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please provide the procedure to do the project 'study of drug resistance of bacteria in milk using penicillin and amoxylin?
Milk samples corresponding to the morning machine milking session (6 am)of 16 cows were collected in the 60 - 90 day post partumperiod. The animals received no pharmacological treatment throughout the sampling period(18). The chemical composition and pH values of the selected samples were normal for bovine milk, with low somatic cell counts (SCC < 400,000 cells/mL) and an acceptable bacterial count for cow’s milk (CFU < 100,000 cfu/mL).
II. Antimicrobial Solutions and Spiked Samples
The drugs used for the preparation of antimicrobial solutions were stored and handled according to the manu-facturers’ instructions before use. All the dilutions were
prepared in 100 mL volumetric flasks at the time the analyses were carried out in order to avoid the possibility of unstable solutions.Antimicrobial solutions were prepared using antimicro-bial-free milk(18), as determined by the Delvotest. The finaldrug concentrations in milk (?g/L) were achieved after serial dilutions so that the volume of the antimicrobial agent solu-tion did not exceed 1% of the volume of the final solution to be analyzed(.
III. ResScreen Test
The system consists of two microbial bioassays using Geobacillus stearothermophilus subsp. calidolactis C-953spores. The microbiological method is based on growth
inhibition of bacteria-test when milk containing residues ofantibiotics.The BT bioassay (Betalactams and Tetracyclines) is composed of a culture medium containing spores of ther-mophilic microorganism, chloramphenicol and bromocresol purple indicator(16). If the milk sample is free of antibiotics and allows bacteria-test growth and changes in color of the acid base indicator (purple to yellow). Otherwise the test will remain the same color.   
Moreover, the BS bioassay (Beta-lactams and Sulfona-mides) use a medium inoculated with a microorganism spore suspension, brilliant black indicator, toluidine blue and trimethoprim. So, the absence of antibiotic residues inmilk causes bacteria-test growth, producing a color change
of indicators from black to amber.The ResScreen system was carried out according to the manufacturer’s instructions. Thus, 50 ?L milk sample was added to individual plates of the BT and BS ResScreenmethods. Plates were incubated in a water bath at 64 ± 1°C for3 (BT ResScreen) and 4 h (BS ResScreen) until the color change of the negative samples had taken place.Visual interpretation was performed independently by 3 trained persons, and was assessed visually as negative and positive; doubtful qualifications were interpreted as positive.
IV. Detection Limits and Cross Specificity
(I) Detection limits
The following substances (Sigma Chemical Co, St.
Louis, MO) were used to determine the ResScreen® system
detection limits:
1. Ten betalactams: amoxicillin, ampicillin, cloxacillin,oxacillin, penicillin-G, cefadroxil, cephalexin, cefopera-zone, cefuroxime and ceftiofur®.
2. Four sulfonamides: sulfadiazine, sulfadimethoxine,sulfamethoxazole and sulfathiazole.
3. Three tetracyclines: chlortetracycline, oxytetracyclineand tetracycline.
The detection limits of the antimicrobial agents were established according to the Codex Alimentarius guide-lines(18). For this purpose, 12 concentrations were prepared
with different levels of each drug. For each concentration, 16replicates were prepared using antibiotic-free milk samples.


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