Chemistry: molecular biology and microbiology – Apparatus – Bioreactor
Reexamination Certificate
2001-03-19
2002-08-06
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Bioreactor
C435S305400, C435S307100, C435S801000, C422S105000
Reexamination Certificate
active
06429008
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of Invention
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.
2. Description of Prior Art
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.
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”.
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.
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.
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.
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° 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.
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.
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.
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.
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.
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.
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.
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
Adler Howard I.
Adler Martha Vogeler
Copeland James C.
Spady Gerald E.
Adler Martha Vogeler
Fay Sharpe Fagan Minnich & McKee LLP
Oxyrase, Inc.
Redding David A.
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