US 3843456 A
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Oct. 22, 1974 HADEN ETAL 3,843,456
DEVICE FOR IMPLEMENTING THE DELIVERY STORAGE AND USE OF MICROORGANISMS Filed March 22, 1973 FIGURE l.
, .mvNTdRs Y ONALD FREAKE FIG R 4. R U E BILLY HARPER HADEN ATTORNEY United States Patent 3.843.456 DEVICE FOR IMPLEMENTING THE DELIVERY, STORAGE AND USE OF MICROORGANISMS Billy Harper Haden, 613 French Road, Rochester, N.Y. 14618, and Ronald Freake, Box 343, RR. 2, Elkhart, Ind. 46514 Continuation-impart of abandoned application Ser. No. 93,231, Nov. 27, 1970. This application Mar. 22, 1973, Ser. No. 343,908
Int. Cl. C12k N US. Cl. 195-139 9 Claims ABSTRACT OF THE DISCLOSURE A device for implementing the delivery, storage and use of microorganisms comprising a microporous membrane having deposited on a surface portion thereof a dry standardized composition basically comprising a predetermined concentration of microorganisms, a microbiological nutrient material and a microbiologically inert colloidal suspending agent. The device is useful, inter alia, in the quality control of microbiological test systems.
CROSS REFERENCE TO RELATED APPLICATIONS This is a continuation-in-part of Application Ser. No. 93,231, filed on Nov. 27, 1970, now abandoned.
BACKGROUND OF THE INVENTION Microbiology, although the oldest branch of laboratory medicine, is still lacking quality assurance. For example, culture media, the substance used to provide nutrients for the laboratory growth and multiplication of microorganisms, varies widely in both composition and performance. As far as existing quality assurance is concerned, most laboratories rely on known and unknown stock bacterial cultures which provide a check on only the accuracy of identification, rather than the ability of the culture media or other test system or reagent to isolate the microorganism. The use of such stock cultures does not, however, provide a sensitive index to minor variations in media formulations and/or preparation, largely due to the variation in inoculum size, which differs greatly from one microbiologist to another and from one transfer means to another.
Moreover, to date no facile means has been developed to store a predetermined quantity of a specific microorganism and to deliver or transfer the same to a test system, reaction vessel or any other activity or process requiring the presence of such microorganisms.
DESCRIPTION OF THE PRIOR ART In the field of microbiology, devices containing standardized microorganism inoculum are practically nonexistent and no direct prior art is known. Related prior art is found in the area of reconstitutable microbiological cultures which employ gels and the like. An instance of such art is found in U.S. Pat. 3,360,440 which provides a reconstitutable hydrogel bacteriological medium homogeneously inoculated with specific quantities and forms of microorganisms for later rehydration to produce a culture medium with desired rigidity. Thus, the object is to provide a hydrogel medium which will support the growth of the specific quantities of microorganisms upon rehydration. For this purpose, nutrients are included in the composition and must necessarily be in amounts sufiicient to promote growth of the microorganisms upon rehydration. Such a hydrogel medium finds no use in determining the quality of microbiological growth supporting mediums based on colony formation since colony formation is insured upon rehydration due to the presence of sufiicient nutrients to provide a culture medium (i.e. growth or Patented Oct. 22, 1974 colony formation). Moreover, fixed quantities of microorganisms cannot be delivered using this system since rehydration produces instantaneous reproduction of the microorganisms. Thus, upon rehydration there is no way of knowing the exact number of microorganisms present.
SUMMARY OF THE INVENTION The present invention consists essentially of a microporous membrane having deposited on at least one surface thereof the dried residue of an aqueous suspension of a predetermined quantity of at least one variety of microorganism, a nutrient material in an amount suflicient to preserve the viability of said microorganism while insuflicient to promote the formation of observable colonies, and a microorganism suspending quantity of microbiologically inert colloidal suspending agent. The porosity of the microporous membrane is sufiiciently small to prevent passage of the microorganism therethrough while sufficiently large to allow nutrients and other chemical solutions to pass through an contact the microorganism. It is preferred that the dried residue, which is preferably the result of a lyophilization process, also include a substance capable of retaining a sufiicient amount of moisture to preserve the viability of the microorganism.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an exploded perspective view of a device of the present invention and a cooperable handle means therefor.
FIG. 2 is a perspective view of a disc device comprising a microporous membrance filter and a nutrient pad afiixed thereto.
FIG. 3 is a vertical sectional view taken along the line 3-3 of FIG. 2.
FIG. 4 is a plan view of a device of the present invention showing colonies or locations of microorganisms developed thereon.
