Abstract:
A process for preparing sterile culture media in unit dosage form which comprises preparing a composition of such media of conventional composition, adjusted or augmented by adding constituents in such manner that after sterilization by ionizing radiation a sterile medium of satisfactory composition is obtained. Additional constituents added are radio protectors, increased quantities of indicators, enzymes preventing the formation of toxic degradation products, vitamins or a combination of any of these.

Description:
BACKGROUND OF THE INVENTION 
     The present invention relates to sterile culture media and to a novel process for the production of same. More particularly, the invention relates to sterile, liquid or solid, culture media in unit dosage form and to their production. Other and further features of the present invention will become apparent hereinafter. 
     The sterilization according to the present invention is effected by means of ionizing radiation. This is done by means of first preparing the culture medium, filling a desired quantity into a suitable container and sterilizing the packaged medium by means of ionizing radiation of suitable intensity and duration. 
     It is preferred to effect the sterilization with gamma radiation. It is also possible to use other types of ionizing radiation, such as X-rays and electron beams of suitable intensity. Care must be taken not to use such radiations which bring about the formation of radioactive products. 
     STATE OF THE PRIOR ART 
     The sterilization of various products by means of gamma radiation is well known and widely used. Hitherto various attempts were made to examine the effect of gamma radiation on culture media, and in most cases deleterious effects were observed. The result was generally that the capability of such irradiated media to support the growth of bacteria was seriously impaired, and this in comparison with heatsterilized culture media of equal initial composition. The effect of gamma radiation on culture media was examined amongst others by: 
     A. H. E. Frey et al, Radiation Research, 28, 663-672 (1966); 
     B. H. E. Frey et al, Radiation Research, 36, 59 - 67 (1968); 
     C. V. L. Chopea, Mutation Res. 8, 25-33 (1969); 
     D. I. H. Blank et al., J. Bac. 30, 21-32 (1935); 
     E. E. C. Pollard et al., Radiation Res. Supp.6, 194-200 (1966); 
     F. J. H. Becking, Misc. Papers, 9 (1971) Landbouwhogschool Wageningen, Netherlands; 
     G. P. C. Kesawan, Radiation Botany 11, 253-281 (1971). 
     In view of the above it was generally assumed that gamma radiation cannot be effectively used for the sterilization of packaged culture media. The deleterious effects of such radiation is generally due to the formation of certain decomposition products and also due to the decrease of the concentration of certain necessary ingredients of the culture medium. In some cases toxic products are formed and these impair or even prevent the growth of microorganisms. Redox potentials are changed and certain indicators commonly used are destroyed by such irradiation. 
     DESCRIPTION OF THE PRESENT INVENTION 
     The present invention relates to a process for preparing sterile culture media in unit dosage form which comprises preparing a composition of such media of conventional composition, adjusted or augmented by adding constituents in such manner that after sterilization by ionizing radiation a sterile medium of satisfactory composition is obtained. Additional constituents added are radio protectors, increased quantities of indicators, enzymes preventing the formation of toxic degradation products, vitamins or a combination of any of these. 
     In spite of the generally accepted assumption that such irradiation cannot be effectively used for the intended sterilization, the above drawbacks are overcome by the process of the present invention. The process is applicable to culture media which can be broadly grouped into &#34;solid&#34; and &#34;liquid&#34;  ones. Culture media can be grouped according to the characteristic constituents of same. The drawbacks of gamma irradiated culture media are eliminated according to the present invention by certain adjustments of the initial composition of said culture media, the nature of the adjustment depending on the composition of the medium. For example, the destruction of certain critical components is prevented by the addition of adequate quantities of radio protectors. Certain constituents, such as vitamins or indicators are augmented so as to retain an adequate concentration after irradiation. The formation of certain toxic products is prevented by adding enzymes like catalase. Certain indicators are maintained at an efficient level by the addition of radio protectors and/or by starting with a higher initial concentration compared with that of conventional culture media. 
     Sterilized culture media were prepared according to the present invention and tested with a wide variety of microorganisms. Satisfactory results, as compared with conventionally sterilized media, were obtained. 
     The process according to the present invention for the manufacture of culture media in unit dosage form comprises essentially preparing a suitable culture medium, adjusted initially, if necessary, by the addition of certain additives to remain suitable after sterilization for the intended use, filling the medium into suitable containers and sterilizing same by means of ionizing radiation. The sterilization is advantageously effected at a dose of about 0.5 to 2.5 Mrad. The equipment for effecting the sterilization according to the invention is comparatively simple and only one process step of sterilization is needed. Furthermore, the manipulation of sterile materials is obviated. 
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     Further features of the invention will become apparent from the following description, which is intended to serve as illustration of the invention and which is to be construed in a non-limitative manner. 
     EXAMPLE 1 
     Triple Sugar Iron Agar 
     A mixture of conventional composition was prepared, with the modification that the phenol red content was increased to 60 mg per liter, as part of the phenol red is decomposed during irradiation. 65 g. dry culture medium were boiled in 1000 ml of water, cooled to 60° C, poured into tubes to obtain the necessary slant and butt. After cooling and formation of the gel, the tubes were stoppered and sterilized by gamma radiation of 1.0 Mrad. The thus obtained culture medium was sterile, it was stored for a number of weeks and tested after sterilization and after storage. This culture medium was tested as to performance with the following bacteria: Salmonella typhi; Salmonella typhimurium; Salmonella enteritidis; Salmonella haifa and other serotypes belonging to Salmonella groups C, G + E, Shigella flexneri 2, Shigella flexneri 4, Shigella flexneri 5, Shigella flexneri 6, Shigella sonnei, Pseudomonas aeruginoss, Pasteurella pseudo-tuberculosis, Vibris cholerae biotype El Tor, E.Coli OSS, Proteus morgani; Proteus vulgaris, Proteus rettgeri, Providence, Citrobacter, Serratia, E.hafniae and  E. cloacae. In all cases identical growth as with conventionally sterilized similar culture media were obtained. 
