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1.
Simplified plate diffusion system for microbial assays of antibiotics   总被引：1，自引：0，他引：1  
A protocol for microbial assays of antibiotics, using the plate diffusion system, is presented. The system is based on the concept that a complete standard curve and assay unknowns can be placed on an assay plate and that 2 plates can be a complete assay with an accuracy and precision essentially equivalent to the official AOAC diffusion procedure. Four antibiotics, bacitracin, chlortetracycline, oxytetracycline, and streptomycin, were used in the design and comparison studies with the AOAC protocol. The coefficients of variation (CVs) for the AOAC design, using 10 replicates, ranged from 1.4 to 10.3% with a mean of 4.5%. The CVs for the single-plate option of the simplified design ranged from 4.3 to 9.6% with a mean of 6.6%; the CVs for the 2-plate option ranged from 2.5 to 6.8% with a mean of 4.3%; the CVs for the 3-plate option ranged from 1.2 to 5.0% with a mean of 3.0%.  相似文献   

2.
The volume of antibiotic solutions in cylinders used for diffusion assays is assumed to have no significant effect on estimation of potency. The size of zones of inhibition from cylinders containing 0.10, 0.20, and 0.30 mL of sample solution was compared with zones of inhibition from cylinders containing 0.20 mL of standard solutions. For zinc bacitracin, chlortetracycline.HCl, oxytetracycline, lincomycin.HCl, monensin Na, neomycin sulfate, K penicillin, streptomycin sulfate, and tylosin, the percent recovery (95-102) was optimum when both standard and sample cylinders contained the same volume (0.20 mL/cylinder). At 0.30 mL/cylinder for sample and 0.20 mL for standard solutions, there was a positive bias in potency of about 50%. At 0.10 mL/cylinder, there was a negative bias of approximately 25% except for neomycin, monensin, and bacitracin. For these antibiotics, the bias was about -50%. For hygromycin B, variation in volume of solution per cylinder has little effect on assay results. Experiments on commercial feeds and premixes gave essentially the same results as for the standard solutions experiments.  相似文献   

3.
An analytical procedure, based on the concept that exposure of bacteria to antibiotics will result in the selection of a resistant population, was developed. Two strains of enteric bacteria, Escherichia coli CS-1 and Enterobacter cloacae B520, which are sensitive to a wide variety of antibiotics, were used as the test organisms. E. coli CS-1 were exposed to 1.00 micrograms antibiotic or antimicrobial/mL; E. cloacae B520 were exposed to 0.01, 0.10, 0.50, 1.00, and 5.00 micrograms/mL. Both organisms developed increased resistance to other antibiotics after exposure to chlortetracycline and oxytetracycline, as measured by the minimum inhibitory concentration (MIC). E. cloacae B520 showed increased resistance to ampicillin, oxytetracycline, and chloramphenicol after exposure to levels as low as 0.10 microgram/mL. Exposure to streptomycin, sulfamethazine, tylosin, bacitracin, flavomycin, virginiamycin, and monensin at levels of 1.00 microgram/mL did not increase the MIC. Exposure to 5.00 micrograms streptomycin, sulfamethazine, tylosin, and monensin/mL increased the MIC of E. cloacae to one of the antibiotic markers. These increased MICs exceeded the 95% confidence limits of the MIC values of the unexposed organisms.  相似文献   

4.
A liquid chromatographic method for the assay of oxytetracycline in premixes and veterinary products is described. Premix samples are extracted with acidified methanol, diluted with mobile phase, and filtered before chromatography on a C-8, reverse phase column. The assay method separates oxytetracycline from epioxytetracycline, tetracycline, and chlortetracycline. Total elution time for oxytetracycline is less than 5 min at 1.5 mL/min. Five spiked premix samples each of 2 and 50 g/lb had a coefficient of variation of 3.5 and 4.5% and a mean recovery of 99 and 104%, respectively. The results of premixes and veterinary products assayed by this method compared closely with those of the same assayed by the official AOAC microbiological method.  相似文献   

