Abstract:
A transport media, particularly for fastidious microorganisms, has a concentration of activated charcoal less than 10 grams per liter of water. This maintains viability of the microorganisms while permitting Gram interpretation of the specimen.

Description:
FIELD OF THE INVENTION 
     The present invention relates to media for transporting microorganisms. 
     BACKGROUND 
     Maintaining the viability of clinically relevant pathogens for subsequent diagnosis is a major concern in the healthcare field, especially in outpatient settings where delay in transit may be expected, and specialized transport media is utilized for this purpose. 
     The use of transport media was first popularized in the 1940&#39;s, when Robert Stuart, a public health microbiologist in Glasgow, Scotland, developed and disclosed to the world a transport medium consisting of sodium glycerophosphate, sodium thioglycollate, methylene blue, agar and calcium chloride. The Stuart media was subsequently improved by C. R. Amies, of the Ontario Public Health Laboratory, who substituted an inorganic phosphate buffer for the glycerophosphate of the Stuart media, and added sodium, calcium and magnesium salts. Both the Stuart and Amies media remain widely used for transporting a wide variety of microorganisms. 
     For fastidious organisms such as  Heomophilus influenza, Neisseria gonorrhoeae  and  Streptococcus pneuemoniae , activated charcoal at the rate of 10 g per liter of water is ubiquitously added to the media, as neither the Stuart nor Amies media provide satisfactory conditions for maintaining viability otherwise. The charcoal is believed to remove toxic substances often found in the specimens, prevent photochemical oxidation reactions, reduce production of potentially toxic hydrogen peroxide and trap free radicals. However, the presence of charcoal particles can interfere with the Gram interpretation of the specimen. Charcoal particles in the smear can mask or even be confused with Gram positive organisms. Since Gram stains are often the initial result available to physicians, this has a significant impact on patient management. Accordingly, it is commonplace for transport media to be maintained in clinical inventory in both charcoal-containing and charcoal-absent variants. 
     SUMMARY OF THE INVENTION 
     The present invention pertains to a transport media which has a charcoal concentration sufficiently low to permit Gram interpretation yet maintains the viability of fastidious microorganisms. This permits a single inventory to be maintained, with commensurate impacts on cost and convenience. 
    
    
     DETAILED DESCRIPTION 
     To prepare transport media according to the invention, various ingredients are solubilized in water. In an exemplary embodiment, components as listed below are suspended in 1.0 liters of distilled water; heated to boiling with frequent agitation to solubilize the ingredients; dispensed into screw-capped tubes; and sterilized at 121° C. for 15 minutes. 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Activated charcoal 
                 0.50 grams 
               
               
                   
                 MOPS Acid 
                 3.15 grams 
               
               
                   
                 MOPS Salt 
                 8.48 grams 
               
               
                   
                 Potassium Chloride 
                 1.00 grams 
               
               
                   
                 Calcium Chloride 
                 1.26 grams 
               
               
                   
                 Magnesium Chloride 
                 5.48 grams 
               
               
                   
                 Sodium Thiosulfate 
                 10.90 grams  
               
               
                   
                 N-Acetyl Glucosamine 
                 0.50 grams 
               
               
                   
                 Betaine 
                 5.00 grams 
               
               
                   
                 Gluten Hydrolysate from maize 
                 0.50 grams 
               
               
                   
                 Agar 
                 7.50 grams 
               
               
                   
                   
               
             
          
         
       
     
     The media was tested in accordance with the procedures set forth in  Quality Control of Microbiological Transport Systems; Approved Standard , CLSI, (formerly NCCLS) M-40A. Strains included  Neisseria gonorrhoeae  (NG),  Haemophilus influenzae  (HI),  Streptococcus pneumoniae  (SPN),  Streptococcus pyogenes  (SP),  P. aeruginosa  (PA) and  S. aureus  (SA) and  E. coli  (EC). Swabs inoculated with NG, HI, SPN were held at 4° C. and those inoculated with SP, PA, SA, and EC were held at room temperature (RT). Organism survival was evaluated by determining CFU/ml at 0, 6, 24 and 48 hours by plating 100 ul of the suspension to appropriate aerobic media. Dilutions with visible growth at 48 hours incubation with a range of 30-300 CFU were evaluated. Combinations were tested in triplicate and compared using a two-tailed student t-Test. For the Gram stain, both swabs were inoculated with 0.5 McFarland suspensions of each strain and held for ½ hour at RT prior to preparing and staining. The results of the testing are set forth below. 
     
