WO1983001581A1

WO1983001581A1 - Prolonged incubation microbiological culture plates

Info

Publication number: WO1983001581A1
Application number: PCT/US1981/001490
Authority: WO
Inventors: Kamal Abdou Youssef
Original assignee: Kamal Abdou Youssef
Priority date: 1981-11-04
Filing date: 1981-11-04
Publication date: 1983-05-11
Also published as: AU7891582A; AU7891581A; JPS59500448A; EP0092547A4; EP0092547A1

Abstract

Apparatus for the culture of aerobic and anaerobic organisms comprising (1)- a dish having a base portion (16) with a wall (18) extending upwardly therefrom, said wall (18) terminating in an upper peripheral edge (20), (2)- a cover portion (22) with a top (24) whose inner peripheral surfaces (23) are adapted to seal with upper peripheral edge (20), and (3)- an annular gasket (10) fitting between upper peripheral edge (20) and inner peripheral surfaces (23), said gasket (10) being permeable to gases but impermeable to particulate matter, whereby gases are allowed to pass into and out of said apparatus, while contaminating organisms are excluded therefrom.

Description

Prolonged Incubation Microbiological Culture Plates

This invention concerns apparatus of the Petri dish type and a contamination-proof, prolonged-incubation sealing gasket therefor.

One of the shortcomings of the Petri dish is that there is no control over the biological or physical characteristics of the air or gas that gets into or out of the Petri dish. Ventilation in a Petri dish is often a desirable feature and is presently provided by the "beading" of the rim of the container section if the dish is made of glass and by the presence of "ridges"

(usually three) either on the cover at its circumference on the inside or on the rim of the container section. These ridges or protrusions ensure the presence of a space or a physical gap between the container section and the cover. This gap, however, is big enough to allow not only dust and suspended particulate matter and (occasionally) dangerous material, e.g., spores of pathogenic fungi, to get in (or out) of the plate but also allows relatively large insects as well, e.g., flies and even mites and small roaches, to creep freely in and out.

The second disadvantage arising out of this structural deficiency of Petri dishes is that it allows culture media and other relatively volatile constitutents to dry or desiccate fairly rapidly.

- Hence, cultures of microorganisms which grow slowly such as mycobacteria (e.g, tuberculosis) and fungi (e.g., dermatophytes and dimorphic fungi) are endangered by the rapidly decreasing water content of the culture medium. It has been difficult to store cultures on ^" agar plates except for a limited time, and the convenience of storing such cultures is therefore not available. The same is true for unused media; i.e., they cannot be stored for a long time. It is quite evident, then, that with presently known Petri dish devices, there can be little or practically no control over the gas entering them or emerging from them or their contents.

There is a definite flow of air into, and out of a Petri dish culture, as occasioned simply by the expansion and shrinkage of the gas under varying temperature conditions. Culture media are usually stored at low refrigerator temperatures and have to be warmed when cultures are made. This inevitabl ^'entails the expansion of air from inside the plate to the outside. When the plates are removed from the warm temperature of the incubator to be examined at cooler room temperature, the air in the plate has to shrink, inviting air from outside into the plate. All these air movements take place on account of change in conditions of a purely physical nature (i.e., temperature changes). On the other hand, cultures of many microorganisms which very actively absorb and/or produce gases generate a relentless fairly strong flow of gases (sometimes with great force, e.g., stormy fermentation by Clostridium welchii) into and out of the culture plates. For example, oxygen goes in and carbon dioxide and sometimes hydrogen sulfide go out.

The prior art relating to this invention includes U.S. Patents No. 3,158,353, 3,184,395, 1,513,360, and 3,326,401. The first of these describes a Petri dish fitted with a pressure-sensitive adhesive which releasably attaches the cover to the dish and hermetically seals the juncture between the dish and its cover, being removable with the cover from the dish. U.S. Patent No. 3,184,395 provides an envelope for aerobic culturing made of two layers of flexible, thermoplastic material having a sealing connection along the edges thereof and a plurality of heat seal partitions extending inwardly from opposite sides of the envelope, both of the layers being impervious to the culture media and at least one of the layers being pervious to air. U.S. Patent No. 1,513,360 describes a closure wherein a test tube is provided with a circumferential groove toward the top thereof with cotton disposed in the groove and compressed by a cap removably disposed over the open end of the tube and over the groove. U.S. Patent No. 3,326,401 shows a closure cap with a top having an opening receiving a plug supported intermediate a gasket abutting the end wall and the top of the container. Thus, none of the above prior art structures suggest a micronized filter gasket of the present type or its equivalent.

It is an object of this invention to provide apparatus of the general class of Petri dishes in different models to suit different purposes according to the material from which they are made.

