Multi-layer microorganism culture testing apparatus
BACKGROUND OF THE INVENTION 
     Field of the Invention 
     This invention relates to an apparatus for use in microorganism culture tests of liquid samples containing given microorganisms, and more particularly, to such an apparatus capable of cultivating microorganisms such as bacteria and eumycetes isolatedly from the liquid sample in a simple manner even when the sample contains antibiotics. 
     Isolation cultures are known in the art. Culture procedures vary depending on the type of sample to be tested. For example, a test on bacteria in blood was carried out by mixing a blood sample with a liquid growth medium. An isolation culture is then carried out in an isolation culture medium such as a blood agar plate and chocolate agar plate. Since it was impossible to directly isolate microorganisms from samples and an extra incubation step was necessary, prior art culturing procedures were rather cumbersome and time consuming. Detection was sometimes impossible when samples contained few microorganisms. 
     Furthermore, in culture tests of blood samples from septicemia patients, for example, the samples contain antibiotics which have been administered to the patients. These antibiotics significantly inhibit the growth of microorganisms in cultures, adversely affecting the test results. A testing apparatus as disclosed in U.S. Pat. No. 3,741,877 failed to provide accurate test results when samples contained antibiotics. There is a need for a testing apparatus capable of providing accurate test results from antibiotic-containing samples. 
     SUMMARY OF THE INVENTION 
     Therefore, an object of the present invention is to provide a multi-layer microorganism culture testing apparatus capable of directly collecting microorganisms from a sample by filtration to allow their culture in isolation without the need for an extra incubation step. 
     Another object of the present invention is to provide a microorganism culture testing apparatus capable of accurate examination without being affected by the presence of antibiotics in the samples. 
     According to a first aspect of the present invention, there is provided an improved multi-layer microorganism culture pad assembly for use in tests of liquid samples containing given microorganisms. A water-absorbing pad is impregnated with a culture medium for culturing the microorganisms and then dried. A filter member having submicron pores of a size capable of substantially preventing the passage of these microorganisms therethrough is bondedly laminated on the water-absorbing pad under sterile conditions. An antibiotic deactivating agent is applied under sterile conditions to the surface of the filter member remote from the water-absorbing pad and/or to the common surface between the filter member and the water-absorbing pad to form a coating thereof, completing the assembly. Preferably, the water-absorbing pad has a water-absorbing capacity substantially equal to the amount of liquid sample to be applied to the pad assembly for testing. The culture medium impregnated and dried in the water-absorbing pad is preferably present in an amount capable of forming a liquid culture medium of an acceptable concentration when dissolved in a liquid sample that has penetrated into the pad through said filter member after application to the pad assembly. The pores of the filter member are sized to pass liquid, but to prohibit the microorganisms from passing therethrough, and preferably, have a diameter of 0.22 to 0.75 microns. The filter member is laminated or bonded onto the water-absorbing pad, and preferably, this bonding is achieved with a hydrophilic high-molecular weight adhesive to ensure passage of liquid from the filter member to the water-absorbing member. 
     The term antibiotic deactivating agent, means an agent capable of deactivating antibiotics likely to be present in a sample, thereby eliminating any inhibition of the growth of microorganisms in the sample by the antibiotics. The antibiotic deactivating agents include agents capable of physically adsorbing antibiotics, agents capable of chemically bonding with antibiotics, and agents capable of decomposing antibiotics. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The above and other objects, features and advantages of the present invention will be more fully understood from the following description taken in conjunction with the accompanying drawings, in which: 
     FIG. 1 is a schematic cross-sectional view exaggeratedly showing a prior art multi-layer microorganism culture pad assembly; 
     FIGS. 2 and 3 are schematic cross-sectional views of different embodiments of the multi-layer microorganism culture pad assembly of the present invention; 
     FIG. 4 is a similar cross-sectional view of the culture pad assembly of the present invention showing colonies grown during cultivation; 
     FIG. 5 is a schematic cross-sectional view of a multi-layer microorganism culture testing apparatus of the present invention; 
     FIG. 6 is a perspective view of the apparatus of FIG. 5, and 
     FIG. 7 is a schematic cross-sectional view of the upper and lower sections of the vessel bonded together by an ultrasonic sealing technique. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The multi-layer microorganism culture pad assembly and the apparatus having the same mounted therein will be further illustrated in connection with a typical example of blood culture by referring to the preferred embodiment shown in the drawings. 
     The multi-layer microorganism culture pad assembly of the present invention includes a laminate of a water-absorbing pad 1 which is previously impregnated with a culture medium for culturing given microorganisms and dried, and a filter member 3 laminated or bonded to the water-absorbing pad 1 and having a number of submicron pores 2 of a size capable of substantially preventing the microorganisms from passing therethrough as shown in FIG. 1. The laminate of the water absorbing pad 1 and the filter member 3 is sterilized by any of the conventional methods. This type of laminate is known in the art as disclosed in the above-mentioned U.S. Pat. No. 3,741,877. This laminate type pad assembly was unsuccessful in culturing blood samples taken from septicemia patients since such samples often contained antibiotics. For the purpose of eliminating any influence by antibiotics on microorganism growth, an antibiotic deactivating agent is applied to the upper surface of the filter member 3 as shown in FIG. 2, or to the common surface between the filter member 3 and the water-absorbing pad 1 as shown in FIG. 3 to form a coating 4 thereof, completing the pad assembly of the present invention. In the drawings, the coating 4 is illustrated in an exaggerated and schematic manner. 
