Patent Publication Number: US-4728608-A

Title: Bacterial screening system with enhanced shelf life

Description:
DESCRIPTION 
     1. Technical Field 
     The present invention relates to assays for the presence of organisms, particularly bacteria, in specimens taken from sources such as biological fluids, water, and food. 
     2. Background Art 
     It is known in the art to use a tetrazolium salt as a component in a system for identifying the presence of bacteria in fluid specimens. Such a system is based upon the reduction/oxidation reactions which occur as bacteria ferment and perform oxidative phosphorylation of high energy nutrients (i.e., carbohydrates) and linking them to indicators which develop color upon reduction or oxidation: 
     
         ______________________________________                                    
In Vivo          Bacterial Assay System                                   
______________________________________                                    
High Energy                                                               
         Low Energy  High Energy Low Energy                               
Carbohydrate                                                              
         Carbohydrate                                                     
                     Carbohydrate                                         
                                 Carbohydrate                             
 ##STR1##                                                                 
                  ##STR2##                                                
                 Bacterial enzymes (if present)                           
 ##STR3##                                                                 
                  ##STR4##                                                
NAD (P)  NAD (P) H   Electron    Electron                                 
Enzymes  Enzymes     Carrier     Carrier                                  
(oxid.)  (red.)      (oxid.)     (red.)                                   
 ##STR5##                                                                 
                  ##STR6##                                                
 ##STR7##                                                                 
                  ##STR8##                                                
Flavoproteins                                                             
         Flavoproteins                                                    
                     Tetrazolium Tetrazolium                              
                     Component   Component                                
(red.)   (oxid.)     (red.)      (oxid.)                                  
 ##STR9##                                                                 
 ##STR10##                                                                
Cytochromes                                                               
         Cytochromes                                                      
(oxid.)  (Red.)                                                           
 ##STR11##                                                                
 ##STR12##                                                                
H.sub.2 O                                                                 
         O.sub. 2                                                         
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     When a tetrazolium compound is reduced, a highly insoluble intensely colored formazan is formed, permitting the presence of bacteria to be identified on direct visual inspection after an incubation period. 
     The in vivo purpose of the cascade-like breakdown of high energy components is to trap the energy stored therein as ATP. Each transfer component has a progressively higher reduction/oxidation potential than the components preceding it in the cascade. In tetrazolium-based assays, the NAD (P) dehydrogenases generally reduced tetrazolium dyes poorly when directly coupled, but when intermediate electron carriers are used, the reduction of the tetrazolium dye to the colored formazan occurs more rapidly. 
     It is also known to use, for the substrate for bacterial growth, Schaedler broth (Difco) which is a solution of tryptic soy broth, proteose peptone no. 3, yeast extract, dextrose, tris(hydroxy-methyl)amino methane, L-cystine, and hemin; the solution has a physiologically typical pH in the range of 7.0-7.5. 
     Prior art tetrazolium-based bacterial assays have typically been plagued by a number of disadvantages, making such assays difficult to employ in individually packaged disposable test kits for mass distribution. First, the shelf-life of tetrazolium solutions is generally short--less than six months even under refrigeration. Second, the sensitivity of such assays of urine specimens is typically not better than about 10 6  colony forming units (CFU)/ml., which is not sufficiently high sensitivity for many clinical purposes. Third, such assays may also produce non-specific background color development, which can interfere with ready, direct visual observation of the colored formazan. 
     DISCLOSURE OF INVENTION 
     The present invention provides a tetrazolium-based system, overcoming disadvantages in prior art systems, for testing for the presence of organisms in a specimen, wherein the overall system, including a mixture of a substrate and an indicator system, is buffered to a pH above approximately 8.0. In a preferred embodiment of the invention, the test system is buffered to a pH of approximately 9.0 and until use is stored as a separate substrate solution, having a high pH and an indicator solution buffered to a pH of approximately 3.3 thereby enhancing the shelf life of the indicator solution. Another embodiment provides a substrate consisting of glucose with at least one amino acid in a buffered solution. 
     DESCRIPTION OF SPECIFIC EMBODIMENTS 
     As described in further detail below, a preferred embodiment of the present invention utilizes separate substrate and indicator solutions which are mixed just before use to form the test system. In the examples following, the specimen is typically a 1 ml fluid specimen, such as urine, which is first passed through a course filter to remove large particles that may otherwise interfere with testing, then through a bacterial filter having a porosity sufficient to permit fluid flow, but which traps and concentrates the bacteria. A 1 ml wash solution of saline, phosphate-buffered saline, tris, or other physiologically reasonable buffer is then passed through the same filters. A pH of the wash ranging from 6-9 and molarity ranging from 0.05 to about 0.2 have been found to be uncritical. Quantity of 0.25 to 0.50 ml of the previously mixed test system reagent is then added above the bacterial filter and about 0.1 ml of the test system is passed through the filter, leaving a fluid head above the filter. The bacterial filter with its fluid head is then incubated at 35°-37° C. for periods up to one-half hour. (Temperatures up to 43° C. have produced acceptable results.) The presence of bacteria is determined by direct visual inspection of the surface of the filter, which under such circumstances develops a color ranging from violet to blue-black. 
     The chemistry described herein is suitable for use in a device, of the type described in co-pending application Ser. No. 709,595, filed Mar. 8, 1985, for an invention of Buck et al., which is hereby incorporated herein by reference. For use in a device of the type therein described, the chambers 16a and 16b of FIGS. 1-3 therein may be used to hold the separate substrate and indicator solutions, which are mixed to form the test system when the device is used. 
    
