CHEMICALLY DEFINED MEDIA FOR THE DETECTION OF MICROORGANISMS

Abstract

The present invention relates to chemically defined culture medium comprising at least glutamine, cysteine and/or cystine, adenine, guanine, aminobenzoic acid, nicotinaminde adenine dinucleotide and an iron salt for the rapid detection of a broad range of microorganisms comprising prokaryotes and eukaryotes.

Claims

1. A method for the culturing of prokaryotes and eukaryotes in a chemically defined cell culture medium, characterized in that the cells are incubated in a cell culture medium comprising glutamine, cysteine and/or cystine, adenine, guanine, aminobenzoic acid, nicotinaminde adenine dinucleotide and an iron salt.

2. Method according to claim 1, characterized in that the cell culture medium comprises one or more saccharide components, one or more amino acids, one or more vitamins or vitamin precursors, one or more salts, one or more buffer components, one or more co-factors and one or more nucleic acid components.

3. Method according to claim 1, characterized in that the cell culture medium comprises 0.01 to 10 g/L glutamine, 0.01 to 3 g/L cysteine and/or cystine, 0.1 to 300 mg/L adenine, 0.01 to 100 mg/L guanine, 0.01 to 10 g/L aminobenzoic acid and 0.01 to 100 gm/L of an iron(III) salt.

4. Method according to claim 1, characterized in that the cell culture medium comprises a gelling agent.

5. Method according to claim 1, characterized in that the prokaryotes that are cultured comprise one or more of the following strains: Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Streptococcus pyogenes, Pseudomonas aeruginosa.

6. Method according to claim 1, characterized in that the eukaryotes that are cultured comprise one or more of the following strains: Candida albicans, Aspergillus brasiliensis and/or Saccharomyces cerevisiae.

7. Method according to claim 1, characterized in that the prokaryotes and eukaryotes comprise at least one fastidious strain.

8. A method for detecting prokaryotes and eukaryotes in a sample by a) Contacting the sample with a chemically defined cell culture medium comprising glutamine, cysteine and/or cystine, adenine, guanine, aminobenzoic acid, nicotinaminde adenine dinucleotide and an iron salt b) Incubating the cell culture medium of step a) c) detecting the presence of the prokaryotes and eukaryotes in the cell culture medium.

9. Method according to claim 8, characterized in that the incubation in step a) is performed for less than 30 hours.

10. Method according to claim 8, characterized in that the presence of the prokaryotes and eukaryotes is detected via their growth or via a change in color or pH induced by the prokaryotes and eukaryotes.

11. Method according to claim 8, characterized in that the medium comprises 0.01 to 10 g/L glutamine, 0.01 to 3 g/L cysteine and/or cystine, 0.1 to 300 mg/L adenine, 0.01 to 100 mg/L guanine, 0.01 to 10 g/L aminobenzoic acid and 0.01 to 100 gm/L of an iron(III) salt.

12. Method according to claim 8, characterized in that the medium comprises a gelling agent.

13. A method according to claim 1, which is for bioburden, sterility, environmental or media fill testing.

14. Cell culture medium supplement comprising glutamine, cysteine and/or cystine, adenine, guanine, aminobenzoic acid, nicotinaminde adenine dinucleotide and an iron salt.

15. Cell culture medium supplement according to claim 14, characterized in that it contains 0.01 to 10 g/L glutamine, 0.01 to 3 g/L cysteine and/or cystine, 0.1 to 300 mg/L adenine, 0.01 to 100 mg/L guanine, 0.01 to 10 g/L aminobenzoic acid and 0.01 to 100 gm/L of an iron(III) salt.

