Abstract:
A single basal growth medium for receiving various substrates for the purpose of rapid identification of any species of non-fermentative Gram-negative bacilli (NFB), wherein the medium is low in organic nitrogen but is supplemented with inorganic nitrogen from an ammonium ion source to enhance NFB growth. The medium also serves to identify members of the family Enterobacteriaceae, cytochrome oxidase positive fermenters, Gram-positive bacilli, Gram-positive cocci, and anaerobes.

Description:
BACKGROUND OF THE INVENTION 
     In the prior art many media have been developed for the identification of bacteria but most prior art media have had limited capabilities. Some, for example, have been used for determining only a single biochemical reaction of an unknown microorganism. Others have been compounded to identify only fermentative or non-fermentative bacteria. 
     A primary problem in the prior art media has been an inordinate incubation time for positive reactions. Prolonged incubation time for bacterial speciation is particularly hazardous in cases of nosocomial infections or post-operative wound infections. 
     Pseudomonas aeruginosa, Acinetobacter anitratus, P. maltophilia, and many other NFB are becoming increasingly recognized as important causes of infection and therefore it is mandatory that they be rapidly isolated and identified. While most commercially available isolation media are applicable for NFB, identification methodology for these organism has been inadequate. 
     Two known prior art media have been developed for the identification of NFB. One is disclosed in U.S. Pat. No. 3,399,115 to Sellers. This medium is capable of identifying only five species of NFB. This medium includes a large amount of organic nitrogen. In general a large amount of organic nitrogen tends to cause additional alkaline byproducts of metabolism so that positive reactions are significantly delayed and in addition unreliable results are obtained. The Sellers patent does suggest the use of inorganic nitrogen. 
     The second medium is disclosed in U.S. Pat. No. 4,048,016 to Otto. The Otto disclosure is essentially that of a non-growth or buffered substrate medium. For inoculation it requires a heavy inoculum, that is, an inoculum of about 10 11  organisms per milliliter. This is obtained by harvesting numerous (50 to 100) bacterial colonies and requires an additional time of 24 hours for NFB identification. 
     SUMMARY OF THE INVENTION 
     Although there are over thirty species of clinically encountered NFB, three of the species account for more than 90% of the clinical isolates. The three common species, Pseudomonas aeruginosa, Pseudomonas maltophilia and Acinetobacter anitratus, and the other twenty-seven species are efficiently and rapidly identified by the basal growth medium according to the invention. 
     The success of the present medium is due to the discovery of the use of a small amount of organic nitrogen supplemented with inorganic nitrogen in the form of an ammonium ion source. It was known that low organic nitrogen does not support adequate growth of NFB but it was surprisingly discovered that the addition of inorganic nitrogen, in the form of an ammonium ion, greatly enhanced NFB growth without producing the additional alkaline byproducts of metabolism that occur with the prior art use of organic nitrogen in amounts sufficient to permit NFB growth. The same results were surprisingly obtained in the identification of other microorganisms to be described. 
     The limited low organic nitrogen is necessary to show the rapid attack of carbohydrates and to show the attack of organic salts and amides without any false positive reactions due to the extremely proteolytic nature of most NFB. The ammonium ion source, however, has been found to be an ideal supplement to provide sufficient NFB growth for identification. 
     Accordingly, it is an object of the present invention to provide an improved basal medium to which substrates may be added whereby virtually all species of NFB and other microorganisms can be identified. 
     It is another object of the invention to provide a basal medium, as described in the preceding paragraph, containing a relatively low amount of organic nitrogen in the form of a low protein degradation product, supplemented with inorganic nitrogen in the form of an ammonium ion. 
     It is still another object of the invention to provide a basal medium, as described in the preceding paragraphs, by which the reaction time of identification of particular NFB and other microorganisms is reduced to a maximum of 24 hours. 
     It is a further object of the invention to provide a basal medium, as described in the preceding paragraphs, in which a relatively light inoculum may be used to accomplish identification of NFB. That is, only one bacterial colony is needed to inoculate from 25 to 30 substrates in a miniaturized multi-compartmented container. That the invention permits the use of the relatively light inoculum saves 24 hours of identification time over the prior art. 
     It is a still further object of the invention to provide a basal medium, as described in the preceding paragraphs, which when combined with an appropriate substrate may be used as a liquid, semi-solid or solid. Agar is used as a gelling agent to reduce the fluidity. 
     It is another object of the invention to provide a basal medium, as described in the preceding paragraphs, by which bacteria other than NFB can be identified. For example, Enterobacteriaceae, cytochrome oxidase positive fermenters, Gram-positive cocci, Gram-positive bacilli, and anaerobes can also be identified with the use of this basal medium and the proper selection of known substrates. 
     Further objects and advantages of the invention may be brought out in the following part of the specification wherein small details have been described for the competence of disclosure, without intending to limit the scope of the invention which is set forth in the appended claims. 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The basal growth medium of the invention is comprised of organic nitrogen in the form of a protein degradation product, inorganic nitrogen in the form of an ammonium salt, any potassium salt, any magnesium salt, yeast extract, an indicator and water. The organic nitrogen may be peptones in general, protein hydrolysate, trypticase, casamino acids, Poly peptone, pancrearic digest of casein. The ammonium salts may be ammonium chloride, ammonium sulfate, ammonium hypophosphate, ammonium hypophosphite, or ammonium orthophosphate, for example. 
     The indicator may be brom cresol purple, brom thymol blue or phenol red. 
     A suitable basal medium is comprised of the following: 
     