    
     EXAMPLE 1 
     A two-part test system that includes (a) an indicator system and (b) a buffered substrate is prepared as follows: 
     
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A. Indicator System                                                       
1. Nitrobluetetrazolium                                                   
                  23.8 mg Calbiochem 202945                               
2. Phenazine ethosulfate                                                  
                  9.8 mg Sigma 13F0041                                    
3. Deionized water QS to                                                  
                  70 ml                                                   
4. Dissolve, adjust pH to                                                 
                  3.3 w/0.1 m HCl                                         
Filter, sterilize and store in the dark at 2-8° C.                 
B. Substrate/buffer                                                       
1. Glucose        750    mg     Sigma                                     
2. Serine         150    mg     Sigma                                     
3. Glutamic acid  255    mg     Sigma                                     
4. Tris base      1500   mg     Sigma                                     
5. Anhydrous sodium carbonate                                             
                  74     mg     Mallinkrodt                               
6. Deionized water QS                                                     
                  75     ml                                               
Filter, sterilize, and store at 2-8° C.                            
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     Substrate/buffer pH is 8.8 to 9.2 when mixed. 
     Before use, the test system is prepared by mixing 1 part of the indicator system with 1 part of the substrate/buffer; pH after mixing is between 8.8 and 9.2. The mixture is stored in the dark at 2°-8° C. 
     Following the procedure set forth above, this test system produced a sensitivity at least as good as 10 5  colony forming units (CFU) per ml, which corresponds to approximately 95% of urine specimens containing pathogens. This is a preferred embodiment of the invention. 
     In comparison to a substrate based on Schaedler broth, it was found that the individual presence of any of hemin, yeast, or peptones in the Schaedler formulation at the high pH levels used here caused non-specific background color development. The above buffered substrate (which eliminates all components of the Schaedler formulation determined to be non-essential, and produces a test system with an exceptionally high pH) yielded dramatic improvements over prior art formulations. Increasing the pH of the test system above 9.2 increased sensitivity but also increased spontaneous color development, resulting in a loss of test specificity. The inventors believe that the elevated pH produces improved test results owing to the optimal activity of many bacterial dehydrogenases at these pH levels, even though these levels are in excess of what one might otherwise expect for a physiological system. 
     EXAMPLE 2 
     A two-part test system is prepared as in Example 1, but the pH of the indicator system and substrate/buffer are formulated to give the test system a more physiologically typical pH ranging between 7.0 and 7.5. This test system produced a sensitivity of about 10 6  colony forming units (CFU) per ml 
     EXAMPLE 3 
     A two-part test system is prepared as in Example 1, but the step of adjusting the pH of the indicator system to 3.3 is omitted. The indicator system and the substrate/buffer were each aliquotted into 0.5 ml quantities in separate vials and sealed. Samples were stored at 2°-8° C. (5° C.), 18°-25° C. (20° C.), 35° C., 45° C. Periodically samples were taken by opening and mixture one vial of indicator system and one vial of substrate/buffer. Dilutions (in urine) of a standard bacterial suspension (produced from a stock freezer culture) were used to evaluate potency of the test reagent. A freshly prepared (within 24 hours) reagent was used as a reagent control. A sterile urine was used as a negative bacterial control and historical documentation of the bacterial strain&#39;s reactivity coupled with use of fresh reagent served as a positive control. Using the Arrhenius accelerated stability method (a product is generally stable twice as long at 5° C. as it is at 15°  C. and four times as long at 5° C. as it is at 25° C., etc.), refrigerated stability was extrapolated. Refrigerated, stability of the test system was found to be less than 6 months, whereas refrigerated stability of the two-part test system prepared in accordance with Example 1 was 2-3 years. 
     It will be apparent that although Example 1 identifies NBT [2,2&#39;,d(p-nitrophenyl)-5,5&#39;diphenyl-3&#39;,(3,3&#39;dimethoxy-4,4&#39;diphenylene)ditetrazolium chloride] as the tetrazolium salt and PES as the electron carrier employed, any suitable tetrazolium salt, such as INT [2(p-iodophenyl)3(peranitrophenyl)5phenyl tetrazolium chloride], TNBT [2,2&#39;,5,5&#39;tetra-(p-nitrophenyl)-3,3&#39;(dimethoxy-4&#39;diphenylene)di-tetrazolium chloride], or MTT, and any suitable electron carrier, such as PMS (phenazine methosulfate) or meldola blue, may be employed.