5.
为明确不同抗生素及其处理方式对绿色型豌豆蚜生物学特性的影响,将盐酸金霉素(chlortetracycline HCl)、氯霉素(chloraomycetin)、盐酸土霉素(oxytetracycline HCl)、青霉素G钾盐(penicillin-G K salt)和硫酸链霉素(streptomycin sulfate)5种抗生素分别按同时喷洒蚕豆植株和蚜虫虫体、只喷洒蚜虫虫体和只喷洒蚕豆植株3种方式处理,了解不同处理下豌豆蚜的发育历期、平均体重、体质量差、相对日均体质量增长率和平均产蚜量等生物学参数变化特征。结果表明:3种处理方式对绿色型豌豆蚜的生物学参数影响大小顺序为:同时喷洒植株和虫体处理喷洒植株处理喷洒虫体处理。5种抗生素对绿色型豌豆蚜的生物学参数影响大小顺序为:盐酸土霉素盐酸金霉素硫酸链霉素氯霉素青霉素G钾盐。经过喷洒蚜虫虫体和植株表面联合作用处理,土霉素对绿色型豌豆蚜的生长发育影响最大,若虫期延长2.25 d,整个世代周期延长3.70 d,体重减轻52.37%,体质量差减小55.84%,相对日均体质量增长率减小53.85%,产蚜量下降79.07%;金霉素处理表现为延长发育历期,青霉素、氯霉素和链霉素为缩短发育历期;经5种抗生素处理后的绿色型豌豆蚜体重均减轻,产蚜量下降。土霉素处理对绿色型豌豆蚜的若虫期、世代历期和相对日均体质量增长率的影响与其他4种抗生素差异显著(P0.05),土霉素和金霉素对平均体重和体重差的影响与其他3种抗生素差异显著(P0.05),但相互间差异不显著(P0.05)。经过喷洒植株表面的间接作用处理,土霉素对绿色型豌豆蚜的生长发育影响最大,若虫期龄期延长1.63 d,世代历期延长3.38 d,体重减轻50.28%,体质量差减小51.49%,相对日均体质量增长率减小41.67%,产蚜量下降75.45%;金霉素的影响作用次之,表现为延长发育历期;青霉素、氯霉素和链霉素为缩短发育历期。5种抗生素处理后绿色型豌豆蚜均体重减轻,产蚜量下降。土霉素处理对绿色型豌豆蚜的若虫期、平均体重和产蚜量的影响与其他4种抗生素差异显著(P0.05),土霉素和金霉素对世代历期和相对日均体质量增长率影响差异显著(P0.05),但相互间差异不显著(P0.05)。经过直接喷洒蚜虫虫体间接作用方式处理,土霉素对绿色型豌豆蚜的生长发育影响最大,若虫期延长0.34 d,体重减轻24.32%,相对日均体质量增长率减小26.32%,产蚜量下降44.23%,其他4种抗生素对绿色型豌豆蚜的生物学参数影响较小。土霉素处理对相对日均体质量增长率的影响与其他4种抗生素差异显著(P0.05),土霉素和金霉素对若虫期和产蚜量的影响与其他3种抗生素差异显著(P0.05),但相互间差异不显著(P0.05)。由不同抗生素的不同处理方式进行多重比较得知,对绿色型豌豆蚜生长发育影响较大的3种处理组合为:土霉素同时喷洒植株和虫体处理组合土霉素喷洒植株处理组合金霉素同时喷洒植株和虫体处理组合,3种作用处理间对绿色型豌豆蚜生物学特性影响差异不显著(P0.05),土霉素同时喷洒植株和虫体处理组合对生物学特性的影响与除土霉素喷洒植株处理组合和金霉素同时喷洒植株和虫体组合之外的其他组合处理差异显著(P0.05)。  相似文献   

6.
An analytical system was developed which can assess the ability of antibiotic/antimicrobial residues (0.01-1.00 ppm) to affect the conjugal transfer of resistance among the Enterobacteriaceae. The donor strain, Escherichia coli RP-4 (Amr Tcr Nmr Kmr Lac+), and recipient strain, E. coli Sc-8632 (Smr Lac-), were incubated together in a 1:9 donor:recipient ratio for 18 h with gentle shaking (50 rpm) in brain heart infusion broth in the presence of residue levels of antibiotics. The mating cultures were serially diluted and spread-plated onto MacConkey agar containing 25 micrograms streptomycin/mL to select the total recipient population of sensitive E. coli Sc-8632 and transconjugants. After an 18 h incubation at 37 degrees C, the plates were replicated onto MacConkey agar containing 25 micrograms ampicillin/mL to select the ampicillin-resistant transconjugant population. Repeatability was good; the average transfer was 51.8%, with a coefficient of variation of 9.3%. Residue levels of tylosin (0.10 and 1.00 ppm) increased the transfer of the ampicillin marker beyond the 95% confidence limits. Oxytetracycline, bacitracin, streptomycin, penicillin, and virginiamycin did not increase the percent transfer. Oxytetracycline at 0.01 ppm decreased the percent transfer. In general, residue levels of antibiotics (0.01-1.00 ppm) did not affect the conjugal transfer of antibiotic resistance.  相似文献   