Description

EXAMPLES

Test Procedure for All Strains

[0189] Filter-sterilized liquid culture media are prepared extemporaneously or stored in 125mL glass bottles in the fridge before use. [0190] Serial dilution of a strain solution stored in HEPES/Glycerol at 80 C. is performed in 0.9% sodium chloride solution. [0191] Each culture medium bottle is inoculated with 20 to 50 CFU/mL. [0192] The inoculated bottles are incubated at 22.5 C.2.5 and 32.5 C.2.5 for 14 days maximum depending on the strain. [0193] Sterile bottles are incubated in parallel as control. [0194] Microorganism development is visually observed in each bottle.

Example 1

[0195] Eight cell strains typically used for testing the growth potential of Tryptic Soy Agar (TSA) are selected for screening the suitability of chemically defined, synthetic medium CHO Medium (Cellvento CHO-100, product item number 1.00899 from Merck-Millipore, Darmstadt, Germany) supplemented with agar-agar as a substitute for said TSA. Growth parameters of CHO-S are measured compared to TSA, item number 1.05458.0500 purchased from Merck-Millipore, Darmstadt, Germany. The pre-cultures are prepared in Tryptic Soy Broth (TSB), item number 1.05459.0500 purchased from Merck-Millipore, Darmstadt, Germany in which they are grown aerobically for 24 hours at 37 C. with the exception of the Aspergillus spp. which is prepared directly by washing off and re-suspending fungal spores from an agar plate. Pre-cultures are diluted in sodium chloride peptone broth and plated out using a spiral plater. The CHO-S Medium is prepared from double concentrated CHO-100 medium with adjustment of the pH to 7.3 using sodium carbonate and the solution is sterile filtrated after addition of the aqueous supplement concentrate. Separately, agar is suspended in water at double concentration (26 g/L) and autoclaved at 121 C. for 20 minutes. The agar solution is then cooled to around 46 C. and mixed with double concentrated CHO-S Medium at the same temperature. Agar plates are prepared after gentle but thorough mixing of the two solutions whilst avoiding extraneous microbial contamination. After surface inoculation the plates are incubated aerobically at 32 C. +/2.5 C. and scored after 24 hours.

[0196] The results are shown in Table 2.

TABLE-US-00002 TABLE 2 CHO-S CHO Medium Tryptic Medium (with Soy Agar (no supple- supple- (Control) ments) ments) Test Cell Comparison Reading Reading Reading Strains Parameter 24 h 24 h 24 h Staphylococcus Count (CFU) 418 561 737 aureus % Recovery 100% 134% 176% ATCC 6538 Growth 4 3-4 3-4 Bacillus Count (CFU) 50 64 84 subtilis % Recovery 100% 128% 168% ATCC 6633 Growth 3 3 3 Escherichia Count (CFU) 564 561 571 coli % Recovery 100% 99% 101% ATCC 8739 Growth 3-4 3-4 3-4 Streptococcus Count (CFU) 20 0 11 pyogenes % Recovery 100% 0% 55% ATCC 21059 Growth 3-4 0 3-4 Pseudomonas Count (CFU) 701 524 693 aeruginosa % Recovery 100% 75% 99% ATCC 9027 Growth 3 4 4 Candida Count (CFU) 306 304 314 albicans % Recovery 100% 99% 103% ATCC 10231 Growth 3 3 3-4 Aspergillus Count (CFU) 25 15 27 brasiliensis % Recovery 100% 60% 108% ATCC 16406 Growth 1 1 1 Streptococcus Count (CFU) 506 4 502 pyogenes % Recovery 100% 1% 99% ATCC 12344 Growth 3-4 3-4 3-4

[0197] Growth is given as the relative diameter of colonies on a scale of 0 (no growth) to 5 (luxuriant growth for the respective cell strains) as read at 24 hours after incubation at 32.5 +/-2.5 C.