     Similar results were obtained by X-ray irradiation of similar dosage. 
     EXAMPLE 2 
     Mueller Hinton Agar 
     A conventional composition was prepared, with the modification of the increase of the agar content by 25 percent. Catalase was added to contain 10 keil units per liter. The addition and change of concentration of these was made so as to obtain the desired gel strength and to eliminate toxic effects due to gamma irradiation. A quantity of 38 g. of this composition (including added agar) was boiled in 1000 ml distilled water and after cooling to 50° C the catalase was added, plates were poured and these were packed into polyethylene sleeves which were hermetically closed. 
     The sealed sleeves containing the agar plates were sterilized at a dose of 1-2 Mrad. The sterilized plates were stored for 6 weeks and tested both after sterilization and storage. 
     The microorganisms tested were: Streptococcus faecalis; N. Meningitidis; Pasteurella pseudo-tuberculosis, Salmonella typhi; Salmonella typhimurium; Shigella boydii, Staphylococcus aureus; Shigella flexneri; Serratia; Vitro cholerae biotype El Tor; Klebsiella; Proteus; E.Coli 055; Pseudomonas seruginosa. With all strains satisfactory growth was obtained on this medium. 
     EXAMPLE 3 
     Fluid Thioglycollate Medium 
     A conventional composition was modified by an increase of the initial thioglycollate content to 1.5 g. per liter, This modification is necessary so as to maintain the redox potential necessary for the growth of anaerobic bacteria. 29.5 g. of the composition were boiled in 1000 ml distilled water, cooled to 60° C. and poured into plastic tubes. These were stoppered and sterilized by means of gamma radiation of 1-2 Mrad. After sterilization and after storage for 6 weeks, the culture media was tested and found to be suitable for bacteriological work. 
     Clostridium perfringens, Strep/hemolyticus group A; Strep. faecallis, Strep/viridans and E. coli were innoculated and satisfactory growth was obtained even with small inocular. 
     EXAMPLE 4 
     Fluid Thioglycollate Medium 
     A medium was prepared as in Example 3, but there was used the regular thiglycollate content to which there was added 1.00 g. cysteine per liter. Similar results were obtained. 
     EXAMPLE 5 
     Brian Heart Infusion 
     37 g. of culture medium were boiled in 1000 ml. distilled water, cooled to 60° C. poured into plastic tubes and stoppered. After gamma sterilization at 2.0 Mrad. the sterile liquid culture medium was stored for 6 weeks and tested after preparation and after storage. It was found to be suitable for all conventional bacteriological work. Streptococcus viridans; Strep. hemolyticus, Strep. faecalis; Salmonella typhimurium; E.Coli; Staph. aureus were grown on the thus prepared medium and growth was entirely satisfactory. 
     EXAMPLE 6 
     Tryptose Agar 
     41 g. of tryptose agar were boiled in 1000 ml distilled water, cooled to 60° C. plates were poured and sealed in polyethylene sleeves. After gamma irradiation at a does of 1.0 to 2.0 Mrad. the sterile culture medium was stored for 6 weeks and tested. It was found to be suitable for all conventional purposes. 
     The following bacteria were grown on this agar and growth was found to be satisfactory: N.meningitidis; Brucella melitensis; E. coli, Shigella boydii; Staphylococcus aureus; Streptococcus faecalis. 
     EXAMPLE 7 
     Agar MacConkey 
     The conventional formulation was increased to 70 mg neutral red per liter and thioglycollate or cysteine was added at a quantity of 0.01 M per liter. 50 g. of the medium were dissolved in 1000 ml distilled water, poured into petri dishes, packed in polyethylene sleeves and sterilized with gamma rays at 0.5-2.0 Mrad. After sterilization and after storage of 4 weeks the medium was tested and found to be suitable for the cultivation of gram negative bacilli. 
     Klebsiella, Herrelea; Serratia, Proteus, Levinea, Pseudomonas aeruginosa; E.coli, Shigella boydii and Salmonella typhimurium were grown and growth was up to standard. 
     EXAMPLE 8 
     C.L.E.D. 
     The conventional formula was modified by an increase of the brom-thymol blue content to 40 mg per liter and by adding 0.01 M thioglycollate or crystamine per liter. 36.2 g. were dissolved in 1000 ml boiling water, cooled to 50° C. and poured into plastic petri dishes and sealed in polyethylene sleeves. After sterilization with gamma rays at 1-2 Mrad. The medium was stored for 6 weeks and tested after sterilization and after storage. It was found to be suitable for all types of bacteriological work. 
     The following bacteria were innoculated and found to yield good growth on this medium: Proteus; Levinea; Pseudomonas aeruginosa; E.coli; Salmonella typhimurium; Klebsiella, Herrellea, Serratia, Shigella boydii, Staph. aureus, Strep, faecalis and Candida. 
     EXAMPLE 9 
     Slide Kit 
     A dip slide kit was prepared, according to Guttmann et al., Brit.Med. J.iii, 343-345 (1967), and the two sides of the plastic slide were coated with C.L.E.D. and with MacConkey agar, respectively. The slide was inserted into a tight, hermetically closed plastic container and this was sterilized by gamma irradiation of 2 Mrad. The culture media was tested immediately after sterilization and a number of each containers were stored for 8 weeks. Satisfactory growth was obtained on both media. 
     Most of the Examples were repeated and instead of gamma rays there were used X-rays or irradiation by electrons, of similar dosage. Similar results were obtained.