     The materials of which the water-absorbing pad 1 are made are not particularly limited as long as they can absorb aqueous solutions. The preferred materials are cellulosic filter paper, cellulosic unwoven fabric, glass fibers and the like. 
     The materials of which the filter member 3 are made are not particularly limited as long as they are inert to the samples tested. Typical examples of the filter materials include polycarbonate, polyamide, cellulose esters and the like, commercially available examples being Millipore membrane filter (trade name, Millipore Corporation), Metricell (trade name, German Instrument Company), Saltrius membrane filter (trade name, Karl Zeiss Corporation), and TOYO membrane filter (trade name, Toyo Filter K.K.). 
     The filter member has a number of submicron pores. If it is important that the filter member functions to isolate any microorganisms in a sample, then a pore diameter of 0.22 microns is most desirable which permits the retention of even Serratia marcescens, the smallest microorganism. In practice, however, all microorganisms tested do not pass through the filter member even when the diameter of pores in the filter member is somewhat larger than the size of the smallest microorganism to be examined. Pore diameters of 0.75 microns or less, and especially 0.45 microns or less are preferred because the filter member having pores within this submicron range can retain or isolate essentially all microorganisms while allowing quick permeation of the sample fluid into the underlying pad. 
     Ordinarily, blood samples taken from septicemia patients contain antibiotics. As described earlier, more accurate results can be obtained from blood cultures by deactivating the coexisting antibiotics. Typically, the antibiotic deactivating agents include: 
     (1) Antibiotic adsorbing agents (manufactured and sold by Marion Laboratories) 
     (a) Cation exchange resins: Polystyrene sulfonate, phenol methylene sulfonate and other types are available. These resins adsorb cationic antibiotics to inhibit their activity. 
     (b) Anion exchange resins: Polystyrene amine, phenol formaldehyde polyamine and other types are available. These resins adsorb anionic antibiotics to inhibit their activity. 
     (2) Chemical bonding agents 
     Sodium amylosulfate (SAS, Searle Diagnostics Inc.): This chemically bonds with a variety of antibiotics of aminoglucosides, etc. such as streptomycin, kanamycin, and polymyxin B to deactivate them. 
     (3) Decomposing agents 
     (a) Penicillinase: Penicillinase is a specific enzyme which decomposes penicillin. 
     (b) Cephalosporinase: Cephalosporinase is a specific enzyme which decomposes cephalosporin and similar antibiotics. 
     The culture media with which the water-absorbing pad is impregnated are not particularly limited as long as they are compatible with the samples to be tested. Preferred are those culture media allowing anerobic and aerobic bacteria to grow or propagate under anerobic and aerobic culture conditions, respectively. A typical composition of the culture medium adapted for blood cultures under anerobic and aerobic conditions is shown in Table 1. A test may advantageously be carried out by preparing two multi-layer microorganism culture pad assemblies having such a culture medium impregnated and applying a sample to each pad assembly. One sample may be kept under anerobic culture conditions and the other under aerobic conditions. 
     
                       TABLE 1                                                     
______________________________________                                    
Typical composition of blood culture                                      
medium (for filtration of 5-ml sample)                                    
Ingredient         Amount (mg)                                            
______________________________________                                    
Trypton (oxoid)    50                                                     
Soy bean pepton (oxoid)                                                   
                   15                                                     
Meat extract (oxoid)                                                      
                   15                                                     
Yeast extract (oxoid)                                                     
                   25                                                     
Liver hydrolyte (oxoid)                                                   
                   5                                                      
Glucose            12.5                                                   
K.sub.2 HPO.sub.4  0.75                                                   
NaCl               20                                                     
NaHCO.sub.3        10                                                     
L-Cysteine hydrochloride                                                  
                   2.25                                                   
p-Aminobenzoic acid                                                       
                   0.25                                                   
Hemin              0.025                                                  
______________________________________                                    
 
    
     The water-absorbing pad having an impregnating culture medium dried may be adhesively bonded to the filter member, preferably using a hydrophilic high-molecular weight adhesive such as polyvinyl pyrrolidone. 
     The thus constructed multi-layer microorganism culture pad assembly of the present invention is not particularly limited in configuration. Most preferably, the pad assembly is circular to match the shape of many implements commonly used in laboratory work, for example, Petri dishes. The size of the pad assembly is also not particularly limited. When 5 ml of a blood sample is applied dropwise onto the pad assembly, the assembly may preferably have a diameter of 60 mm with the pad ranging from 2 mm to 3 mm in thickness. 