7.
The agar well technique compares favorably with the cylinder plate assay in accuracy, sensitivity, and precision. It is more flexible and more rugged, and growth of seed organism is not inhibited. The wells are precision cut with Grafar gel punch assembly with sets (6) of 10, 7, 5, 4, and 3 mm cutters. The wells are easily and rapidly filled with short, disposable Pasteur pipets fitted with rubber bulbs. The smaller wells are filled with capillary pipets. The diameter of the well (independent of volume) appears to be a function of concentration. For every decrease in the diameter size of the well, concentration can be increased, at least to the next higher level of the standard response line. Extracts of chlortetracycline containing as much as 3.2 mu-g/ml can be analyzed if the 3 mm wells are used and with no sacrifice in accuracy or precision. This works especially well for antibiotic premixes. The technique has been used successfully for penicillin, streptomycin, chlortetracycline, oxytetracycline, oleandomycin, and tylosin.  相似文献   

8.
The manual and automated turbidimetric assays and a modified official plate assay for chlortetracycline (CTC-HCl) in feed were collaboratively studied. Three feed samples (swine feed, 100 g CTC-HCl/ton; premix I, 20 g each of CTC-HCl and sulfamethazine/lb, and 10 g penicillin/lb; and premix II, 50 g CTC-HCl/lb) were analyzed at 2 dilutions. Twelve laboratories conducted the plate assay; 8 laboratories the manual turbidimetric method; and 7 laboratories, the Autoturb analysis. Within a method, there was no significant difference between dilutions. Between methods, there was a significant difference between the manual turbidimetric plate assays only for swine feed. However, the same sample dilutions or the average values of the 2 dilutions for both methods showed no statistical difference. Among the collaborators, the slope of CTC-HCl standard curve varied between about 2.0 and 3.0 for the plate method. The turbidimetric assay has been adopted as official first action for feeds containing larger than or equal to 20 g CTC-HCl/lb.  相似文献   

9.
The Bacillus stearothermophilus disk assay for penicillin in milk (AOAC official method) was adapted for the determination of ampicillin in fish muscle. The method was evaluated in 2 species of cultured fish: channel catfish and striped bass. Recoveries of ampicillin ranged from 99 to 104% when muscle specimens from both species were spiked at concentrations of 0.025-1.00 micrograms/g. The lower limit of determination (LOD) was 0.025 micrograms/g. The assay was applied to monitor the elimination of ampicillin from the muscle of striped bass after intravascular administration (dosage of 10 mg/kg body weight). The mean concentrations in the muscle declined from 1.160 micrograms/g at 2 h to 0.063 micrograms/g at 18 h. The half-life of ampicillin in the muscle was 3.6 h. Ampicillin concentrations were below LOD at 24 h. No inhibitory activity was observed in the muscle of control fish.  相似文献   

10.
Recovery studies in which chlortetracycline hydrochloride (CTC-HCI) standard was added to cattle and swine feed supplements at 4.09-9.99 g/ton showed lower antibiotic recovery turbidimetrically (80.6-98.7%) than by the AOAC modified standard as in 38.179(d) (91.2-98.7%) and the plain buffer as in 38.179(b) (93.8-133.0%) methods. Three feeds fortified with a commercial premix at the levels of 5.0 and 10.0 g CTC-HCI/ton showed an overall CTC-HCI recovery of 87.6-110.6% by manual turbidimetric assay. Results were 89.1-108.7% by the AOAC inactivated feed diluent standard and 95.4-125.4% by the plain buffer methods. For some sample extracts (as in cattle feed) the use of heat to stop bacterial growth in the turbidimetric method caused formation of a precipitate. Cooling of cultures to room temperature and rapid reading of sample turbidity followed by standard curve concentrations minimized this interference. The manual turbidimetric assay of low levels of CTC-HCI in feeds appears to offer advantages over other methods.  相似文献   