DESCRIPTION OF THE PREFERRED EMBODIMENTS Basically the devices of the present invention comprise a matrix in the form of a microporous membrane upon which an accurately measured, predetermined quantity of a microorganism or microorganisms have been deposited along with a nutrient material and a suspending agent as will be described hereinafter. The matrix or microporous membrane is a thin structure comprising a biologically inert and chemically pure cellulosic or other polymeric substance which contains pores having specific and uniform sizes. The pores of these membranes, or filters as they are often called, vary in size about from 0.01 to 14 or more micrometers in diameter, the porosity or pore size being selected to prevent the passage of particulate materials or microorganisms therethrough. Obviously, solutions of chemicals can readily pass through such membranes.
In the present invention a membrane is selected having a porosity sufiiciently small to prevent the passage therethrough of the microorganism or microorganisms being used while sufiiciently large to allow nutrients and other chemical solutions, such as antibiotic solutions or other chemical solutions generally associated with microbiological systems, to pass through and contact the microorganism or microorganisms. Since the smallest microorganism is about 0.22 micrometer in diameter, a membrane having a pore size of about from 0.01 up to 0.2 micrometer may advantageously be used. However, it is to be understood that when larger microorganisms such as yeasts are utilized and a higher solution flow rate through the membrane is desired, a membrane having a pore size larger than 0.2 micrometer and up to about 2.0 micrometers may be utilized.
When used as a quality control means, the basic procedure or process of using the devices of the present invention comprises contacting the composition, growth media, agar gel, test plate, solution, or the like which is to be tested with such a device containing at least a minimum inoculum quantity of the microorganism as will be discussed more fully hereinafter. This standardized composition, hereinafter referred to as the standard, comprises basically a standardized subculture of a known strain of microorganisms which has been stabilized with a colloidal suspending agent. The device containing the standard is then used to inoculate the microbiological reagent, medium or the like to be checked, and after incubation under predetermined conditions the actual colony count on the membrane is compared to the expected count.
The initial step in preparing the standard for the device is to make a series of subcultures of the selected microorganism from a stock culture in order to adapt the microorganism to the stabilized culture medium and to establish growth conditions in the medium for the particular microorganism involved. In each of the subculturing steps, an accurately measured volume or aliquot of the previous culture is used to inoculate the next medium. This aliquot is taken during the so-called log phase when the microorganism is exhibiting its maximum viability and growth. When growth conditions are established and it has been determined that the strain being used is not a mutant variety, then a measured volume of the working culture is transferred to a measured volume of suspending medium and, using a precision micro delivery apparatus, a microaliquot of the standard is transferred to the device, which transfer will be more fully described hereinafter.
The device of the present invention may contain any of the wide variety of known microorganisms such as, for example, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus faecalis, Diplococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Escherichia 601i, Proteus mirabilis, Salmonella sp., strain coli, yeast, fungi and the like.
A microbiological nutrient material is also included in the standard. It has been found that a small amount of nutrient material must be present in order to preserve the viability of the microorganism upon prolonged storage. The presence of this small amount of nutrient material has been found to preserve viability for well over one year. There should not be sutficient nutrient material present to promote observable colony formation during storage or upon rehydration. This is necessary in the application of the present invention as a quality control means since growth is desired only where the medium being tested is capable of sustaining growth. Where fixed numbers of microorganisms are to be manipulated, the amount of nutrient material is such that the exact number of viable microorganisms in the standard is known at the time of use. Therefore, the present invention requires an amount of nutrient material which is sufiicient to preserve the viability of the microorganism While insuificient to promote microbiological growth (i.e. the formation of colonies). Nutrient material includes one or more compounds capable of supporting microbiological viability and includes generally those compounds which are nitrogen sources and particularly proteins such as the peptones, foetal calf serum, soybean protein, mixtures thereof and so forth.
As the suspending agent, it has been found that any of the microbiologically inert colloidal materials may be used. As the term is used herein a microbiologically inert material is a material which in no way exerts a deleterious effect on the microorganism being used. In order to facilitate the accurate delivery of the standard, it is also preferable that the suspending agent be of a character, and used in a concentration, such that it does not form a gel. Among the inert colloidal agents found useful in the present invention are the acid polysaccharides having esterified or free carboxyl groups such as the pectins, pectic araban and galactan, seaweed polysaccharides such as the carragheenans and the alginates. Other classes of inert colloidal agents useful in the present invention comprise gelatin and similar protein degradation products, inert natural gums, the cellulose gums such as methylcellulose and colloidal silica.
The concentration of suspending agent depends on the particular gel forming or thickening qualities of the agent selected. In general, however, sufiicient suspending agent is used so that when the microorganism subculture is in the form of an aqueous suspension, from about 0.1% to 1.0% by weight of suspending agent is present.