Another object of this invention is to provide the plates with suitable filters to allow biological and physical control over the gases and other matter living or dead getting into^' or out of the plates, hence a greater control over the contents of the apparatus commonly referred to as the Petri dish.

In accomplishment of the above-stated objects of the invention, there is provided a filter gasket for

O FI a Petri dish, or other culture-containing apparatus, consisting of an open-celled foamed plastic or fibrous paper core substrate having on at least one side, but preferably on all sides, an air-permeable but particulate-impermeable film, membrane, or laminate. There is also provided a culture-containing apparatus comprising a dish portion including a base and a wall associated with the dish's rim, with a gasket secured between the rim of the dish and the cover to seal the juncture thereof. Preferably, the novel gasket extends along the peripheral edge of the top and along the upper edge of the rim. The gasket can be attached to either the cover or the dish with a suitable adhesive. Figure 1 is a top plan view of the gasket of the invention with parts broken away.

Figure 2 is a cross-sectional view of the same taken along line 2-2 of Figure 1.

Figure 3 is a longitudinal sectional view of the same taken along line 3-3 of Figure 1.

Figure 4 is a sectional view through a Petri dish assembly incorporating the gasket of the invention.

Referring now to the drawings, gasket 10 is formed of an open-celled core 12 made (e.g.) of plastic or paper having secured on at least one side a skin or membrane 14 which is permeable to gas but impermeable to bacteria and other particulate matter. Preferably, skin 14 will cover core 12 on all -sides as shown in Figures 2, 3, and 4. Suitably, gasket 10 will be rectangular or rounded in cross section and about 3 to 5 mm thick and wide.

Core 12 suitably is made from soft foam sheets such as polyurethane foam a few millimeters in thickness (3 to 5 mm) with an open cell structure. Other examples of suitable plastics for the core materials are available

O PI under the trade names "Acropor" and "Versapor" from the Gel an Company.

Skin 14 can be made from a variety of substances in the form of sheets, films, or membranes which are porous enough to allow the passage of air and other gases sufficiently to sustain the growth of microorganisms and cells inside the plate, but hinder bacteria and other microscopic particles, such as dust, pollen, etc., from gaining access to the inside of the plate. Such membranes cut down on the rate of evaporation from containers protected by them, since the^* permeation of water vapor through them is restricted in some degree. Skin 14 may suitably have submicronic pores, 0.2 micron and smaller being known as absolute-sterility pores, even 0.45 micron pores being adequate to prevent contamination and allow gaseous permeation while at the same time cutting down on the rate of evaporation of water. These membranes hinder bacteria, spores, or other particulate matter from gaining access into the containers once the container is sealed with a suitable sealant or lubricant as the case may be.

Skin 14 can be manufactured from a very wide variety of natural or synthetic materials, organic and inorganic (or a blend thereof) , either with a submicronic porosity or from intimately woven fibers thereof. Such substances may include cotton (virgin or chemically treated) , wool, artificial silk, nylon. Teflon, polyester, etc. Polyethylene can be used in the form of sheets of intimately woven fibers, e.g., as available from the DuPont Company under the trade name "Tyvek," or in the form of polyethylene-coated paper having the desired porosity. Nitrocellulose is also a suitable material for such submicronic porous material sheets. For economic reasons, "Tyvek" and cellophane and cellophane-plastic blends are the most practical to use and are readily available. All these membranes are very thin, are measured in terms of mils rather than millimeters, and vary from 6 to 10 mils in thickness. The sheets of skin 14 and sheets of the core

12 of the filter are laminated either by heat and compression or by some suitable adhesive such as methyl cellulose, polyvinyl alcohol, certain polymers (e.g., polyethylene oxide polymer), gums (such as dextrin), or resins or a miscible blend thereof. Heat lamination with compression is the easiest and most practical.

Figure 4 shows a Petri dish provided with a gasket 10. The dish can be of any shape and made of any suitable material. It includes a base 16 with a wall 18 extending upwardly therefrom. Wall 18 is formed with an upper peripheral edge 20. A cover 22 fits over edge 20 of the dish and extends downward loosely parallel to wall 18. Preferably, cover 22 will have a top 24 whose ^' inner peripheral surfaces 23 are adapted to seal with edge 20. Gasket 10 is an annulus fixed to cover 22 on the inside and to the wall 26 of cover 22 by heat sealing or by means of an adhesive such as a silicone cement or an acrylic glue. The adhesive used need not be gas permeable. It is also possible to have cover 22 equipped with a circumferential "bed" on the "inside" of about 3 to 5 mm in width to hold mechanically "snugly fitting" the gasket which may be in the form of a ribbon instead of a die-cut gasket and without the need for any glue or adhesive- and without wasting of material in the die-cutting process. The depth of the bed may vary to match the thickness of the gasket. A sealant or lubricant 30 is placed on the upper edge 20 of base 16 to form a sealing engagement with gasket 10. The sealant can be petroleum jelly or a silicone jelly. Dish 10 can be manufactured in glass (with a flat brim surface, bead-free) or from plastic or polycarbonate in different heights 10, 15, 20, 25, 30 millimeters or higher and in different diameters, e.g., 60, 90, 120, 150 millimeters, etc., to suit different purposes. It is also possible to manufacture the dishes with the base divided into 2, 3, 4 sections, or more, as selected for differential diagnostic work or with a grid marking to help in differential counts.