11.
A multiclass method has been developed for the determination and confirmation in honey of tetracyclines (chlortetracycline, doxycycline, oxytetracycline, and tetracycline), fluoroquinolones (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, and sarafloxacin), macrolides (tylosin), lincosamides (lincomycin), aminoglycosides (streptomycin), sulfonamides (sulfathiazole), phenicols (chloramphenicol), and fumagillin residues using liquid chromatography tandem mass spectrometry (LC-MS/MS). Erythromycin (a macrolide) and monensin (an ionophore) can be detected and confirmed but not quantitated. Honey samples (approximately 2 g) are dissolved in 10 mL of water and centrifuged. An aliquot of the supernatant is used to determine streptomycin. The remaining supernatant is filtered through a fine-mesh nylon fabric and cleaned up by solid phase extraction. After solvent evaporation and sample reconstitution, 15 antibiotics are assayed by LC-MS/MS using electrospray ionization (ESI) in positive ion mode. Afterward, chloramphenicol is assayed using ESI in negative ion mode. The method has been validated at the low part per billion levels for most of the drugs with accuracies between 65 and 104% and coefficients of variation less than 17%. The evaluation of matrix effects caused by honey of different floral origin is presented.  相似文献   

12.
A turbidimetric method is described for determination of tylosin in animal feeds containing urea. This method includes several modified or new steps to existing turbidimetric and AOAC plate assays that improve the extraction of tylosin, remove interferences from feeds, free tylosin activity, concentrate tylosin from low-level feeds, and reduce variability of assay results. A larger analytical sample size has been incorporated into the assay to decrease variability of assay results. A methanol-phosphate buffer extraction solution has replaced the hot buffer and methanol extraction solution. A hydrolysis step, which is not contained in the AOAC plate assay, was developed to free tylosin from the tylosin urea adduct that forms over time in feeds containing urea. A disposable C18 column was used to concentrate tylosin from feeds at levels less than 15 ppm. By increasing the analytical sample size from 25 to 100 g, the coefficient of variation for 12 weighings of cattle feed was reduced from 28.4 to 9.3%. Average recoveries from cattle rations containing tylosin at levels of 8, 10, and 100 ppm were 94, 94, and 91%, respectively.  相似文献   

13.
Tetracycline (TC) specific luminescent bacterial biosensors were used in a rapid TC residue assay sensitized to meet the EU maximum residue limit (MRL) for TC residues in poultry muscle tissue (100 microg kg(-1)) by membrane-permeabilizing and chelating agents polymyxin B and EDTA. Sensitivities of 5 ng g(-1) for doxycycline, 7.5 ng g(-1) for chlortetracycline, and 25 ng g(-1) for tetracycline and oxytetracycline were reached. Except for doxycycline, the MRLs of these tetracyclines include their 4-epimer metabolites. In the biosensor assay, all four 4-epimers showed induction capacity and antimicrobial activity, and antimicrobial activity was also observed in the inhibition assay, although with lower efficiency than that of the corresponding parent compound in both assays. The biosensor assay is an inexpensive and rapid high-throughput screening method for the detection of 4-epimer TC residues along with their parent compounds.  相似文献   

14.
Seventeen laboratories evaluated the pyridine extraction method and neomycin-sensitized agar for the determination of zinc and MD bacitracin in swine and broiler rations at 10 and 100 g/ton. The method was also applied to the analysis of 2 premixes labeled 50 g/lb (MD bacitracin) and 40 g/lb (zinc bacitracin). Bacitracin activity was determined on each of 2 days with 2 dilutions on each day. No significant difference was found between dilutions within a day or between days for each sample. The type of bacitracin or type of feed did not significantly affect results. The difference in results between MD and zinc bacitracin in premixes approached significance. The large coefficients of variation for premixes (ca 13%) and complete feeds (ca 15--30%) indicate operational problems. The main difficulty was evaporation of pyridine. Some laboratories were not able to evaporate it completely, whereas others lost bacitracin activity, probably due to high temperature of drying. The pyridine extraction method as in 42.200 and 42.204 should be discontinued.  相似文献   

15.
Results are compared for the microbiological analysis of chlortetracycline using the AOAC method and a modified method applicable to potencies above 50 g/ton. Two modifications are presented: substitution of a pH range of 4.0-4.5 instead of the specified pH of 4.5 for the plating solution, and substitution of extraction by shaking instead of the blending procedure. There were no significant differences in results between the AOAC method and the modified method.  相似文献   

