilized.
[0033] The biocatalytic oxygen reducing agents suitable for use in
the invention are non-toxic to microorganisms. Being catalysts,
they are dynamic and highly efficient at reducing the oxygen in the
trapped headspace in the specially designed culture dish. The
biocatalytic oxygen reducing agents use substrates that are
commonly found in microbiological media and that are natural to
microorganisms to effect this reaction. The products produced from
this reaction are also natural and non-toxic to microorganisms. The
use of the biocatalytic oxygen reducing agents makes possible the
opening and closing of this dish several times and the agents
continue to reduce the oxygen trapped in the headspace after each
occurrence.
[0034] The culture dish ("OxyDish.TM.") containing the oxygen
reducing agent provides a means to work with microorganisms free of
the complications and expense of anaerobic bags, jars, incubators,
or chambers. Each dish is light in weight and is designed to be
stacked without crushing the solid (agar or gelatin-based) medium
in the lower dishes in the stack. The dishes can be made of low
cost materials, preferably plastic, are designed to be readily
molded, are sterilizable, and preferably can be disposed after use.
Because of the incorporation of a biocatalytic means of removing
oxygen, an enlarged headspace is possible. This enlarged headspace
relieves the moisture condensation problems encountered with the
Brewer Lid.
[0035] Moreover, the dish top of the culture dish in certain
embodiments of the present invention, has a small dome or cavity
designed to contain an anaerobic gas (such as CO.sub.2) generating
pad or indicator strips to show the anaerobic state within the
headspace of the closed culture dish. A variation of this dish
design provides for additional removal of moisture from the dish as
needed by placing pores in the bottom of the dish base. This
feature prevents the build-up of excess condensate inside the dish
which leads to flooding of the agar media surface. The pores are
too small to let molten agar media flow out of the dish, yet they
provide an exit for moisture. An oxygen intruding into the dish
through these pores must pass through the media containing the
oxygen reducing agent. This intruding oxygen is removed before it
can diffuse to the top layer of media or into the headspace where
it would interfere with growth of anaerobic microorganisms.
[0036] The culture dish, i.e., "OxyDish.TM." of the present
invention, is designed for automated preparation of agar or
gelatin-based media plates necessary for commercial production.
When in the upright position, the dish can be readily filled with
molten medium (such as a molten agar or gelatin-based media)
without the sealing ring contacting the medium surface. When stored
or used, the dish is placed into an inverted position. In this
position, a seal (i.e. a media-ring seal) is formed by the contact
of the sealing ring of the dish top with the media surface
contained in the dish bottom when the media surface comes to rest
on the sealing ring. This creates a headspace defined by the media
surface, the inside wall of the sealing ring, and the inside top of
the dish lid.
[0037] Furthermore, when the culture dish is utilized with the
oxygen reducing agent such as a biocatalytic oxygen reducing agent,
the oxygen reducing agent in the media reacts with the oxygen
trapped in that headspace. This reaction renders the headspace
sufficiently low in oxygen such that microorganisms affected by the
presence of oxygen can grow on the media surface typically within
24 to 48 hours when the dish is incubated at 35.degree. C. to
37.degree. C. in an aerobic incubator. Any oxygen that intrudes
into the dish around the media ring-seal or through the plastic is
removed by the action of the reducing agent. The catalytic reducing
agent facilitates the design and function of this dish.
[0038] The media suitable for use in the present invention includes
any solid type media which can be inverted to form a media
ring-seal. Solid media generally consists of liquid media which
have been solidified ("gelled") with an agent such as agar or
gelatin. Examples of other known suitable gelling agents include
alginate, gellan gum ("Gelrite.TM.") and silica gel ("Pluronic
Polyol F127.TM."). The solid type media is of such a composition to
support growth of anaerobes, microaerophiles and facultative
aerobes.
[0039] Further, the culture dish, i.e., "OxyDish.TM.", of the
present invention, is designed in certain embodiments so that it
can be stacked in a stable configuration. The dish top has a
stacking ring that interlocks with the adjacent dish top below it.
The dish bottom, when the assembled dish is inverted and placed in
a sealed position, rests (i.e., nests) between the two adjacent
dish tops. The functionality of the dish to establish and maintain
an anaerobic environment is preserved and protected in the stack.
The stackability of the culture dish increases the efficient use of
incubator space. Stackability is also important for the mechanized
filling of these dishes and shipment of dishes or of finished
pre-made, plates to the microbiologist or end user.
[0040] The culture dish of the present invention, simplifies
handling anaerobes by the microbiologist or laboratory technician.
The culture dish, i.e., "OxyDish.TM.", can be opened and closed
several times while continuing to generate an anaerobic environment
in the closed position. The specially designed culture dish
significantly increases the microbiologist's efficiency by reducing
and simplifying the number of manipulations required to work with
anaerobes. Furthermore, the microbiologist can now treat each
culture dish and its microbial contents individually. This allows
the microbiologist to make decisions based on his observations of
each isolate or treatment, rather than having to wait for the
slowest growing isolate in a group of culture dishes present in a
sealable jar, bag, etc. In addition, the self-contained,
environmentally controlled culture dish provides a secure
environment for the microbe inside.
[0041] The foregoing has outlined some of the most pertinent
objects of the invention. These objects should be construed to be
merely illustrative of some of the more prominent features and
applications of the intended invention. Many other beneficial
results can be attained by applying the disclosed invention in a
different manner or by modifying the invention within the scope of
the disclosure. Accordingly, other objects and a more detailed
understanding of the invention may be had by referring to the
drawings, the detailed description of the invention and the claims
which follow below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] The following is a brief description of the drawings which
are presented for the purposes of illustrating the invention and
not for purposes of limiting the same.