         ______________________________________One of the above ammonium salts                   0.5 to 1.5 gm.Any potassium salt      0.1 to 0.3 gm.Any magnesium salt      0.1 to 0.3 gm.Yeast extract           0.2 to 1.0 gm.One of the above organic nitrogen compounds                   0.2 to 1.0 gm.Agar                    0.00 to 60.0 gm.One of the above indicators                   0.002 to 0.060 gm.Water to make           1000 ml.______________________________________ 
    
     It has been found that amounts in excess of the maximum of the above ranges are of no benefit. 
     To the above basal medium, substrates ranging from 2.0 to 25.0 grams are added, and the pH adjusted depending on the type of indicator and substrate used. The pH range preferably is varied between 6.2 and 7.8. For example, a substrate to be acidified is adjusted to pH 7.0±0.1 when phenol red is used. For brom cresol purple the pH is adjusted to 7.5±0.2. Substrates that are alkalinized are adjusted to pH 6.5±0.2 when phenol red is used and pH 7.0±0.2 when brom cresol purple is the indicator. 
     Examples of subsrates that can be alkalinized in the above medium to identify NFB are salts of dicarboxylic acids; hydroxy acids, e.g. tartrate; sugar derivative, e.g., saccharates; ring amino acids, e.g., phenylalanine; amides, e.g., glutamine; and certain nitrogen compounds including allantoin and asparagine. It was also surprisingly found that the hyrolysis of gelatin was accomplished by the alkalinization of it as a substrate in the basal medium. 
     When the basal medium is used in the alkalinization of the above organic salt and amide substrates, 0.1% by weight of dextrose is added. The added dextrose balances the slight alkaline shift caused by the bacterial attack of the organic nitrogen and yeast extract in medium. A positive identification of an NFB is made when alkalinization occurs as shown by the indicator change in a particular substrate. 
     Examples of substrates that can be acidified in the above medium to identify NFB are poly-alcohols, e.g., glycol, M-inositol, and D-Mannitol; disaccharides, e.g., cellobiose and lactose; pentoses, e.g., L-arabinose, D-xylose, D-ribose and rhamnose; heptoses, e.g., D-fructose; and hextoses, e.g., glucose. A positive identification of an NFB is made when acidification occurs as shown by the indicator change in a particular substrate. 
     The basal medium of this invention may be of three general consistencies. One is in the form of a liquid to make a broth, accomplished by not including agar. The other two are semisolid and solid. These are achieved by adding agar: for the semisolid 1.0 to 1.3% by weight and for the solid above 1.4% by weight. 
    
    
     The following example is presented as a typical basal medium of the invention: 
     
         ______________________________________Ammonium phosphate    1.0    gm.Potassium chloride    0.2    gm.Magnesium sulfate     0.2    gm.Yeast extract         0.5    gm.Casitone              0.5    gm.Agar                  15.0   gm.Phenol red            0.020  gm.Water to make         1000   ml.______________________________________ 
    
     To this basal medium the appropriate substrates may be added so as to, in an appropriate test apparatus, identify all species of NFB, and in particular the three common species. 
     EXAMPLE 1 
     A series of agar slants of the above basal medium were prepared with the following substrates: citrate, dextrose, L-arabinose, lactose, D-mannitol, acetamide, tartrate, gelatin and starch. 
     To make the agar slants each of the above compounds was weighed and added to one liter of cold distilled water. The pH was adjusted to the proper starting point for the substrate, and the ingredients were steamed to melt the agar. The medium was dispensed into test tubes and autoclaved for 15 minutes. After autoclaving the medium was cooled to approximately 50° C. and a filter sterilized substrate was added to provide a final concentration of 1% by weight of the basal medium. The completed combinations were then cooled in a slanted position and solidifed. 
     Each of the slants was inoculated with a single 24 hour colony of an NFB and then incubated for 24 hours at 35° C. in an air incubator. After 24 hours of incubation the reactions were read in the tubes and the results tabulated in Table 1. From the results it can be seen that most NFB produced positive reactions in 24 hours. 
     
                                           TABLE 1__________________________________________________________________________Non-fermentative bacilli (NFB)Test    P. cepacia         P. aeruginosa                P. maltophilia                       P. putida                            P. acidovorans                                    A. odorans__________________________________________________________________________*Acetamide   -     +      -      -    +       +*Citrate   +     +      +      +    -       +*Gelatin    +.sup.w         -      +      -    -       -*Tartrate   +     -      -      +    +       +**Dextrose   +     +      +      +    -       -**L-Arabinose   +     +      -      -    -       -**D-Mannitol   +     +      -      -    +       -**Starch   -     -      -      -    -       -**Lactose   +     -      -      -    -       -__________________________________________________________________________ + = positive within 24 hours; +.sup.w = weak positive within 24 hours; - negative through 72 hours of incubation. *started with acid pH, when positive became alkaline. **started with alkaline pH, when positive became acid. 
    
