Definitions

In this specification, these words have these specific meanings:

- An antibiotic is a chemical substance, produced by microorganisms -which has the capacity, in dilute solu¬ tion, to inhibit the growth of or to destroy bacteria and ^' other microorganisms.

A microorganism is a minute living organism, usually microscopic in size. A pathogen is a disease-producing microorganism.

Background

Ever since the discovery by Sir Alexander Fleming that the fungus Pencillium notaturn produced a substance in¬ hibitory to -the growth of the human pathogen, Stanhylococ- cus aureus, the search for antibiotics has consumed vast amounts of time and money.

Thousands of antibiotics produced by icroorgan- - isms have been identified. Most of them, unfortunately, are toxic not only to pathogens, but also to the human hosts that harbor the pathogens, and only a few dozen antibiotics have found their way into medical practice. The search continues for new antibiotics which will control particu¬ lar pathogens, especially those which are resistant to known antibiotics. There is also a need for antibiotics which are eff ctive -against animal and even plant pathogens. There are several classical methods for searching for and isolating microorganisms which produce antibiotics. In one method,, soil is sprinkled onto the surface of a solid nutrient medium, such as gelled agar, in a petri dish and individual colonies of microorganisms are encouraged to grow. Circular areas clear of other growth around some of the colonies indicate that an antibiotic is inhibiting the growth of neighboring colonies. This is how antibiotic- producing colonies are identified. Each such colony must

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^ then be tested for its ability to inhibit the pathogen . against which an antibiotic is sought. To test a colony, one ma -remove a plug of agar containing the active colony and place the plug on a second plate of nutrient medium 5 which has been "seeded", or uni ormly inoculated with the pathogen against which one seeks an antibiotic. A clear area surrounding the plug indicates that the antibiotic produced is effective against the pathogen.

This method has a shortcomingi For the anti- biotic to be evident in the first place, it must be effec¬ tive against neighboring bacteria cultured from, and present in, the original soil sample. Each antibiotic has its own - particular spectrum of organisms against which it is effec¬ tive. If an organism present in the soil sample produces an antibiotic effective against the pathogen, but not against neighboring soil-derived colonies, its e fectiveness will be undetected. If an antibiotic producer is a slow grower, its neighbor may grow rapidly ^" enough to overwhelm and mask the antibiotic effect. Most pathogens, being obligate parasites and many times fastidious, are often vulnerable to weak antibiotics, the very antibiotics least likely to be toxic to the pathogen host. Yet, it is the weak antibiotics- which are most likely to be undetected by this method, for the reasons discussed. A further shortcoming of this method is the ^' time and space requirement for two-step culturing, first against neighboring colonies, and then against the pathogen. When thousands of tests are to be made these constraints are seriously limiting. In another test method, an unknown sample of micro¬ organisms, as for example from the soil, is grown directly upon a nutrient medium which has been preseeded wi-ch a path¬ ogen, and clear areas are sought in the medium, indicating antibiotic action against the pathogen directly. In this 5 case, it is required that the new antibiotic producer thrive in the same environment as the pathogen - at the same temp¬ erature, pK, aerobicity, and so forth. Pathogens, however,

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frequently require exotic media and often require a special pH or redox potential which may not be acceptable to the culture of the very microorganisms which produce the sought- for antibiotic. There is, therefore, an even larger risk of passing up the most promising new candidate microorganisms. In this method, temperature is a major problem since most human pathogens require a temperature of 37°C and most soil microorganisms grow best at about 25 C. Additionally, if the pathogen is a strict anaerobe (cannot grow in the presence of oxygen) , aerobic antibiotic-producing organisms effec¬ tive against the pathogen will not be found.

As indicated previously, soil is the source mater¬ ial for almost all antibiotic-producing organisms. The only practical way to find new antibiotics is to conduct massive soil screening programs'. The methods described above, and with minor permutations thereof, have been em¬ ployed since the search for antibiotic-producing microorgan¬ isms began. Many potentially valuable antibiotics have surely been missed in these screening procedures, since the procedures rely on the ability of the antibiotic-producing microorganism either to inhibit its neighbors, or to pro¬ duce an antibiotic when cultured under conditions optimal for the growth of the pathogen, rather than optimal for the antibiotic-producing microorganism. Under these constraints, cost of finding a new antibiotic-producing organism of potential value is becoming economically impractical. There is, then, an urgent need for an effective and efficient method for screening source materials (.e.cj _* soils) for microorganisms capable of inhibiting specific pathogens. This invention is a unique device and method for culturing microorganisms which fills this need. Two cul¬ tures, j§.c[. a pathogen and mixed soil microorganisms, are grown sequentially in the same vessel, each on its preferred medium. The two media in the vessel are disposed, sandwich- like, back-to-back. Antibiotics produced on one side of t media sandwich diffuse through it and inhibit growth (cause a clear area) on the pathogen side of the sandwich.