16.
The microtiter plate system for turbidimetric assay of chlortetracycline (CTC) and oxytetracycline (OTC) levels in feeds uses a 96 well microtiter plate, a multichannel pipette, and an ELISA reader to measure turbidity. Feeds are extracted for both tetracyclines using AOAC extraction systems. For CTC, the range of the standard curve is 0.001-0.005 microgram CTC/mL; for OTC, the range is 0.004-0.016 microgram OTC/mL. Repeatability of CTC assays, as shown by the coefficient of variation (CV), ranged from 0.54 to 5.65% for same-day assays and from 2.01 to 9.39% for assays on different days. For OTC, CVs ranged from 2.69 to 10.01% for same-day assays and 3.24 to 9.08% for different-day assays. Average recoveries for CTC were 108.7% for same-day assays and 106.8% for different-day assays; for OTC, average recoveries were 112.4% and 106.5% for same-day and different-day assays, respectively.  相似文献   

17.
建立了同时测定有机肥料中磺胺类、四环素类和喹诺酮类13种抗生素残留的高效液相色谱-串联质谱法,并对样品的前处理方法、仪器分析参数进行了优化。结果表明,13种抗生素在50~1 000 ng/mL间线性关系良好,相关系数均达到0.999以上,检出限在0.02~0.04 mg/kg,样品加标回收率在69%~111%之间,具有良好的重现性。该方法具有灵敏度高、准确度高等优点,可用于有机肥料中13种抗生素残留的同时检测。运用该方法在有机肥料实际检测中发现恩诺沙星、诺氟沙星、土霉素等抗生素残留。  相似文献   

18.
The potentiality of using a luminescent Escherichia coli strain for the specific detection of tetracycline residues in raw bovine milk was investigated. The sensor cells contain a reporter plasmid carrying the bacterial luciferase operon of Photorhabdus luminescens under the control of the tetracycline responsive control region from transposon Tn10. Incubation of the cells with the sample containing tetracyclines increases the light emission of the sensor cells. The most sensitive tetracycline detection was achieved in 120 min and by using CDTA as a chelating agent in the assay. Heat-treatment of milk before the assay decreased the variations in background luminescence signals and in tetracycline-induced luminescence between different milk samples. The detection limits for tetracycline, oxytetracycline, chlortetracycline, doxycycline, methacycline, demeclocycline, and minocycline were between 2 and 35 ng/mL. Nontetracycline antibiotics did not significantly interfere with the detection of tetracyclines.  相似文献   

19.
A liquid chromatographic method was developed for the determination of nicarbazin (4,4'-dinitrocarbanilide.2-hydroxy-4,6-dimethylpyrimidine) in chicken feed. Ground feed was extracted with hot dimethylformamide, filtered, and then cleaned up on an alumina column. The nicarbazin was eluted from the column with ethanol and quantitated using a reverse phase C-18 column, with a methanol-water mobile phase and ultraviolet detection at 344 nm. Recoveries at a typical use level of 100 micrograms/g feed averaged 98% with a standard deviation of 3%. Samples fortified at levels as low as 0.1 micrograms/g were analyzed with 92% recovery. The detection limit is 1 ng, and the response is linear between 4 and 1000 ng. Feed additives in combination with nicarbazin do not interfere with recovery.  相似文献   

20.
A microbial competitive receptor assay for detecting residues of antibiotic families in milk was studied collaboratively by 13 laboratories. The drugs and levels (ppb) tested in this study include penicillin G, 4.8; cephapirin, 5.0; cloxacillin, 100; tetracycline, 2000; chlortetracycline, 2000; oxytetracycline, 2000; erythromycin, 200; lincomycin, 400; clindamycin, 400; sulfamethazine, 75; sulfamethoxazole, 50; sulfisoxazole, 50; streptomycin, 1000; novobiocin, 50; and chloramphenicol, 800. In this method, microbial cells added to a milk sample provide specific binding sites for which 14C or 3H labeled drug competes with drug residues in the sample. The 14C or 3H binding to the specific binding sites is measured in a scintillation counter and compared with a zero standard milk. If the sample is statistically different from the zero standard, it is positive. The assay takes about 15 min. The binding reaction occurs between the receptor site and the drug functional group, so all members of a drug family are detected. In this case, beta-lactams, tetracyclines, macrolides, aminoglycosides, novobiocin, chloramphenicol, and sulfonamides, including p-aminobenzoic acid (PABA) and its other analogs, are detectable. The incidence of false negative determinations among samples is about 1%; the incidence of false positives is about 3%. For negative cases, the relative standard deviations for repeatability ranged from 0 to 5% and for reproducibility from 0 to 6%. For positive cases, relative standard deviations ranged from 0 to 13% for repeatability and from 0 to 14% for reproducibility. The method has been adopted official first action.  相似文献   

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