SUMMARY

[0198] Speed of growth, represented by colony size after 24 hours, and percent recovery, are all in good comparison with TSA but only after addition of the supplement components glutamine, cysteine/cystine, adenine, guanine, aminobenzoic acid, nicotinamide adenine dinucleotide and iron salts to the CHO medium. This is the case for almost all the bacteria tested including the gram positive organisms (both the Streptococcus pyogenes cell strains, Staphylococcus aureus and Bacillus subtilis) as well as the gram negative cell strain Pseudomonas aeruginosa. Indeed, this positive effect can also be seen for the eukaryotic cells tested: including an improvement of percent recovery of Aspergillus brasiliensis as well as a slight increase in the colony size of the Candida strain tested. Thus, both the prokaryotes and the eukaryotes tested show significant growth improvement and/or colony size to make the medium comparable with tryptic soy agar.

Example 2

[0199] The following 49 strains of prokaryotes and eukaryotes are selected for testing as they can be environmentally relevant or are isolated from real environmental samples.

[0200] Results of positive growth, as identified visually within 14 days of incubation at the given temperatures are shown in Table 3.

[0201] Inoculum refers to the numbers of cells inoculated into the TSB or CHO S medium. The preparation of the CHO-S medium is described in Example 1.

TABLE-US-00003 TABLE 3 CHO-S TSB Medium Inoc- 22.5 32.5 22.5 32.5 Teststrain ulum C. C. C. C. B. clausii ATCC 700160 10 + + + + B. pumilus WT ARDS6V56 92 + + + + B. subtilis ATCC 6633 39 + + + + B. subtilis WT C-J 20 + + + + C. albicans ATCC 10231 200 + + + + E. coli ATCC 8739 22 + + + + E. coli ATCC 25922 35 + + + + P. aeruginosa ATCC 9027 96 + + + + S. typhimurium ATCC 14028 185 + + + + S. aureus ATCC 25923 38 + + + + Ral. pickettii ATCC 27511 59 + + + + Ral. pickettii WT PQK 26 + + + + Sph. paucimobilis ATCC 29837 34 + + + + S. aureus ATCC 6538 34 + + + + S. epidermidis ATCC 12228 16 + + + + S. epidermidis WT CJ 64 + + + + S. hominis ATCC 27844 33 + + + + Steno. maltophilia ATCC 13637 36 + + + + Strept. pyogenes ATCC 12344 32 + + + + Ser. marcescens WT Bb 38 + + + + Acinetobacter lwoffii ATCC 42 + + + + 17925 Aspergillus brasiliensis ATCC + + + + 16404 Aspergillus niger Umweltisolat + + + + Aspergillus sydowii DSM 63373 + + + + Penicillium commune ATCC + + + + 10428 Sphingomonas paucimobilis WT + + + + AP-W Sphingomonas parapaucimobilis + + + + WT M Pantoea sp. WT MT -1661 39 + + + + Achromobacter sp. WT C-2929 209 + + + + Clostridium sporogenes ATCC 21 + + + 11437 Clostridium sporogenes ATCC 23 + + 19404 Debariomyces hansenii DSM >50 + + + + 3428 Kocuria rhizophila ATCC 9341 40 + + + + Methylobacterium extorquens 27 + + + + ATCC 43645 Methylobacterium extorquens 24 + + + NBRC 15911 Methylobacterium fujisawaense 54 + + + Wild Type Methylobacterium mesophilicum 16 + + + ATCC 29983 Methylobacterium ssp. WT CJ 31 + + + + Micrococcus luteus ATCC 10240 40 + + + + Micrococcus ssp. lylae 5 + + + + Rentschler Wild Type Paenibacillus lautus Wild Type 27 + + + + Propionibacterium acnes ATCC 35 + + + 6919 Streptococcus pyogenes ATCC 30 + + + + 21059 Exophiala sp. WT MT-1570 >50 + + + + Burkholderia sp. WT C-2424 66 + + + + Leifsonia sp. WT C-2946 64 + + + + Corynebact. tuberculostearicum 25 + + + + WT Bacillus halodurans WT 32 + + + + Bacillus okuhidensis WT C-3745 42 + + + +

[0202] The results show that all the prokaryotes and eukaryotes tested grow well in TSA and in the chemically defined cell culture medium according to the present invention.