It has been found that the water soluble alginates are particularly advantageous for use as suspending agents in the present invention. These substances are naturally occurring polysaccharides found in algae. The water soluble sodium salt, called algin, is the most important of such alginates. It has been found that about from 0.1% to 0.5% by weight of sodium alginate is advantageously used as a suspending agent in the present invention with 0.5% by weight the preferable concentration of sodium alginate in the standard subculture of microorganism.
In its dried state, the standard preferably includes a substance capable of retaining a sufiicient amount of moisture in the standard to preserve viability of the microorganism. Such a substance prevents loss of moisture essential to viability during the drying process and in the dried state. Such substances which are useful therefor include various carbohydrates such as the polysaccharides, glucose and sucrose. Glucose is particularly useful in this regard.
In quality control use, it is contemplated that a series of standard devices be used to test batches of media, agar gel and the like, and that the minimum number of microorganisms necessary to initiate growth be established. This minimum number is termed the isolating coefficienfor the particular system or medium being checked. For example, a new batch of medium may be prepared and tested with a series of devices of the present invention containing between S and colonies per standard. Under predetermined growth conditions, the medium will initiate and support microorganism growth only when a sufiicient number of such microorganisms are present in the standard. Thus, by using a series of standards, devices having known different numbers of microorganisms, the isolating coeflicient of a medium can be readily identified by noting the standard, among those in the series which show colony growth under the predetermined conditions, which has the smallest number of microorganisms. The quantity of microorganism constituting the isolating coefiicient may also be termed the minimum inoculum."
Referring now to the specific embodiments of the present invention illustrated in the drawings, the device shown in FIG. 1 is in the form of a micropore membrane disc 11 the upper surface of which has a hydrophilic center portion 13 and a hydrophobic continuous peripheral edge portion 12.
The peripheral edge portion 12 may be given its bydrophobic character by treatment with microbiologically inert hydrophobic chemicals, such as silicone oils, parafiin waxes and the like, or by heating to thereby eliminate the porosity and destroy any hydrophilic character the treated surface portion may have had. The center portion 13 of the upper surface of the disc 11 may be made hydrophilic by treatment with an inert surface active agent solution such as a 0.1% solution of tris(polyoxyethylene) sorbitan monooleate. Such treatment is advantageous in that it causes a microorganism suspension appied to the surface portion 13 to disperse evenly over the entirety of the surface portion 13 of the disc 11, and the hydrophobic peripheral surface portion 12 serves to contain the suspension therewithin prior to drying of the suspension.
As shown in FIG. 1, the disc 11 may be detachably mounted on the end portion of a handle or carrier means which may take the form of an elongated flat strip of semi rigid paper or plastic foil. Such a carrier means 10 facilitates removal and placement of the disc 11 in processing and use.
FIGS. 2 and 3 show a micropore membrane disc 21 the upper surface of which has a hydrophilic center portion 23 and a peripheral edge portion 24. The portion 23 is covered with the dried remains of a microorganism culture suspension 33. The membrane disc 21 is laminated by an inert adhesive means to a paper matrix 22 of the same diameter as the disc 21 but thicker, such that the paper can be impregnated with a nutrient medium solution and dried. This embodiment serves as a self check on the viability of the microorganisms as will be later explained in the examples. 7
FIG. 4 shows a disc device 41 of the type described in FIG. 1 after contact with a microorganism growth supporting medium, followed by incubation and development of colonies of microorganisms 43 thereon.
Although the preceding description relates to a disc shaped device, there is no intention to restrict such device to a circular configuration. For example, the device may display a square shaped configuration.
It is contemplated that the device of the present invention have utility and apply to areas other than quality control or assurance particularly where small numbers or countable amounts of microorganisms are involved. For example, the stabilized standard provides a unique and facile means of delivering a fixed quantity (i.e. a known exact quantity) of microorganisms to a laboratory or production facility.
The following examples are illustrative of the present invention and are not intended to limit the scope thereof.
EXAMPLE 1 Gm. Peptone 0.5 Sodium alginate 0.5
Sterile, distilled water to make 100.0 ml.
The subculture was incubated overnight at 37 C.
The next morning, 10 ml. of the subculture was thoroughly mixed for 2 minutes using a vortex mixing device, and after a 3 minute wait to allow the air bubbles to disperse, a second tube of the above culture medium was inoculated with 0.01 ml. of the first subculture. The second subculture was incubated for 8 hours at 37 C. A third subculture was made in the manner described above and allowed to incubate overnight at 37 C.