[0043] FIG. 1 is a cross-sectional view of a two-part culture dish
for growing anaerobic microorganisms shown in separated or exploded
relation.
[0044] FIG. 2 is a cross-sectional view of the assembled culture
dish of FIG. 1 shown in what is generally referred to as a first or
upright position.
[0045] FIG. 3 is a cross-sectional view of the assembled culture
dish shown in what is generally referred to as a second or inverted
position.
[0046] FIGS. 4A is a top view of a first component or bottom dish
of the culture dish.
[0047] FIG. 4B is a side elevation view of the bottom dish.
[0048] FIG. 4C is a bottom view of the bottom dish.
[0049] FIG. 4D is an enlarged view of the side wall of the bottom
dish.
[0050] FIG. 5A is a top view of the second component or dish top or
cover of the culture dish.
[0051] FIG. 5B is a side elevational view of the dish top.
[0052] FIG. 5C is a bottom view of the dish top.
[0053] FIG. 5D is a sectional view of the dish top taken generally
along the lines 5D-5D of FIG. 5C.
[0054] FIG. 6 is a side elevational view of two assembled culture
dishes stacked vertically in an upright position.
[0055] FIG. 7 is a side elevational view of three assembled culture
dishes stacked vertically in an inverted position.
[0056] FIGS. 8A and 8B are photographs exhibiting growth of
anaerobic organisms in the culture dish of the present
invention.
[0057] FIG. 9 is a cross-sectional view of another preferred
embodiment of a base or bottom dish of the culture dish
assembly.
[0058] FIG. 10 is a cross-sectional view of another preferred
embodiment of a lid or cover of the culture dish assembly.
[0059] FIG. 11 is an enlarged cross-sectional view of multiple
culture dish assemblies stacked in a first orientation.
DETAILED DESCRIPTION OF THE INVENTION
[0060] The culture dish of the present invention is designed to
meet the strict requirements of anaerobiosis while simplifying
handling by the microbiologist. As shown in the drawings, a culture
dish 10 includes two separately configured parts. A first component
or dish bottom 12 receives a culture media 14 and a dish cover or
top 16 defines a second component of the culture dish. The dish
bottom and dish top selectively cooperate to define a culture dish
for growing microorganisms. Together, the dish bottom 12 and dish
cover 16 define in a first position or orientation a covered Petri
culture dish as shown in FIG. 2. This first position will be
referred to as an upright position. When inverted (FIG. 3), the
dish bottom and dish cover alter their cooperative configuration to
define a second position or orientation that forms an enclosed
chamber or head space 80 in which anaerobic microorganisms 20 can
be cultivated.
[0061] The structural and functional details of the dish bottom 12
will be described with reference to FIGS. 1-3, and more
particularly with reference to FIGS. 4A-4D. The dish bottom is
comprised of a generally planar base 22 and a circumferentially
continuous side wall 24 extending generally orthogonally from the
base. For purposes of discussion only, the side wall will be
described as extending upwardly from the base as illustrated in
FIGS. 1, 2, and 4. It will be recognized, however, that any
directional description is merely for purposes of simplifying an
understanding of the present invention.
[0062] In addition, the dish bottom 12 has a first or inner surface
26 that faces inwardly over the base and side wall toward the
cavity defined by the cup-shaped arrangement of the base and side
wall. Likewise, a second or outer surface 28 faces away from the
cavity and encompasses the exterior surfaces of the base and side
wall. The inner surface of the side wall is preferably divided by a
lip 30 into first and second portions 32, 34. The lip 30 can be
optionally provided on the inner surface 26 of the side wall of the
dish bottom and acts as a guide for indicating the fill height of
the culture media 14. The first portion of the side wall defines an
upper rim 36 and the second portion 34 joins the upper rim to the
base 22. As illustrated, the base and side wall are shown as a
one-piece construction such as a molded arrangement, although other
equivalent configurations can be used without departing from the
scope and intent of the invention.
[0063] The dish bottom 12 can be of any convenient dimension, and
is usually circular so that this dimension is referenced as a
diameter. Typically, the diameter of the dish bottom is about eight
(8.0) to fifteen (15.0) cm. The depth of the dish bottom 12 defined
by the height of the side wall as it extends upwardly from the base
can vary and is generally about 0.8 to 1.8 cm. In certain
embodiments (FIG. 4C), the base 22 of the dish bottom 12 can be
divided into two, three, four or more sections 38 by sectional
dividers, grid markings or other indicia 40 to enhance differential
diagnostics of microorganisms (FIG. 4C).
[0064] The dish cover or top 16 (FIGS. 1 and 5A-5D) is sized to fit
or conform over the dish bottom 12. The dish cover includes a top
wall 50, first and second side walls 52, 54, and a seal ring 56.
The top wall is disposed at approximately mid-height of the outer
side wall 52 for reasons which will be described in greater detail
below. In a manner similar to the dish bottom, the side walls 52,
54 are disposed generally orthogonal to the top wall and are
themselves radially spaced apart by a gap or recess 58. The side
walls are joined along one end by an interconnecting wall 60 to
define an inverted, generally J-shaped configuration when the
culture dish components are disposed in the first or upright
position (FIGS. 1 and 2).