       
         
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 % Recovery 
               
               
                   
                 Initial 
                 Storage Time/Conditions 
               
             
          
           
               
                 Test Organism 
                 Count 
                 24RT 
                 24REF 
                 48RT 
                 48REF 
               
               
                   
               
             
          
           
               
                   Haemophilus influenzae  ATCC 10211 
                 330 
                 31  
                 34 
                 19 
                 18 
               
               
                   Neisseria gonorrhoeae  ATCC 43069 
                 428 
                 0 
                 11 
                 ND 
                 ND 
               
               
                   Streptococcus pneumoniae  ATCC 6305 
                 298 
                 ND 
                 ND 
                 11 
                 18 
               
               
                   Streptococcus pyogenes  ATCC 19615 
                 92 
                 ND 
                 ND 
                 70 
                 86 
               
               
                   Propionibacterium acnes  ATCC 6919 
                 222 
                 ND 
                 ND 
                 39 
                 43 
               
               
                   Prevotella melaninogenica  ATCC 25845 
                 314 
                 0 
                 48 
                  0 
                  7 
               
               
                   Bacteroides fragilis  ATCC 25285 
                 276 
                 ND 
                 ND 
                 93 
                 84 
               
               
                   Peptostreptococcus anaerobius  ATCC 27337 
                 205 
                 7 
                 40 
                  1 
                 14 
               
               
                   
               
               
                 24RT = 24 hours room temperature storage 
               
               
                 24REF = 24 hours refrigerated storage 
               
               
                 48RT = 48 hours room temperature storage 
               
               
                 48REF = 48 hours refrigerated storage 
               
               
                 ND = no data 
               
             
          
         
       
     
     A second series of tests was carried out, with the charcoal amount varied to 1.0 grams per liter, the results of which being as follows: 
     
       
         
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 % Recovery 
               
               
                   
                 Initial 
                 Storage Time/Conditions 
               
             
          
           
               
                 Test Organism 
                 Count 
                 24RT 
                 24REF 
                 48RT 
                 48REF 
               
               
                   
               
             
          
           
               
                   Haemophilus influenzae  ATCC 10211 
                 204 
                 25  
                 30 
                 12 
                 15 
               
               
                   Neisseria gonorrhoeae  ATCC 43069 
                 412 
                 0 
                 19 
                 ND 
                 ND 
               
               
                   Streptococcus pneumoniae  ATCC 6305 
                 172 
                 ND 
                 ND 
                 10 
                 27 
               
               
                   Streptococcus pyogenes  ATCC 19615 
                 36 
                 ND 
                 ND 
                 39 
                 76 
               
               
                   Propionibacterium acnes  ATCC 6919 
                 319 
                 ND 
                 ND 
                 63 
                 44 
               
               
                   Prevotella melaninogenica  ATCC 25845 
                 827 
                 0 
                 83 
                  0 
                 12 
               
               
                   Bacteroides fragilis  ATCC 25285 
                 333 
                 ND 
                 ND 
                 59 
                 88 
               
               
                   Peptostreptococcus anaerobius  ATCC 27337 
                 782 
                 3 
                 39 
                  0 
                 26 
               
               
                   
               
             
          
         
       
     
     In both tests, because of the relatively low charcoal concentration, Gram, interpretation of the samples was possible. Surprisingly, in view of the relatively low charcoal concentrations, under refrigerated conditions, the various microorganisms remained viable at levels deemed useful according to CLSI M-40A. 
     A third series of tests was carried out, comparing the exemplary transport media with the charcoal amounts varied to 1.0 grams and 0.5 grams for both ATCC and clinical strains. 
     The results of such testing for both the ATCC and clinical strains are set forth below. 
     
