| A test kit designed for detecting residual antibiotics in milk by microbial inhibitions assays is widely used as the simplest operation. Indicator bacteria currently used in test kits is almost Bacillus stearothermophilus. Thermophilic bacteria were isolated from high-temperature environment, which grew on low nutrition, produced acid in a short time and were sensitive to commonly used antibiotics. The isolated strains were used for the test kit by microbial inhibition assays.The results were as follows:1) Isolation of thermophilic bacteria:Water and mud samples were collected form heat vent of washing plant in Jin’an, Fuzhou. Water and mud samples were blended together and treated with centrifugation. The mixed liquid coated on solid medium, incubated at 55℃ in homoeothermic incubator.8 single colonies were obtained according to characteristics of colonies.2) Identification of thermophilic bacteria:Based on the low nutrition, production of acid in a short time and sensitivity to commonly used antibiotics, one thermophilic stain FZ was isolated from above 8 strains. Strain FZ was identified as Anoxybacillus by 16S rDNA sequence analysis, morphological characteristics of colony, thallus and spore.3) Test kit preparation and parameters optimization:Based on the single-factor experiment, the embedding of nutrients was optimized to 0.3% glucose,0.2% peptone,0.1% beef extract. Three kinds of color indicators were investigated and bromocresol purple was selected as the best; The optimal embedding spores number was about 5×107cfu/mL; The volume of milk added was 50-80μL.4) Detection limits of antibiotics:The detection limits of antibiotics were as follows:Penicillin, ampicillm:4μg/L;amoxicillin:5μg/L; tetracycline, oxytetrac-ycline, doxycycline:100μg/L; streptomycin, gentamicin:200μg/L; kanamycin:1-000μg/L; sulfonamides:100μg/L; sulfadiazine:200μg/L; erythromycin:40μg/L.5) The evaluation of test kit. The good homogenicity was obtained according to the better concordance between detection results.25 milk samples purchased randomly in market were detected and two doubtful samples were only detected by the test kit made by ourselves, other test results were consistent with the commercial test kits (Delvotest tese kit of ECLIPSE50)... |