[0065] The dish cover has a first or inner surface 70 that
generally faces the dish bottom when the individual components of
the culture dish are assembled. A second or outer surface 72
extends over the exterior of the dish cover.
[0066] The seal ring 56 projects outwardly or downwardly from the
inner surface 70 of the top wall 50. The ring is circumferentially
continuous and located along the radial periphery of the top wall.
It also interconnects along its outer radial edge with the inner or
second side wall 54. The seal ring has a planar seal face 78 that
cooperates with the culture medium 14 in a dish bottom to define an
anaerobic environment for growing microorganisms when disposed in
an inverted position (FIG. 3) and as will be described in greater
detail below.
[0067] The recess 58 in the dish cover is defined between the first
and second side walls. When the dish cover 16 is assembled with the
dish bottom 12 and placed in an upright position (FIG. 2), the side
wall 24 of the dish bottom is received in the recess 58 of the dish
cover. The recess can vary in width depending upon the overall size
and configuration of the dish cover and dish bottom.
[0068] Further, when the cover 16 is joined with the dish bottom 12
and positioned in an upright position, the height of the side wall
52 of the dish cover is sufficient to keep the planar seal face 78
of the seal ring 56 from contacting the medium surface 14. This
allows the freshly poured plate with molten agar, to cool and
solidify before the media surface of the dish bottom 12 can rest on
the seal ring in the dish cover 16 when the assembled culture dish
10 is inverted (FIG. 3). This feature also provides a means for
producing finished plates in a continuous manner by mechanized
means on a conveyor belt for large scale commercial production
while maintaining aseptic conditions.
[0069] When dish bottom 12 is filled with solidified media 14 and
the assembled culture dish 10 is inverted, the solidified media
surface will come into contact with the seal ring 56 of the dish
cover 16 forming a media-ring seal along the planar seal face 78
(see FIGS. 3 and 7). In this inverted configuration of the culture
dish, an oxygen reducing agent present in the media 14 will remove
all of the oxygen that is trapped in the head space of the enclosed
chamber 80 formed between the surface of the solidified media and
the inner surface 70 of dish cover. The assembled culture dish can
be incubated aerobically in the inverted position while producing
an internal anaerobic environment for the growth of anaerobic
microorganisms 20.
[0070] In addition, in certain embodiments of the invention, a
raised area, dome or cavity 82 is present in the dish cover 16
(FIG. 1). Specifically, projecting outward from the top wall 50 of
the dish cover is a dome 82 designed to contain an anaerobic gas
(CO.sub.2, etc.) generating pad or an indicator strip (not shown).
The dome 82 is comprised essentially of a dome side wall 84 and a
dome top wall 86, although a circular dome is the more preferred
embodiment. It is understood by those skilled in the art that domes
or cavities of alternative shapes and sizes can be utilized with
equal success.
[0071] In accordance with the illustrated embodiment, strengthening
ribs 90 are peripherally spaced along side wall 52. The ribs are
preferably equally spaced about the circumference of the dish cover
and protrude radially outward from the exterior surface of the side
wall. The ribs provide additional rigidity and strength to the dish
cover which is particularly helpful when the culture dishes are
stacked in either the upright or inverted positions as shown in
FIGS. 6 and 7.
[0072] The dish cover 16 can be of any convenient diameter so long
as it mates with the dimensions of the dish bottom 12. Typically,
the dish cover 16 is approximately nine (9.0) cm to sixteen (16.0)
cm in diameter. The seal ring 56 can be of any desired diameter and
is generally about seven (7.0) cm to fourteen (14.0) cm in diameter
and is centrally positioned relative to the side wall 52 of the
dish cover. The overall radial dimension of the seal ring 56 can
vary with a preferred radial dimension being about two-tenths (0.2)
cm to one-half (0.5) cm.
[0073] Moreover, in some embodiments, the base 22 of the dish
bottom 12 contains an indicator ring 100 (FIG. 4A). The area or
annulus 102 between the indicator ring 100 of base and the side
wall 24 identifies the area on the media surface 14 that the seal
ring 56 will occupy when the assembled culture dish 10 is inverted.
This area is not to be used for culturing microorganisms. Microbes
present in this area will swarm around the seal ring 56 when the
dish top is placed in contact with the culture media.
[0074] The difference in the height of the side wall 52 of the dish
cover in relation to the height of the side wall 24 of the dish
bottom can also vary, with a height differential of about one-half
(0.5) cm being preferred. The fill height 110 in FIG. 1 and FIG. 4D
is the distance from the base 22 of the dish bottom 12 to the
surface level of the culture media 14 and is variable. Typically,
this height can be two-tenths (0.2) cm to four-tenths (0.4) cm. The
dimension from the top of the culture media 14 surface, which is
generally indicated by the inner lip 30 to the top edge of the dish
bottom is (D) and is determined by the relationship D=A-C. (see
FIG. 4D, wherein (A) is the total height of the side wall 24 of the
dish bottom 12 and (C) is the fill height of the culture
media).
[0075] The seal ring 56 inside the dish cover extends a distance
downward from the dish cover 16 equal to (E) in FIG. 5B which is
determined by the relationship E=B-(Cmax+0.1 cm), where (B) is the
total height of the side wall 52 of the dish cover and (Cmax) is
the maximum fill height of the culture media 30. This assures that
the seal ring 56 clears the culture medium surface by approximately
one-tenth (0.1) cm when the dish bottom 12 is filled to its maximum
level and the assembled culture dish 10 is in its upright position
(FIG. 2).