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Even though an antibiotic is too weak to inhibit neighbor colonies, it will inhibit the pathogen growing on the other- side of the media sandwich. Even though an antibiotic pro¬ ducer grows more slowly than its neighbors, the antibiotic action on the pathogen is evident.

DESCRIPTION OF THE DRAWINGS

Figure 1 is an exploded cross-sectional view of the Antibiotic Testing Vessel.

Figure 2A is a schematic cross-sectional view of the Antibiotic Testing Vessel. Figure 2B is an enlarged view of a part of Figure 2A.

Figure 3 is a schematic cross-sectional view of the Antibiotic Testing Vessel, showing bacterial cultures growing on a layer of agar in Compartment A, and a clear area in the seeded medium of Compartment B.

^' SUMMARY

A porous support member perpendicular tσ the axis of the flat cylinder in which it is fastened divides a flat cylinder into two dish-like halves; An upper compartment A and a lower compartment 3. The porous member is temporarily sealed by a removable plate on the compartment 3 side of it. Lids are provided for both sides of the cylinder.

A solid nutrient agar medium is melted, poured into compartment A, allowed to harden, and microorganisms o £>e tested are inoculated upon it and the plate incubated under a chosen set of environmental conditions. Alterna¬ tively, the microbial inoculum (j≥.σ. soil) may be mixed with the melted and cooled nutrient medium, poured into compartment A, and allowed to harden. After growth, the cylinder is inverted. The plate which seals the screen is removed; the porous support member prevents the gelled agar in compartment A from sagging.

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A second medium, melted, cooled (but still liquid)

. and seeded with a pathogen, is poured into compartment 3, where it gels. (The pathogen may be spread onto the surface of the medium in compartment B -rather than incorporated into it.) The inoculated pathogen is cultured in an environment favorable to its proliferation. That is, such variables as pH, temperature, oxygen concentration, and the like are controlled optimally.

An antibiotic generated by a culture in compartment A will diffuse through the agar layer in which it is growing, through the porous member supporting the agar gel, and into the pathogen-containing agar in compartment B, causing a clear area therein, which may be readily observed.

PREFERRED EMBODIMENT

-A preferred embodiment is illustrated schematically in the drawings.

In Figure 1, 10 represents the porous support mem¬ ber which divides cylinder 11 into upper compartment A and lower compartment B. Seal 12, shown detached from the assem- bly, will snap into position to seal support member 10 and hold liquid agar poured into compartment A while it gels. 13 and 14 are lids which fit over the ends of the cylinder.

Figure 2A shows the Antibiotic Testing Vessel (ATV hereafter) assembled. Figure 2B shows a construction for holding seal 12 in place against a shoulder to provide clearance between seal 12 and support member 10.

The sole purpose of support member 10 is to sup¬ port the sheet of agar once it has gelled. The holes in the support member are as large, and the material between the holes is as thin, as is consistent with that purpose.

Seal 12 must allow clearance between its- surface and support member 10 to allow a continuous sheet of gel to form, so that support member 10 will become embedded in, and thereby support, the body of the hardened .gel when the seal is removed.

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Preferably, the ATV is injection molded of inex¬ pensive, transparent plastic, after which it is gas sterilized and packaged in a sterile package.

In Figure 3, the ATV is shown assembled, with colonies growing in compartment A, one of which has produced an antibiotic, and a corresponding clear area above it in the medium of compartment 3, in which a _. ..pathogen has been cul¬ tured.

Experiment 1 A fifty gram sample of soil is vigorously shaken with 100 ml of physiological saline and dilutions are prepared using the same solution. One ml of a 1/10,000 dilution of the soil suspension is added to melted nutrient agar which has been cooled.to 47 C. After mixing, the still-liquid agar is poured into compartment A of an ATV and allowed to harden into an agar plate. A similar plate is prepared in a second ATV using Marine Agar. Plates are also prepared in a third and a fourth ATV from the same original soil suspension, but using a 1/1,000,000 dilution, and Tomato Juice Agar and Try tic Soy Agar, respectively. 'Examination of the plates after incubation for 48 hours at 22°C reveals that the icrobial colonies which develop on the different media differ, as expected, in response to the different nut¬ rients supplied in the different agar media. The seal is removed from the bottom of each ATV, it is inverted, and a thin layer of melted and cooled nut¬ rient agar, previously inoculated with a saline suspension of Sarcina lutea, is poured into compartment 3. The agar is allowed- to harden, and the ATV's are incubated a second time at 35 C for 24 ^' -hours. Several clear areas in the ,5. lutea seeded agar indicate inhibition by antibiotics produced by the colonies in the agar of compartment A directly below the clear zones.