Using the third subculture, a working culture was prepared by again adding 0.01 ml. thereof to 10.0 ml. of the stabilized culture medium and incubating for exactly eight hours at 37 C. A dilute, stabilized bacterial suspension was then prepared by transferring 0.01 ml. of the working culture to 15.0 ml. of a suspending/lyophilizing medium having the following composition:
Peptone 0.5 Sodium alginate 0.5 Glucose 7.5
Sterile, distilled water to make 100.0 ml.
Using a precision micro delivery apparatus (B. Braun Catalog No. 71012) 0.02 ml. of the above dilute bacterial suspension was added to a 12 mm. circular disc of a membrane filter comprising mixed esters of cellulose and having the following characteristics:
Prior to adding the aliquot of suspension to the disc, a one millimeter edge portion thereof was hydrophobed by heating so that the membrane edge was in etfect sealed. The suspension added to the disc formed a circular pattern 11 mm. in diameter and was freeze dried, after which the disc was placed in a vial and retained under vacuum.
EXAMPLE 2 Discs prepared in accordance with the procedure in Example 1 were placed on agar gel plates with the surface of the disc having the microorganism deposited thereon away from the agar gel. The plates were incubated at 37 C. for twelve (12) hours, and thirty (30) colonies appeared on the surface of the disc. Additional discs were treated in the same way and the colony count was found to vary from 27 to 33 colonies.
EXAMPLE 3 Discs prepared in accordance with the procedure in Example 1 were attached to nutrient media pads comprising 1 mm. thick S. & S. 470 filter paper impregnated with the following nutrient composition and dried:
Percent by weight Beef extract 0.3 Sodium chloride 0.5
EXAMPLE 4 Examples 1 and 2 were repeated except that Staphylococcus aureus was used in place of the E. coli. Reproducibility similar to that described in Example 2 was obtained.
EXAMPLE 5 Examples 1 and 2 were repeated except that M. lysodeikticus was used in place of the E. coli. Reproducibility similar to that described in Example 2 was obtained.
EXAMPLE 6 Examples 1 and 2 were repeated except that the volume of suspending/lyophilizing medium was increased to 200 ml. The colony count obtained using such discs varied from 13 to 16.
EXAMPLES 7-9 Example was repeated except that the following suspending agents were used in the culture media:
Concentration Example Agent (percent; by weight) 7-..- Gelatin 0.1 Foetal calf serum.-. 1. O 9 Colloidal silica 0. 1
Results obtained were comparable to that described in Example 1.
What is claimed is:
1. A device for use in determining the quality of microbiological growth supporting media and in implementing the quantitative delivery, storage and use of microorganisms comprising a microporous membrane having deposited on at least one surface thereof the dried residue of an aqueous suspension of a predetermined quantity of at least one variety of microorganism, a nutrient material in an amount suflicient to preserve the viability of said microorganism while insufficient to promote the formation of observable colonies, and a microorganism suspending quantity. of a microbiologically inert colloidal suspending agent and having a porosity sufficiently small to prevent the passage ofsaid microorganism therethrough while sufficiently large to allow nutrients and other chemical solutions to pass through and contact said microorganism.
2. A device as in Claim 1 wherein said suspending agent is selected from the group consisting of water soluble alginates, gelatin, natural gums, colloidal silica, cellulose gums, acid polysaccharides, and seaweed polysaccharides.
3. A device as in Claim 1 wherein said suspending agent is sodium alginate.
4. A device as in Claim 1 wherein said suspending agent is present in said aqueous suspension in a concentration of about from 0.1% to 1.0% by weight.
5. A device as in Claim 1 wherein said dried residue additionally comprises a substance capable of retaining a sufiicient amount of moisture in said residue to preserve the viability of said microorganism.
6. A device as in Claim 5 wherein said substance capa- 8 Me of retaining a sufiicient amount of moisture in said residue comprises glucose or similar substance.
7. A device as in Claim 1 wherein said membrane is made hydrophilic by treatment with a surface active agent. 8. A device as in Claim 1 wherein said membrane has a hydro-phobed edge portion. 9. A device as in Claim 1 wherein said membrane is removably aflixed to a handle means.
References Cited UNITED STATES PATENTS 3,360,440 l2/l967 Haab et al l03.5 R
A. LOUIS MONACELL, Primary Examiner R. J. WARDEN, Assistant Examiner U.S. Cl. X.R. 195-4035 R UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent 34843 .456 Dated October 22 1974 lnventor(s) B-.H. Had-en. et 3.1.
It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:
In Column 2, Line 20 "an" should be and. In Column 2, Line 31 "membrance" should be --membrane--.
In Column 4, Line 69 "appied" should be applied. In Column 6, Line 32 "37%" should be --37--.
Signed and Sealed this fourteenth I) 3) of October 1975 [SEAL] Attest:
RUTH C. MASON C. MARSHALL DANN Arresting Officer Commissioner ofParents and Trademarks