[0076] The distance between the top edge of the side wall 24 of the
dish bottom and the upper extreme of the inner surface 70 of the
dish cover 16 when the culture dish is oriented in the upright
position is (F) in FIG. 6. In a preferred embodiment, F is
determined by F=B-A, where (B) is the total height of the side wall
52 of the dish cover 16 and (A) is the total height of the side
wall 24 of the dish bottom 12.
[0077] The depth of the headspace or enclosed chamber 80 below the
media surface 38 formed when the assembled culture dish is inverted
to form a media-ring seal is determined by the dimension (G) in
FIG. 7. This dimension can vary depending on the size of the dish
top 16, but typically ranges between two-tenths (0.2) cm to
one-half (0.5) cm. The dimension (H) in FIG. 7 is the height of the
top wall from an upper edge of the dish cover. It is determined
according to the following relationship H=E-G, where (E) is the
height of the inner side wall 54 of the dish cover and (G) is the
height of the headspace 80.
[0078] The dish cover preferably includes one or more cut-out areas
112 (FIG. 5B) in a portion of the side wall 52. These cut-out areas
112 facilitate the grasping and separation of the dish bottom 12
from the dish cover in an assembled culture dish 10. The cut-out
areas may be variably or constantly spaced from each other in the
side wall 52 of the dish cover. As shown, one preferred arrangement
has two cut-out areas 112 in the side wall 52 that are equidistant
from each other. Likewise, the particular configuration of the
cut-out areas may vary without departing from the scope and intent
of this invention.
[0079] Moreover, in the preferred embodiment of the invention, the
assembled culture dishes 10 are designed to be stacked one on top
of another. A dish bottom 12 of one assembled culture dish is
nestled between stacked dish covers 16 (see FIG. 6) in the upright
positions. In this regard, each dish cover has a stacking ring or
protruding rib 120 around the upper edge of the dish cover (FIG.
1). While the diameter of the stacking ring 120 can vary, it is
generally about one-half (0.5) mm to one (1.0) mm less than the
overall peripheral diameter of the dish cover 16. This provides an
outer radial ledge 122 upon which the bottom edge of the side wall
52 of an adjacent dish cover rests when placed either in an upright
(FIG. 6) or inverted position (FIG. 7). The projection of the
stacking ring 120 is preferably about one-half (0.5) mm to one and
one-half (1.5) mm in height. The stacking ring 120 prevents an
adjacent dish cover from sliding laterally and upsetting the
stacked arrangement (see, for example, FIGS. 6 and 7).
[0080] Similarly, the stacking ring 120 on the dish cover 16
radially contains or nests an adjacent dish bottom 12 when stacking
is desired in an upright position (see FIG. 6). The stacking ring
120 defines a radial inner ledge 124 to impede slide-out of the
enjoining dish bottom 12. The stacking ring is preferably one-half
(0.5) mm to (1.5) mm in width.
[0081] The minimum fill height (Cmin) to which the dish bottom 12
can be filled with culture media 14 and have the media surface rest
on the seal ring 56 when the dish is in an inverted position is
determined by Cmin=A-E, wherein (A) is the total height of the side
wall 24 of the dish bottom 12 and (E) is the height of the inner
side wall 54 of the dish cover. If the fill height of the culture
media 14 is below this level, then the upper rim of dish bottom 12
rests in contact with the inner surface 70 of the dish cover 16
rather than the media surface 14 resting on the seal ring 56 of the
dish cover when the assembled culture dish is inverted. In this
situation, there is no seal formed between the seal ring 56 and the
medium surface 14. The sealed headspace 80 is not formed. This
condition renders the assembled culture dish 10 useless for one
designed purpose of the culture dish which is to provide a
self-contained environment for the isolation and growth of
microaerophiles and anaerobes.
[0082] A variant of the culture dish contains one or more
perforations or pores 132 in the dish bottom 12 for the purpose of
controlling moisture inside the headspace 80. The sizes of the
pores 132 can vary but are usually about one-tenth (0.1) cm to
three-tenths (0.3) cm in diameter. The number of pores 132 can vary
from one (1) to eighty (80) or more and their location can be
grouped or evenly spaced. The pores may be covered with an adhesive
film (not shown) such as Mylar.TM. which retards the passage of
oxygen and can be sterilized in place when the dish is sterilized.
This film can be removed after the culture dish 10 is filled and
before it is incubated. The pores provide a means to reduce the
water content of the media during incubation in a controlled
manner. This reduces the condensate that forms inside the assembled
culture dish 10. Any oxygen infiltrating into the assembled culture
dish 10 through these pores 132 must pass through the media 14 to
get to the media surface where the microbes 20 are planted. The
media 14 contains the biocatalytic oxygen reducing agent and
optionally one or more substrates that removes the oxygen before it
can reach the surface by this route.
[0083] The culture dish 10 is designed to be easily manufactured by
known injection molding techniques. The dish top 16 and dish bottom
12 have no features that prevent them from being ejected from a
mold. The materials of construction can vary but are preferably
polystyrene, polycarbonate, or polystyrene-acrylonitrile. These are
clear thermoplastics that are inexpensive, easy to mold,
sterilizable by ethylene oxide or radiation, resilient to handling
and resistant to chemical substances used in microbiological media.
Styrene-acrylonitrile has the lowest oxygen permeability of the
three thermoplastics mentioned. All of the parts are preferably
transparent to permit observation of the anaerobic culturing
process. However, pigments or dyes may be added to the polymeric
materials in order to produce different shades or colors. Further,
as it is understood by those skilled in the art, ultra-violet light
absorbers and other additives can be added to produce culture
dishes having the properties desired by the end user.