     To facilitate testing, the above-mentioned pad assembly may be mounted in a container of a suitable configuration and size. A typical example of such a container is shown in FIGS. 5, 6 and 7. As seen from the cross-sectional view of FIG. 5, the container consists of a cylindrical vessel 6 having the pad assembly mounted therein and a cap 8 in loose fit on the vessel. It is necessary for effective cultivation that a gap 9 be left between the vessel 6 and the cap 8 to allow airflow. The vessel 6 has a circumferential recess 7 formed at a predetermined height from the bottom so that the pad assembly is firmly held in the recess at the predetermined height. The pad assembly may be mounted in the vessel by placing it in press fit with the recess. Preferably, the vessel 6 is divided into upper and lower sections 10 and 11 which define the recess at their intersection when mated. In this case, the container may be assembled by placing the upper section 10 on the lower section 11 with the pad assembly interposed therebetween, and securing the upper and lower sections to each other. The pad assembly is then rigidly clamped between the upper and lower sections. The distance of the recess 7 from the bottom of the vessel may be determined such that, when a given volume of sample fluid is applied to the pad assembly and excess fluid drips from the pad assembly, the excess fluid that accumulates in the bottom of the vessel does not rise up to the level of the pad assembly positioned in the recess. 
     Securing or bonding of the upper and lower sections 10 and 11 with the pad assembly clamped therebetween may be carried out by a variety of techniques. For example, the upper and lower sections may be fused to each other by ultrasonic or heat sealing after the pad assembly is interposed between them with no adverse affection to samples to be tested. Alternatively, they may be bonded to each other by applying a solvent capable of dissolving the material of which the vessel is made, or an adhesive comprising such a solvent having the material of which the vessel is made dissolved therein, to the mating edges of the upper and lower sections to thereby partially dissolve the vessel material, eventually forming an integral bond. Of course, the solvent should not inhibit or retard the growth of microorganisms, the preferred examples being methylene chloride and xylene. The material of which the vessel 6 and the cap 8 are made, whether the same or different, may be semi-transparent or preferably transparent for visual observation from the outside. Other requirements are satisfactory hardness, ease of molding and low cost. Typical examples of the vessel and cap forming materials include polystyrene, polyethylene, hard polyvinyl chloride, polycarbonate, methacrylic resins and the like. In case that the upper and lower sections 10 and 11 is bonded by an ultrasonic sealing technique, the upper and lower sections 10 and 11 are provided with mating circumferential flanges 13 and 14, respectively, as shown in FIG. 7. 
     The lower section 11 of the vessel may be provided with at least one vent 12 for allowing air to escape from the interior of the lower section 11 when sample fluid is applied to the pad assembly (see FIG. 5). However, the vent 12 need not necessarily be formed for the sake of the adequate lower space under the pad assembly. 
     The multi-layer microorganism culture testing apparatus of the present invention shown in FIGS. 5 and 6 may be used as follows when it is applied, for example, to blood culture. Prior to starting the culture, a liquid sample, i.e., blood is rendered anticoagulative by adding an anticoagulant thereto. A hemolytic agent such as saponin is aseptically added to the sample to fully hemolyze the erythrocytes. The thus treated sample is applied dropwise to the pad assembly in the culture testing apparatus. The microorganisms in the blood sample are isolated or retained by the filter member 3 while the remaining fluid passes through the pores in the filter member 3 to the water-absorbing pad 1 where it dissolves the dried impregnating culture medium to form a liquid culture medium. As described above, for a given volume of sample fluid applied, the water-absorbing pad 1 has an adequate water-absorbing capacity and the culture medium is present in an amount capable of forming a liquid culture medium of a practically acceptable concentration. Thus, according to the present invention, the microorganisms retained above the filter pores will be given an optimum culture medium. Cultivation is carried out under adequate conditions. A culturing test may advantageously be carried out using two sets of testing apparatus each receiving a given volume of sample blood, one being kept under aerobic conditions and the other under anerobic conditions. The thus cultured microorganisms form colonies 5 on the filter member 3 as shown in FIG. 4. The use of the pad assembly of the present invention shown in FIG. 2 or 3 eliminates any adverse influence by antibiotics on the cultivation of microorganisms because the antibiotic deactivating coating adsorbs and removes antibiotics in the blood. When the sample fluid is applied to the pad assembly, excess fluid drips from the pad to the bottom of the container. This prevents the pad from falling down while the purged air may escape through the vent 12, if present. 
     As is clear from the foregoing, the multi-layer microorganism culture pad assembly and the testing apparatus having the same mounted have the following advantages. 
     (1) Direct isolation culture is allowable without the need for an extra incubation step as required in prior art cultures, and the culture procedure is considerably simplified. 
     (2) Since the impregnating culture medium is present in the water-absorbing pad in a dry state, it is highly stable. Conversion of the culture medium from the dry state to the ordinary liquid state may be controlled through adjustment of the water-absorbing capacity of the pad and the amount of the culture medium impregnated. The culture medium becomes restored to the liquid state in which it is ordinarily used after receiving the necessary volume of fluid to ensure consistent culture conditions. 
     (3) Microorganisms are detectable even at low concentrations. 
     (4) No extra steps are necessary even when samples contain antibiotics.