The antibiotic-producing cultures are.isolated. Further tests indicate that different antibiotic-producing

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cultures develop in the different media used in the four ATV's

. although the same soil sample was used in each of them. .

. This experiment shows that varying environmental conditions will cause various antibiotics to be produced

\ from the same source of mixed microorganisms.

Experiment 2

A test similar to Experiment 1 is conducted using only Tryptic Soy Agar. Different ATV's are inoculated with ^' the same soil slurry, and are incubated at different temp- eratures: 10°C, 20°C, 25°C, 30°C, 35°C, and 45°C. The types of colonies which develop at the different temperatures are similar, but some of the types which proliferate when incubated at 35°C and 45°C are absent on the plates incu¬ bated at 10°C and 20°C. Sarcina lutea is again employed as the test or¬ ganism against which an antibiotic is sought, as in Experi¬ ment .

In this experiment similar colonial types produce clear zones of inhibition in the S>. lutea-seeded agar. The clear zones are of greater diameter at the higher temperatures. This shows greater antibiotic production by this particular microorganism at the higher temperatures. The experiment shows how the ATV can be used to characterize optimal con¬ ditions for antibiotic production.

Experiment 3

In this experiment, one ATV of agar is seeded with soil as in Experiment 1 and incubated under anaerobic con¬ ditions. A second, similarly prepared ATV is incubated under aerobic conditions. Both ATV's are incubated at 22°C. A different flora develops on the two plates. Some types of colonies are present on both plates, while other types are present only on one or the other ATV.

As in Experiment 1, each ATV is inverted, the seal removed, and S_. lutea is cultured in compartment 3. The

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number and types of colonies inhibiting the S _, . lutea are different on the two ATV's. One antibiotic-producer is present only on the aerobic plate. Another producer is present only or. the anaerobic plate. This e'xperiment shows the usefulness of the ATV in detecting antibiotic producers under different environ¬ mental conditions. To obtain the same information by con¬ ventional testing ^' means would require a prohibitively com¬ plex procedure.

Experiment 4

To detect microorganisms capable of producing antibiotics effective against cancer cells, the procedure is as follows: The appropriate nutrient medium and micro¬ organism inoculum is cultured in compartment A, as above. After growth of the microorganisms in compartment A has started, the plate is inverted, the seal is removed, and a semi-solid agar suspension of cancer cells is poured into compartment 3. The cells are incubated under appropriate conditions, and are observed under a microscope for prolif- eration. Evidence of an antibiotic effective against the cancer cells will be evident by a paucity of cells, or evi¬ dence of inhibited proliferation of the cancer cells, in areas of the semi-solid gel directly above the antibiotic- producing organism.

Experiment 5

An agar medium is mixed with a sample of a waste water, the components of which might be useful; for example, a dairy waste. A solid agar plate of the mixture is prepared in Compartment A of an ATV, and is inoculated with a soil sample, as in Experiment 1.

After incubation, the plate is inverted and the seal removed. An agar medium, seeded with a microorganism which requires methionine for growth, is poured into compart¬ ment 3 and allowed to harden. After a second incubation

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under conditions optimal for the methionine-dependent micro¬ organism, the plate is examined for colonies. Colonies of ^" methionine-dependent microorganisms growing in compartment B indicate that colonies, on the agar in compartment A direc- tly beneath them produce methionine. The size of the colonies in compartment B is a measure of the quantity of ■ methionine produced by the microorganisms in compartment A beneath them.

This experiment shows how the ATV is used to iso- late microorganisms which can produce valuable, biologically useful chemicals other than antibiotics, using a specific substrate (in this case, dairy waste) as an energy source. The microorganism, once isolated, may be exposed to a mutagen in hopes of increasing its productivity, the improved mu- tants being isolated by the same ATV technique. Conven¬ tionally employed technology would entail multitudinous, expensive, and time-consuming chemical analysis to accomplish the same ends.

It will ^' be clear to those skilled in the art of microbiology that the ATV can be used in- these many other ways to detect and isolate microorganisms useful for many . purposes. The terms and conditions used in the foregoing experiments and explanation are only exemplary of the in¬ vention, and are not to be construed as limiting the scope thereof, which is defined below in claims.

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