[0084] The assembled culture dish 10 can be opened by one of three
methods:
[0085] A) The assembled and sealed culture dish 10 is placed
upright on a bench top. The dome 82 on the dish top 16 is
depressed. The flexing of the dish top 16 causes the media-ring
seal to part releasing and allowing the dish bottom 12 to come to
rest on the bench top.
[0086] B) The assembled and sealed culture dish 10 is gently struck
onto the bench surface. This action breaks the media-ring seal
which in turn releases the dish bottom 12 and allows the dish
bottom 12 to come to rest on the bench top.
[0087] C) The assembled and sealed culture dish 10 is placed
upright on a bench top. The side walls 52 of the dish top 16,
between cut-out areas, are gently flexed. This action causes the
media-ring seal to part and releases the dish bottom 12 to come to
rest on the bench top.
[0088] The media-ring seal can be reformed simply by placing the
dish top 16 over dish bottom 12 and re-inverting the assembled
culture dish 10. Gravity will cause the dish bottom 12 containing
the solidified media to come into contact with the seal ring. The
substrate and/or oxygen reducing agent present in the media 14 will
once again remove all of the oxygen trapped in the head space
80.
[0089] A second preferred embodiment is illustrated in FIGS. 9-11.
Where possible, like numerals will identify like components with a
primed suffix, while new components will be identified with a new
numeral. Moreover, it will be appreciated that the culture dish
assembly of this second preferred embodiment operates in
substantially the same manner, i.e., having first and second
orientations in which the dish components cooperate with one
another.
[0090] A first dish or base 12' has a substantially planar wall 22'
and a side wall 24' extending therefrom. The side wall has a slight
outward taper as it proceeds outwardly from the wall 22'. A culture
medium or agar 14' is received in the cup-shaped first dish
component (FIG. 11). As described above, the fill height of the
culture medium is important so that an effective seal can be
achieved between the dish components and the second or inverted
orientation. A rib 150 extends outwardly from the generally planar
base in a direction generally opposite that of the side wall 24'.
In the preferred arrangement, the rib 150 is circumferentially
continuous and is radially dimensioned and located to seat on a
terminal end of the second leg of the second dish component or lid,
as will be described in greater detail below.
[0091] The second dish component 16', also referred to as a cover,
lid, or top, is sized to fit and conform over the dish bottom. It
includes a wall 50' that is substantially planar and first and
second side walls 52', 54'. Radially interposed between the planar
wall 50' and the second leg 54' is the seal ring 56' defined by a
planar, annular seal face. As will be appreciated from a review of
FIG. 11, it is apparent that the first and second legs 52', 54' of
the lid have tapered conformations. In particular, they define a
generally inverted U-shaped configuration. The second leg 54'
tapers radially outward from its interconnection with the seal
region 56' to an interconnecting portion 152. The interconnecting
portion 152 has a radial dimension that supports the rib 150
extending from the first dish. A raised protrusion 154 assists in
centering a dish base when disposed in stacked relation on the lid
of an adjacent culture dish assembly. Moreover, a support region
156 is disposed radially outward of the protrusion and is adapted
to receive the terminal end 158 of the first leg of an adjacent
lid. In this manner, assembled dish components in the first
orientation can be stacked one atop the other (FIG. 11). The side
wall 24' of the base is then received between the diverging first
and second legs 52', 54' of the lid. The degree of taper is
sufficiently controlled so that region 152 is located for
supporting the rib 150 of an adjacent base and region 156 of the
interconnecting portion provides suitable support for the first leg
of the lid. Moreover, the protrusion 154 serves a centering
function to facilitate stacking of the dish components.
[0092] In this arrangement, the circumferential side wall defined
by the first leg 52' of the lid is also preferably
circumferentially continuous. Although the cutouts may facilitate
handling of the lid, warpage could occur during the molding
operation, thus it will be appreciated that for more precise
control of the manufactured component, a circumferentially
continuous side wall may be desired in some instances.
[0093] Although not illustrated, it will be appreciated that the
dish components of the second preferred embodiment also cooperate
in the second orientation, or inverted relation in a manner similar
to that described above. Thus, the seal face 56' will suitably seat
or seal against the surface of the agar 14'. The space between the
first and second legs 52', 54' of the lid allows the side wall 24'
of the base to move inwardly toward the interconnecting portion
between the legs. This movement is stopped by engagement between
the seal face and the agar to define a sealed headspace between the
culture medium and the generally planar wall 50'.
[0094] While the culture dish of the invention has been shown and
described herein as being particularly adapted for use in circular
form, it is not desired or intended to thus restrict the scope and
utility of the improvements by reason of such specific embodiments
since the apparatus may be of various shapes and sizes without
departing from the invention. In addition, it is also contemplated
that certain specific descriptive technology used herein shall be
given the broadest possible interpretation consistent with the
disclosure.
[0095] The biocatalytic oxygen reducing agents suitable for use in
the invention include known biocatalytic oxygen reducing agents
such as glucose oxidase and catalase and the oxygen scavenging
bacterial cell membrane fragments disclosed in U.S. Pat. No.
4,476,224 entitled "Material and Method for Promoting the Growth of
Anaerobic Bacteria", issued Oct. 9, 1984 to Howard I. Adler, Oak
Ridge, Tenn., one of the co-inventors of the present invention. The
'224 patent is incorporated herein by reference.
