Microsoft word - bio k 331.doc

I – INDICATIONS
TOTAL ANTIBIOTIC - kit is designed for the rapid detection of antibiotics in milk and meat samples. Due to its simplicity, short assay time and multiresiduality, the kit is suitable for screening assays both in laboratories and in food industries. Time required for the assay : 3 h at 65°C. II – PRINCIPLE OF THE TEST

It is a growth inhibition test of Bacillus Stearothermophilus var. Calidolactis, included in a suitable
agar medium. In the assay conditions, the microbial growth yields a colour change of the medium. The
presence of antibiotics in the samples inhibits the bacterial growth and no colour change is observed.
III – KIT COMPONENTS
3 microplates with 8-wells strips breakable (288 analyses) Extraction buffer concentrate 10x, one bottle of 100 ml. IV – DETECTION LIMITS IN MATRIX
Detection limit is the minimum amount of antibiotic being able to inhibit the bacterial growth completely in the assay conditions (3h at 65°C), so that the medium colour remains unchanged. Anti-microbial milk meat Dicloxacillin 0.015 µg/ml 0.075 µg/gr. - Streptomycin 3.500 µg/ml 17.50 µg/gr. Dihydrostreptomycin 5.000 µg/ml 25.00 µg/gr. Bio-X Diagnostics – Site du Complexe des Postes – 49, rue J. Wauters – 5580 Jemelle – Belgique Bio-X Diagnostics – Site du Complexe des Postes – 49, rue J. Wauters – 5580 Jemelle – Belgique Tél : 0032(0)84.32.23.77 - Fax : 0032(0)84.31.52.63 E-mail : [email protected] (26/12/06) Tél : 0032(0)84.32.23.77 - Fax : 0032(0)84.31.52.63 E-mail : [email protected] - Erytromicin 0.500 µg/ml 2.500 µg/gr. VII – INTERPRETATION OF THE RESULTS
- Tetracycline 0.100 µg/ml 0.500 µg/gr. Oxytetracycline 0.100 µg/ml 0.500 µg/gr. After the 3 hours incubation take the plate from the thermostatic water bath and allow to cool 1-2 min. Chlortetracycline 0.100 µg/ml 0.500 µg/gr. Remove the sealing tape and empty the plate knocking it on an absorbent paper. Knocking again the plate, after leaving it for 1-2 minutes upside down. Turn the plate up and read the results by observing the medium color in the wells (best reading is obtained by observation in sunlight or light-bulb). If during the incubation the sample is fallen on the bottom of the well, a best reading is obtained taking the strip from the frame and read the results observing sideways the color of the medium in the wells. Negative samples and controls: medium colour turns from purple to yellow. - Chloramphenicol 5.000 µg/ml 25.00 µg/gr. Positive samples: medium colour remains unchanged (purple). V – PREPARATION AND STORAGE OF SAMPLE
VIII- NOTES
Milk samples: no preparation is required. In case that after the 3 hours the negative controls should not have turned to yellow, check the Milk samples should be tested as soon as possible after sampling, and should be stored at 4°C for no longer incubation temperature and prolong the incubation time. time than 48 h. Spoiled milk samples may yield false negative results. All the possible colour shades between yellow and purple which may give rise to doubt are due to Meat samples: dilute the provided extraction buffer 1:10 with distilled water (1 part of buffer + 9 parts of a decrease in the bacterial growth rate caused by the presence of inhibition factors in the samples distilled water). In a tube weigh 0.75 g of meat, mincing it with a lancet. Add 3 ml of working extraction (such as antibiocs substances just below the detection limits). These samples should be considered Shake vigorously, then leave at room temperature for 12-16 hours (overnight) or at 37°C for 2 hours. Use the Samples giving positive result in the assay can be further investigated for the presence of clean supernatant directly in test. Meat samples can be stored at 4°C for 24 h before analysis, and at -20°C for penicillins. By treatment of samples with an excess of penicillinase, the penicillins become inactive and the same samples result negative in the repeated analysis. In practice, to 1 ml of positive sample add 50 µl of penicillinase 10 U/ml, incubate 10 minutes at room temperature and VI – ASSAY IMPLEMENTATION
Should the medium change colour inside a well before use, discard that well. First of all, predispose a thermostatic water bath (equipped with cover) at the stable temperature of 65°C. According to the number of assays to be run, remove from the microplate the required amount of strips fitted into the frame, taking care not to uncover the unused strips (put the unused strips again in the bag and store Predispose one or more negative controls (milk samples or meat extracts without antibiotics) for each test Use a single disposable pipette tip for each sample to avoid cross-contaminations. Using a precision micropipette: place 50 µl of negative control in the relative wells place 50 µl of each sample in the relative wells. Close the microwells with the sealing tape, taking care that all the wells are tightly sealed up. Place the microplate in the water bath pre-warmed at 65°C, leaving it floating on the water surface (cover the bath in order to avoid cooling). Incubate for 3 hours at 65°C. Particular care should be taken in checking the incubation temperature, since it is very important for a good performance of the assay. Carefully control that the thermostat is able to keep the temperature accurate and constant (65 ± 2°C). Bio-X Diagnostics – Site du Complexe des Postes – 49, rue J. Wauters – 5580 Jemelle – Belgique Bio-X Diagnostics – Site du Complexe des Postes – 49, rue J. Wauters – 5580 Jemelle – Belgique Tél : 0032(0)84.32.23.77 - Fax : 0032(0)84.31.52.63 E-mail : [email protected] (26/12/06) Tél : 0032(0)84.32.23.77 - Fax : 0032(0)84.31.52.63 E-mail : [email protected]

Source: http://www.biox.be/UDTData/13/UDTEnglishInsert/BIO%20K%20331.pdf