ࡱ> )` RBxbjbj7|{{;0000'''8(DX(IFh)h)~)~)~)e+N./|EEEEEEE$1IhK>EQ0%+@e+00E00~)~)FB3B3B30j0R~)~)EB30EB3B3@|"A~)\) @, t'1dAAF0IFvAK1K$AKA0#070B3E0 Q0S#0#0#0EE2^#0#0#0IF0000$''000000  Total Antibiotics in Milk Test Kit RND99026 Product Description REAGEN"! Total Antibiotics in Milk Test Kit provides an easy and simple test method for detecting total antibiotics in milk. Compared with current commercially available kits,. the unique features of the kit are: A quick spore growth assay (results can be read in about 3 hours). Detection of wide range of antibiotics in milk. High reproducibility. High sensitivity and low detection limit. Procedure Overview The method is based on the inhibition of a standard microbial growth recommended by international organizations. The kit is presented in a microplate format where each well contains medium agar spread with bacillus spores. When the plate (or a strip of wells) is incubated at 65(C, these bacillus spores germinate and the bacillus grows so that acids are produced, which result in the pH value changes. The color of the pH indicator in the medium changes from purple to yellow. If milk samples contain antibiotics at higher concentration than the detection limit, the bacillus will not grow and therefore the medium color remains unchanged. Kit Contents, Storage and Shelf Life REAGEN"! Total Antibiotics in Milk Test Kit has the capacity for 96 determinations for product Reference #: RND99026-01 and 288 determinations for product Reference #: RND99026-02, including negative and positive controls. Return any unused plates/strips/wells to the plastic bag provided in the original package and reseal them. The shelf life is 6 months when the kit is properly stored. RND99026-01 Kit ContentsAmountStorageMicrotiter Plate96-well plates (8 wells x 12 strips)2-8(CMilk Negative Control Powder1 tubeRoom TemperatureMilk Positive Control Powder1 tubeRoom TemperatureMilk Negative Control Diluent1.5 mL2-8(CMilk Positive Control Diluent**1.5 mL-20(CAluminum Seal2 SheetsRoom TemperatureQC Certificate1Na RND99026-02 Kit ContentsAmountStorageMicrotiter Plate3x96-well plates (8wells x 12 strips)2-8(CMilk Negative Control Powder3 tubesRoom TemperatureMilk Positive Control Powder3 tubesRoom TemperatureMilk Negative Control Diluent3 tubes X 1.5 mL2-8(CMilk Positive Control Diluent**3 tubes X 1.5 mL-20(CAluminum Seal6 SheetsRoom TemperatureQC Certificate1Na Upon arrival, immediately store the Milk Positive Control Diluent at -20(C. See storage temperature for other components. After reconstitution of the negative and positive controls, aliquot 0.1mL into separate tubes and store in freezer at -20(C. Sensitivity AntibioticDetection Limit (ppb)MRL (ppb)Penicillin G24Ampicillin34Amoxicillin44Oxacillin2030Cloxacillin3030Gentamycin30100Tylosin5050Ceftiofur50100Oxytetracycline50100Tetracycline50100Sulfamethoxazole200500 Required Materials Not Provided With the Kit 65(C Incubator (heat block or water bath) 100 (L pipettes, tips, and tubes Reagent Preparation Preparation of Negative Control and Positive Control Reconstitute the Milk Negative Control Powder with 1.5 mL of Milk Negative Control Diluent. Reconstitute the Milk Positive Control Powder with 1.5 mL of Milk Positive Control Diluent. Vortex tubes and mix well. Be sure the milk mixture is homogenous and no clumps are present. After the initial reconstitution, it is recommended to aliquot the negative and positive controls into 0.1 mL aliquots and stored at -20C freezer for future use. Failing to do so may result in inaccurate readings. ELISA Testing Protocol Cut the foil sheet covering the wells using a razor blade and take sufficient strips to be used by pressing up from the bottom of the wells. Be very careful not to damage the foil covering the remaining wells. The remaining plate/wells should be placed back in the provided plastic bag and stored at 2-8(C immediately to prevent loss of spore activity. Peel off adhesive foil covering and add 50 (L of milk sample to the well. Add the negative and positive controls (50 (L each) in parallel. Make sure there is no cross-contamination during the sample addition. Seal the wells/plates carefully with the adhesive foil sheet provided in the kit. Incubate the wells/plates at 65(C till the negative control sample turns yellow (approximately 23 hours, see each lot qualification certificate for reference). It is recommended to use a hot water bath for faster incubation time. A heated dry incubator may take an additional 30 minutes more in addition to the lot incubation time. Do not let water enter the wells if you use water bath for incubation. Be sure to seal the wells with the provided foil sheet to prevent water leakage. Do not take the samples out before the time indicated in the QC Certificate to avoid inconsistency in color change. Do not incubate the sample longer than the time indicated in the QC Certificate, otherwise the detection sensitivity could be reduced. Result Analysis Take out the wells/plates after incubation. Allow the outside of the wells/plates to dry by placing them on top of paper towel if water bath is used. Visually observe individual well color changes by placing a piece of white paper behind the wells. The color on the side of the well is more reliable than that on the top or bottom. Occasionally, bubbles may form on one side of the well during incubation. Look at the other side for color changes. Further analysis and repeats may be required for those with intermediate color changes between negative and positive controls. Samples that turn yellow, yellow with a hint of red, or yellow with a hint of purple are considered negative for antibiotics at the indicated limits. Samples that are completely purple or reddish-purple are considered positive for antibiotics at the indicated limits. Always use the control wells as reference points when determining the results. Troubleshooting If the color does not change 15 minutes after the recommended incubation time as indicated in the QC sheet, check the wells to see if they are sealed properly. Also, check the incubation temperature. The incubation temperature should be at 65(C with minus or plus 1 degree. Use different thermometers to double check on the exact temperature. If you accidentally let water enter the wells, use new wells and start incubation again. If the color does not change into reddish-yellow or purple-yellow for negative control, check the storage condition of plates/strips/wells. The activity of the spores may be reduced if plates/strips/wells were frozen or improperly stored.  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