[0096] The '224 patent is directed to a method of removing
dissolved oxygen from a nutrient medium for anaerobic bacteria
through the use of sterile membrane fragments derived from bacteria
having membranes which contain an electron transport system which
reduces oxygen to water in the presence of a hydrogen donor in the
nutrient medium. It is known that a great number of bacteria have
cytoplasmic membranes which contain the electron transport system
that effectively reduces oxygen to water if a suitable hydrogen
donor is present in the medium. Some of the bacterial sources
identified in the '224 patent include Escherichia coli, Salmonella
typhimurium, Gluconobacter oxydans, and Pseudomonas aeruginosa.
These bacterial membranes have been highly effective in removing
oxygen from media and other aqueous and semi-solid
environments.
[0097] The same oxygen reducing effects produces by the cell
membrane fragments from the bacterial sources indicated above, are
also present in the membrane of mitochondrial organelles of a large
number of higher non-bacterial organisms. More particularly, a
great number of fungi, yeasts, and plants and animals have
mitochondria that reduces oxygen to water, if a suitable hydrogen
donor is present in the medium. Some of the sources of oxygen
reducing membranes from these mitochondria are: beef heart muscle,
potato tuber, spinach, Saccharomyces, Neurospora, Aspergillus,
Euglena and Chlamydomonas. The process of producing the useful
mitochondrial membrane fragments involves the following steps:
[0098] 1. Yeast, fungal cells, algae and protozoa, having
mitochondrial membranes containing an electron transfer system
which reduces oxygen to water, are grown under suitable conditions
of active aeration and a temperature which is conducive to the
growth of the cells, usually about 20.degree. C. to 45.degree. C.
in a broth media. Alternately, mitochondria may be obtained from
the cells of animal or plant origin.
[0099] 2. The cells are collected by centrifugation or filtration,
and are washed with distilled water.
[0100] 3. For the preparation of crude mitochondrial membrane
fragments, a concentrated suspension of the cells is treated to
break up the cell walls and mitochondria. This is accomplished by
known means, for example, by ultrasonic treatment or by passing the
suspension several times through a French pressure cell at 20,000
psi.
[0101] 4. The cellular debris is removed by low speed
centrifugation or by microfiltration (cross-flow filtration).
[0102] 5. The supernatant or filtrate is subjected to high speed
centrifugation (175,000.times. g at 5.degree. C.) or
ultrafiltration.
[0103] 6. For the preparation of material of higher purity, the
cells of step 2 are suspended in a buffer containing 1.0 M sucrose
and are treated by means which break up the cell walls or membranes
but leave the mitochondria intact. This is accomplished by known
means, for example, by ultrasonic treatment, passage through a
French pressure cell at low pressure, enzymatic digestion or high
speed blending with glass beads.
[0104] 7. The cellular debris from step 6 is removed by
differential centrifugation or filtration.
[0105] 8. The supernatant or retentate from step 7 is passed
through a French Press at 20,000 psi to break the mitochondria into
small pieces.
[0106] 9. Mitochondria debris from step 7 is removed by
centrifugation at 12,000.times. g for approximately 15 minutes or
by microfiltration.
[0107] 10. The supernatant or filtrate from step 9 is subjected to
high speed centrifugation (175,000.times. g at 5.degree. C.) or
ultrafiltration.
[0108] 11. The pellet or retentate from step 5 (crude mitochondrial
fragments) or the pellet or retentate from step 10 (purified
mitochondrial membrane fragments) are resuspended in a buffer
solution at a pH of about 6.0 to about 8.0. A preferred buffer
solution is 0.02 M solution of
N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES).
[0109] 12. The membrane fragments in the buffer solution are then
passed under pressure through a filter having openings of about 0.2
microns.
[0110] 13. The suspension is then stored at about -20.degree. C.
for later use or it may be freeze dried.
[0111] Furthermore, while many solidified medium do not require the
addition of a hydrogen donor in order for the enzyme system present
in the membrane fragments to reduce the oxygen present in the
product to water, when synthetic medium or medium failing to
contain a hydrogen donating substance are utilized, the addition of
a hydrogen donor (i.e., an organic substrate) may be necessary in
order for the membrane fragments to perform their oxygen removing
functions. Suitable hydrogen donors are lactic acid, succinic acid,
alpha-glycerol phosphate, formic acid, malic acid, and where
available, their corresponding salts.
[0112] The present invention is further illustrated by the
following examples. It is to be understood that the present
invention is not limited to the examples, and various changes and
modifications may be made in the invention without departing from
the spirit and scope thereof.
EXAMPLES
Example 1
Growth of Anaerobic Microorganisms Using the Culture Dish, i.e.,
"OxyDish.TM." of the Present Invention and a Biocatalytic Oxygen
Reducing Agent
[0113] Nutrient agar is supplemented with sodium formate (15 mM),
sodium succinate (30 mM), sodium lactate (45 mM) and cysteine
(0.025 g/100 ml). A biocatalytic oxygen reducing agent,
EC-Oxyrase.RTM. (Oxyrase, Inc., Mansfield, Ohio) is added to cooled
(45.degree. C. to 50.degree. C.) but molten sterile medium to give
a final concentration of 5 units/ml. 20 ml of the above mixture is
soon introduced into the bottom part of a culture dish, i.e.,
"OxyDish.TM.". The top part of the culture dish, is placed over the
filled bottom part to prevent contaminants from entering the dish.