       
         
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Charcoal amount 
                 Charcoal amount 
               
               
                   
                 varied to 1.0 grams 
                 varied to 0.5 grams 
               
               
                   
                 Storage Time (hrs.) 
                 Storage Time (hrs.) 
               
             
          
           
               
                 Test Organism 
                 Storage Conditions 
                 6 
                 24 
                 48 
                 6 
                 24 
                 48 
               
               
                   
               
             
          
           
               
                   
                 % Viability compared to Baseline for ATCC 
               
               
                   
                 strains 
               
             
          
           
               
                   S. pyogenes  ATCC 19615 
                 RT 
                 53 
                 34 
                 15 
                 86 
                 55 
                 52 
               
               
                   S. pneumoniae  ATCC 6305 
                 Refrigerated 
                 80 
                 93 
                 74 
                 64 
                 64 
                 10 
               
               
                   P. aeruginosa  ATCC 27853 
                 RT 
                 82 
                 TNTC 
                 TNTC 
                 143 
                 TNTC 
                 TNTC 
               
               
                   S. aureus  ATCC 25923 
                 RT 
                 104 
                 67 
                 40 
                 72 
                 85 
                 88 
               
               
                   E. coli  ATCC 25922 
                 RT 
                 110 
                 TNTC 
                 TNTC 
                 211 
                 TNTC 
                 TNTC 
               
               
                   N. gonorrhoeae  ATCC 49221 
                 Refrigerated 
                 61 
                 2 
                 1 
                 13 
                 34 
                 1 
               
               
                   H. influenzae  ATCC 10211 
                 Refrigerated 
                 90 
                 59 
                 12 
                 56 
                 33 
                 53 
               
             
          
           
               
                   
                 % Viability compared to Baseline for clinical 
               
               
                   
                 strains 
               
             
          
           
               
                 
                   S. pyogenes 
                 
                 RT 
                 195 
                 31 
                 13 
                 53 
                 34 
                 31 
               
               
                 
                   S. pneumoniae 
                 
                 Refrigerated 
                 71 
                 74 
                 13 
                 140 
                 103 
                 34 
               
               
                 
                   P. aeruginosa 
                 
                 RT 
                 40 
                 TNTC 
                 TNTC 
                 61 
                 TNTC 
                 TNTC 
               
               
                 
                   S. aureus 
                 
                 RT 
                 38 
                 21 
                 12 
                 135 
                 264 
                 348 
               
               
                 
                   E. coli 
                 
                 RT 
                 259 
                 TNTC 
                 TNTC 
                 TNTC 
                 TNTC 
                 TNTC 
               
               
                 
                   N. gonorrhoeae 
                 
                 Refrigerated 
                 20 
                 3 
                 0 
                 67 
                 5 
                 0 
               
               
                 
                   H. influenzae 
                 
                 Refrigerated 
                 52 
                 17 
                 17 
                 54 
                 15 
                 5 
               
               
                   
               
               
                 TNTC = too numerous to count 
               
             
          
         
       
     
     In the third series of tests, there was again a significant reduction in particles with improved visualization of organisms and background staining and except for  Streptococcus pneumoniae , there was no statistical difference in the ability of the two media to maintain the viability of the clinical and ATCC isolates. The discrepancy in viable counts observed for  S. pneumoniae  in the third series of tests is likely a reflection of the fastidious nature of the organisms or variability in the colony counts and not a result of the reduced charcoal in the media. 
     The 0.5 g/l and 1.0 g/l embodiments therefore each constitute a transport medium which has a charcoal concentration sufficiently low to permit Gram interpretation yet maintains the viability of fastidious microorganisms. This permits a single inventory to be maintained, with commensurate impacts on cost and convenience. 
     Whereas but two embodiments of the invention are herein described, various modifications thereto are contemplated. 
     For example, whereas specific ingredients are listed, other analogues could be substituted therefore. For example, buffers other than MOPS acid and salt could be utilized. As well, osmolarity can be maintained otherwise than via Betaine, and salts other than NaCl, KCl and MgCl could be utilized for permeability control. Further, the various components could be provided in varying amounts. 
     Accordingly, the invention should be understood as limited only by the accompanying claims, purposively construed.