     EXAMPLE 2 
     Table 2 is a listing of the identification results of 521 NFB identified with the basal medium of the invention. The medium and particular substrates were inoculated and incubated substantially as outlined above in Example 1. From the results in Table 2, it can be seen that virtually 100% of the three most common species of NFB were identified within 24 hours. 
     Other basal medium substrates were added after 24 hours to effect the identification of the less frequently encountered NFB, such as Alcaligenes odorans, P. acidovorans, and P. putida. The less commonly encountered NFB could also be identified by initial inoculation of additional substrates. 
     
                       TABLE 2______________________________________521 NFB IDENTIFIED WITH LOW PROTEINDEGRADATION PRODUCT SYSTEM               CUMULATIVE %       %       IDENTIFIED WITHIN         OF                    More than orORGANISM      TOTAL     24h    48h  equal to 72h______________________________________Pseudomonas aeruginosaNonpigmented  9         88     100Pigmented     65        98     100P. maltophilia         7         100Acinetobacter anitratus         7         100A. Iwoffi     3         100Flavobacterium IIb         1         57     100P. stutzeri   1         0      71   100Alcaligenes odorans         1         0      100P. acidovorans         1         0      75   100P. cepacia    1         0      75   100Moraxella sp. 1         0      100P. putrefaciensLess than     1         100P. putida Less than         1         0      100P. denitrificansLess than     1         0      0    100P. pseudoalcaligenesLess than     1         0      100P. diminuta Less than         1         0      100P. vesicularisLess than     1         0      0    100Kingella kingaeLess than     1         0      100Flavobacterium IIaLess than     1         0      100Bordetella bronchisep-ticaLess than     1         0      100CDC IIk Less than         1         0      100K 988 Less than         1         0      0    100______________________________________ 
    
     EXAMPLE 3 
     Table 3 is a representative listing of additional bacteria tested on the basal medium described above. This group of bacteria includes members of the family Enterobacteriaceae and cytochrome oxidase positive fermenters. It can be seen that these test organisms also give position reactions in 24 hours. Similar rapid reactions have also been noted with Gram-positive bacilli, Gram-positive cocci, and anaerobes. 
     
                                           TABLE 3__________________________________________________________________________Bacteria    Klebsiella          Enterobacter                 Proteus                      Serratia                            PasteurellaTest     pneumoniae          agglomerans                 morganii                      marcescens                            multocida__________________________________________________________________________**ARABINOSE    +     +      -    +     -**DEXTROSE    +     +      +    +     +.sup.w**SUCROSE    +     +      -    +     +.sup.w**LACTOSE    +     -      -    -     -**INOSITOL    +     +      -    +     -*GELATIN -     -      -    +     -*UREA    +     -      +    -     -*GLUTAMINE    +     -      -    +     -__________________________________________________________________________ + = positive within 24 hours; +.sup.w = weak positive within 24 hours; - negative through 72 hours of incubation. *started with acid pH, when positive became alkaline. **started with alkaline pH, when positive became acid. 
    
     A kit for use in identifying all NFB will contain a multiple array of the basal media in poured agar, dehydrated, or frozen form in a compartmented container or an identification strip. Each compartment will contain the basal medium. It will also contain a different selected substrate and the proper indicator, as known in the art. Additional tests, such as Beta-Galactosidase, hydrogen sulfide production, Voges-Proskauer, ornithine decarboxylase, lysine decarboxylase, arginine decarboxylase, deoxyribonuclease, and other tests deemed necessary will be added to extend the range of organisms identified by such a kit identification system. 
     An example of the contents of such a kit is as follows: 
     
         ______________________________________One of the above ammoniumsalts           0.05 to 0.15 By weight %Any potassium salt           0.01 to 0.03 &#34;Any magnesium salt           0.01 to 0.03 &#34;Yeast extract   0.02 to 0.10 &#34;One of the above organicnitrogen compounds           0.02 to 0.10 &#34;Agar            0.00 to 6.0  &#34;One of the above properlyselected indicators           0.0002 to 0.006                        &#34;Water, in properpercentages by weight,e.g. about 0.1 ml to 0.2 ml.A selected substrate ineach compartment for aparticular NFB  0.2 to 2.5   By weight %______________________________________ 
    
     The invention and its attendant advantages will be understood from the foregoing description and it will be apparent that various changes may be made in the form, construction and arrangements of the parts of the invention without departing from the spirit and scope thereof or sacrificing its material advantages, the arrangements hereinbefore described being merely by way of example. I do not wish to be restricted to the specific forms shown or uses mentioned except as defined in the accompanying claims, wherein various portions have been separated for clarity of reading and not for emphasis.