The agar in the bottom part cools to ambient temperature and
solidifies. The covered dish is left standing to permit excess
moisture to escape. At this point the dish may be sealed by
inverting it to bring the agar surface in the dish bottom into
contact with the ring inside the dish top.
[0114] A suspension of anaerobic microorganisms is spread on the
surface of the agar medium that contains the biocatalytic oxygen
reducing agent and its substrates. The dish is sealed by inverting
it. The dish is then placed into an aerobic incubator at 35.degree.
C. to 37.degree. C. for 24 to 48 hours. Several dishes are stacked
to form a stable column of dishes.
[0115] Assembled dishes can be handled and viewed at any time
without breaching the seal and losing the anaerobic environment
inside the trapped headspace. In this way, a particular culture
dish, i.e., "OxyDish.TM." can be selected at the earliest time when
the microbial isolate has grown sufficiently for selection.
[0116] Using this technique with the culture dish, i.e.,
"OxyDish.TM." and a biocatalytic oxygen reducing agent, the
following microorganisms have been grown:
[0117] Clostridium tertium, C. difficile, C. perfringens, C.
cadaveris, C. acetobutylicum, Bacteroides thetaiotaomicron, B.
fragilis, B. distasonis, Escherichia coli, Fusobacterium varium, F.
mortiferum, F. necrophorum, Peptostreptococcus magnus, P.
anaerobius, P. nigra, P. intermedius, Lactobacillus casei, L.
acidophilus, Eubacterium lentum, Bifidobacterium breve, and
Streptococcus fecalis.
Example 2
Measurement of Oxygen Depletion in the Headspace of the Culture
Dish Effect of the Present Invention by the Biocatalytic Oxygen
Reducing Agent
[0118] A hole is drilled in the base of the culture dish, i.e.,
"OxyDish.TM." and a gas tight septum is inserted. The base is then
filled with 20 ml of agar containing a biocatalytic oxygen reducing
agent. The bottom is sealed to the top by inverting the assembled
dish and incubating it at 37.degree. C. Periodically, 50 ul samples
of the gas in the headspace of the dish are sampled by inserting
the tip of a 100 ul gas tight Hamilton syringe through the septum
in the base of the dish. These samples are introduced into an
Oxygen Sensor (IT Corporation) and the concentration of oxygen
remaining in the headspace is determined. Using this method it has
been determined that all measurable oxygen, less than 10 pp
billion, is removed from the head space in two to eight hours
depending on the concentration and configuration of the
biocatalytic agent used. It has also been determined that the dish
can be opened, resealed and, after a suitable incubation period,
the head space again becomes anaerobic.
Example 3
Multiple Opening and Closing of the Culture Dish of the Present
Invention and Reestablishment of Anaerobic Environment
[0119] A culture dish, i.e. "OxyDish.TM." of the present invention
containing a nutrient agar and the biocatalytic agent is streaked
with an anaerobic organism (Bacteroides thetaiotaomicron or B.
fragilis) covering two quadrants of the dish. After 24 hours of
incubation at 37.degree. C. the dish is opened, growth of the
anaerobe is observed and a small quantity of organism is streaked
on the third quadrant of the dish. The dish is resealed and after
24 hours of incubation at 37.degree. C., the dish is reopened and
growth is observed in the third quadrant. A small amount of growth
from the third quadrant is streaked on the fourth quadrant. The
dish is resealed and after a 24 hour incubation at 37.degree. C. it
is reopened and growth is observed in the fourth quadrant of the
dish.
Example 4
Rapid Anaerobiosis of the Agar Layer Containing a Biocatalytic
Oxygen Reducing Agent as Indicated by Methylene Blue.
[0120] An agar medium was made that contained water, 50 mM sodium
lactate, and 2.5 mg/ml of methylene blue. In the oxidized state
methylene blue is blue in color. In the reduced state it is
colorless. The agar was melted and cooled to 45.degree. C. It is
blue in color. EC-Oxyrase.RTM. is added at 5 units/ml and 20 ml is
delivered to the bottom part of a culture dish, i.e.,
"OxyDish.TM.". As soon as the agar has solidified, about 5 minutes
later, the culture dish, i.e., "OxyDish.TM." is sealed by inverting
it. At this time the agar layer is blue in color. The culture dish,
i.e., "OxyDish.TM." is incubated at 37.degree. C. and observed
periodically. Soon after sealing the culture dish, i.e.,
"OxyDish.TM.", the agar layer begins to lighten in color. Within 30
minutes to 45 minutes of being put into the incubator, the medium
is nearly white in appearance, but with a light blue tinge of
color. By 60 minutes of incubation the agar layer is white, which
indicates that the agar layer is anaerobic shortly after the
addition of EC-Oxyrase.RTM. to the medium.
Example 5
Use of Methylene Blue Strip to Indicate Anaerobiosis in the Culture
Dish
[0121] A small rectangular piece of filter paper impregnated with
methylene blue at an alkaline pH is fixed to the inside of the dome
in the top of the culture dish, i.e., "OxyDish.TM.". The dish
bottom contains nutrient agar and a biocatalytic oxygen reducing
agent. The dish is sealed by inverting it thereby causing the agar
surface to rest on the ring. After incubation at 37.degree. C. for
8 hours or more, the blue color disappears from the filter paper.
This indicates that the headspace of the culture dish, i.e.,
"OxyDish.TM." has become anaerobic.