Furthermore, the dishes may be circular or square-shaped, or rectangular. Gasket 10 will be die cut from a sheet of selected material in the same shape as the dish.

A particularly suitable gasket is made from a sheet of 3 to 5 mm thick filter paper (made from cellulose or fiberglass) coated with a 1 mil low-density polyethylene film having a low air permeability and low moisture transmission. In actual controlled experiments, gasket 10 has proved to cut down on the evaporation rate from culture media by approximately 50% within the growth cycle of a slowly growing microorganism, thus protecting the culture against desiccation, against drop in its pH, and against contamination from the ambient atmosphere, while at the same time protecting the ambient atmosphere and the laboratory worker from biologically hazardous materials, e.g., spores of pathogenic microrganisms.

Among the advantages of the present invention are:

1. Contamination of the culture with particulate matter or otherwise is minimized or eliminated. In actual experiments, the microbial contamination of sterile nutrient media dispensed in sterile filter plates was actually and remarkably 0% following two weeks of incubation at room temperature. In a control group of dishes containing the same

OMPI nutrient media and incubated under the same conditions of atmosphere, temperature duration, etc., but not equipped with the filters, the latter control plate cultures abounded with bacterial and fungal contaminant colonies.

2. The nutrient medium in the dish of Figure 4 did not appreciably dry within an observation period of three weeks, while the medium in the control dish without gasket 10 shrank and dried to less than half its original size.

It will be appreciated that modifications may be made in the illustrative embodiments of my invention within the scope of the subjoined claims. Thus, the specific size, shape, and configuration of the containers and dishes may be changed, any appropriate culture media may be employed, and the apparatus may be used for the culturing of any microorganisms or, if desired, of tissue cells.

OMPI

Claims

1. A container for the aerobic or anaerobic culture of microorganisms, including tissue cells, comprising a dish having a base and a wall extending upwardly from said base and an upper peripheral edge on said wall, a cover for said dish, said cover including a top having inner surfaces adapted to contact the peripheral edge.of the dish wall at the juncture between said dish and the said cover, a gasket between the said dish and said cover and disposed along said juncture, said gasket comprising a core of open-celled material, a. membrane on at least one side of said gasket formed of a material having submicronic pores allowing access of gas into and out of said container but preventing access therein of bacteria and other particulate material.

2. The container of claim 1 wherein said gasket is secured to the inner surfaces of said top and sealing means are disposed on said upper peripheral edge in sealing contact with said gasket.

3. The container of claim 1 wherein said gasket is secured to said upper peripheral edge and sealing means are disposed along the outer periphery of said inner surfaces in sealing contact with said gasket.

4. The container of claim 1 wherein the top of said cover has a depending circumferential wall that fits over the rim of the dish.

5. The container of claim 1 wherein said core is formed of foamed plastic.

OMPI

6. The container of claim 1 wherein said core is formed of fibrous polyethylene paper.

7. As an article of manufacture, a gasket comprising an inner core of open-celled material having on at least one side thereof a membrane having pores of submicronic size, said membrane being permeable to gases but impermeable to bacteria and other particulate matter.

8. The gasket of claim 7 wherein said core is formed of foamed plastic.

9. The gasket of claim 8 wherein said core consists of polyurethane.

10. The gasket of claim 7 wherein said core is formed of fibrous polyethylene paper.

11. The gasket of claim 7 wherein said membrane has pores having a diameter ranging from about 0.2 to about 0.45 micron.

12. The gasket of claim 7 wherein said membrane has a thickness ranging from about 6 to about 8 mils.

13. The gasket of claim 7 wherein said membrane is adhered to said core by means of an adhesive.

14. The gasket of claim 13 wherein said member is laminated onto said core.

15. The gasket of claim 14 wherein said membrane is heat laminated onto said core.

O PI

16. The gasket of claim 14, wherein said member is compressively laminated onto said core.