Example 6
Use of Glucose Oxidase and Catalase as the Biocatalytic Oxygen
Reducing Agent
[0122] Sterile Nutrient agar (Difco), supplemented with 1%i
glucose, is cooled to 45.degree. C. and 1 unit of sterile filtered
glucose oxidase/ml, and 1 unit of sterile filtered catalase (Sigma
Biochemicals, 1994 Catalog, p 221 and p 478) is added. 20 ml of
this medium is deliver into a culture dish, i.e., "OxyDish.TM.".
After the agar has solidified, a small quantity of Bacteroides
fragilis is streaked on the surface of the agar and the dish is
sealed by inverting it. After 48 hours of incubation at 37.degree.
C., growth of the anaerobic microorganisms is observed on the
surface of the agar medium.
Example 7
Use of a Filter Pad with Carbonate to Generate CO.sub.2 in the
Headspace
[0123] A piece of filter paper saturated with a 1% sodium
bicarbonate solution and then dried is fixed to the inside of the
dome in the culture dish, i.e., "OxyDish.TM." top. This filter
paper is then covered by a 0.2 u membrane filter. The dish bottom
is filled with 20 ml of Nutrient Agar (Difco) and a biocatalytic
oxygen reducing agent, EC-Oxyrase.RTM. at 5 units/ml and
substrates. The agar surface is inoculated by streaking with a
small amount of Clostridium acetobutylicum, a microorganism that
requires CO.sub.2 for rapid colony development. Immediately before
sealing the dish, one drop of 0.1 N HCl is placed on the membrane
filter. The dish is sealed by inverting it and placed into a
37.degree. C. aerobic incubator. After 24 hours of incubation,
growth of C. acetobutylicum can be observed indicating that
CO.sub.2 was released from the sodium bicarbonate impregnated
filter paper into the headspace of the culture dish, i.e.,
"OxyDish.TM.".
Example 8
Relief of Moisture Through Pores in the Dish Bottom
[0124] Seventy-six holes of different sizes (large: 0.101 inch,
medium: 0.086 inch, and small: 0.059 inch) are drilled into a
culture dish bottom. The dish is filled with 40 ml of 1.5% agar. A
standard dish cover or a Brewer Lid is fitted onto each dish
bottom. The complete dish is weighed. The covered dish is incubated
at 37.degree. C. and the weighed at timed intervals. The loss of
weight is taken as due to the loss of moisture, since the
solidified agar is 98.5% water by weight. The relative weight loss,
net of control weight loss, is as follows:
1 Pore Size 24 hrs. 48 hrs. Small 6% 11% Medium 9% 14% Large 13%
24%
[0125] This shows that the drying of the agar layer can be
controlled during incubation by the number and size of pores put
into the dish bottom. For all size holes, the molten agar did not
escape through these holes. The Brewer Lid covered dishes exhibited
no moisture build up within the trapped headspace for those dishes
with holes in them.
Example 9
Comparison of Present Invention With Standard Methods for Growing
Anaerobes
[0126] The inoculum is prepared by selecting colonies of particular
microbes from Wilkens-Chalgren blood agar plates. A loopful of
growth is suspended in Brucella broth to a density of about
1.5.times.10.sup.8 colony forming units per mL. The suspended
microorganisms are put into wells of a replicator block. Sterile
replica or pins are dipped into the wells of the block. The
replicator pins are stamped onto the surface of agar medium
(Wilkens-Chalgren blood agar). Each pin is calibrated to deliver
about 1.times.10.sup.5 colony forming units per spot. This
procedure is repeated to inoculate a controlled pattern of spots
onto agar plates containing increasing amounts of antibiotic.
Appropriate control plates, that do not contain antibiotics, are
included. After a short time, the spots dry, the culture dish, i.e.
the OxyDish, is sealed and incubated aerobically. Standard plates,
not containing the oxygen reducing agent, i.e. Oxyrase.RTM., and
substrates, are incubated in anaerobic jars or chambers. After 48
hours of incubation at 35.degree. C. the plates are scored for
growth. The presence of growth on a plate containing antibiotic
indicates that the particular microbe is resistant to the level of
antibiotic in that plate. In this way, one can determine the
antibiotic susceptibility profile of a large number of microbial
specimens.
[0127] With respect to the rate and intensity of growth of a number
of difficult anaerobes using the culture dish of the present
invention and the brocatalytic oxygen reducing agent compared to
the standard methods, it was found that anaerobic microbes grew
faster and to a greater density with the present invention compared
to standard anaerobic methods. As shown below, this observation was
particularly noticeable for difficult to grow anaerobes.
2 Microbe Standard Method OxyDish Method Clostridium dificil 1+ 3+
Clostridium 4+ 4+ perfringens Clostridium 1+ 3+ cadaveris
Bacteroides 3+ 4+ rhetaiotaomicron Bacteroides 3+ 4+ distasonis
Fusobacterium 2+ 4+ varium Fusobacterium 2+ 4+ mortiferum
Fusobacterium 1+ 3+ necrophorum Peptostreptococcus 2+ 3+ magnus
Peptostreptococcus 1+ 3+ anaerobius Peptostreptococcus 1+ 3+ negra
Bifidobacterium 1+ 3+ breve Prevotella 2+ 3+ intermedia
[0128] The invention has been described with reference to the
preferred embodiments. Obviously, modifications and alterations
will occur to others upon reading and understanding the preceding
detailed description. It is intended that the invention be
construed as including all such alterations and modifications
insofar as they come within the scope of the claims and the
equivalents thereof.
* * * * *