Collaborative studies on the Salmonella/microsome mutagenicity assay.

Although the Salmonella/plate test has been used extensively, a collaborative study was undertaken to determine the interlaboratory reproducibility of this microbial mutagenicity assay. Four laboratories participating in the study have completed testing, under code, of 61 carcinogens and noncarcinogens. All chemicals were tested both with and without metabolic activation in Salmonella typhimurium strains TA1535, 1537, 1538, 98, and 100. The metabolic activation systems used were derived from the livers of both uninduced and Aroclor 1254-induced Fischer rats, B6C3F1 mice, and Syrian hamsters. Analysis of the results on 23 of the chemicals tested in 3 of the participating laboratories showed that 8 were negative when tested in all laboratories and 13 were positive. Two chemicals gave positive results in 2 laboratories; the same 2 chemicals were negative when tested in the third laboratory.     

Analysis of fat-soluble vitamins. XIX. Collaborative studies on the chemical assay for vitamin D concentrates

In 1971, a chemical method for the assay of vitamin D in concentrates containing only vitamin D was collaboratively studied by 14 laboratories, using 6 different samples from 2 European manufacturers. On the basis of these results, the laboratories were divided into 2 groups: 5 with significant laboratory biases of greater than or equal to 2%, and 9 laboratories with nonsignificant bias. The 9 laboratories were subdivided into 2 groups which differed significantly as to reproducibility within laboratories. The reproducibility between laboratories, expressed as a standard deviation in per cent with 95% confidence limits, was 1.2% (confidence range 0.6-7.3) and 4.7% (confidence range 2.4-29.3) for 3 and 6 laboratories, respectively. A second collaborative test was performed in 1974, using 12 vitamin D resin samples in oil from 3 United States manufacturers, to compare 2 chemical vitamin D assay methods (with and without maleic anhydride) and to compare results from the chemical and biological methods; 9 laboratories participated in the chemical method study and 3 in the rat bioassay study. The correlation of results of the chemical method including maleic anhydride treatment and the rat bioassays was satisfactory. The reproducibility of the chemical method was about the same as that in the first collaborative test.     

Determination of procaine and related local anesthetics. II. Collaborative study.

The ion-pairing chromatographic method reported previously for the isolation and spectrophotometric determination of the local anesthetics, alone and in combination, was studied collaboratively. Three solutions were assayed. One containing procaine as the single active component gave an average recovery of 99.8 plus or minus 1.7%. A mixture of procaine and tetracaine gave results of 99.3 plus or minus 1.6 and 98.9 plus or minus 8.82%, respectively. A third solution containing procaine and propoxycaine assayed 100.0 plus or minus 1.5 and 99.1 plus or minus 1.9%, respectively. It was shown that low results for tetracaine were due to loss during the final evaporative step. The method for samples containing tetracaine should be studied further. The other methods have been adopted as official first action.     

Collaborative study of effect of light on cadmium and lead leaching from ceramic glazes.

A limited interlaboratory study was carried out to determine the effect of lighting conditions on the release of cadmium from ceramic glazes by 4% acetic acid. Cadmium release increased with increased exposure to light. Further interlaboratory study on plates manufactured under controlled conditions showed that if temperature, intensity of illumination, and time of exposure are specified, reproducible results for leaching of both lead and cadmium can be obtained. A modification of the official AOAC method, 25.031-25.034, to increase sensitivity was collaboratively studied by 8 laboratories. Each received 6 solutions of lead in 4% acetic acid (3 sets of blind duplicates at the 0.1, 0.5, and 0.8 mug/ml levels) plus a reagent blank. Average recoveries were 0.1006, 0.5056, and 0.8194 mug/ml, respectively, with coefficients of variation of 11.4, 3.3, and 2.8%, respectively. The proposed modification has been adopted as official first action, and a parameter for exposure to light during extraction has been included in the method.     

基于Blackboard平台的网络协作学习探究

Blackboard作为一个应用广泛的网络教学平台,在促进协作学习方面具有显著的优势。根据协作学习的要求,结合Blackboard平台的功能特性,提出了基于Blackboard平台的协作学习模式,以期能更好地开展网络协作学习。     

Some principles of microbiological turbidimetric assays of antibiotics.

Turbidimetric methods for determining the potency of antibiotics are inherently more accurate and more precise than are comparable agar diffusion procedures, but assays conducted in liquid media are subject to degradation from less than ideal conditions to a much greater extent than are diffusion methods. The relationships between test organisms, antibiotics, and assay concentrations are discussed. A valid assay procedure must produce a linear response with an adequate slope (-0.4 to -1.2) by the test organism to increasing concentrations of drug; such linear response normally occurs over a limited range of concentrations. Criteria used to select photometers that offer the greatest advantages to analytical microbiologists are described, with guidelines for the most effective use of the chosen instrument.     

Collaborative study of a column chromatographic method for bendroflumethizide and cyclothiazide.

Bendroflumethiazide and cyclothiazide are eluted from a sodium carbonate column with chloroform-acetic acid (98+2) and are measured directly by ultraviolet spectrophotometry. The method was collaboratively studied by 8 analysts. The average per cent recovery and standard deviations for simulated mixes of bendroflumethiazide and cyclothiazide were 99.61+/-0.78 and 99.3+/-1.97. The method has been adopted as official first action for the determination of bendroflumethiazide.     

Comparison of studies on saccharin and sodium nitrite.

A review of long term animal studies of saccharin and sodium nitrite was undertaken to assess the effect of variability of selected protocol elements on the results obtained. These elements were divided into 4 general categories: design, including selection of test animals, basal diet, dosage form and doses of test substance, route of administration, and duration of exposure; observations, including gross observations during life and at necropsy, clinical tests, and histopathology; performance, including conduct of the test and animal husbandry; and analytical procedures, including chemical and statistical analyses. Because many of the protocol elements are not fully discussed in study reports, it was often impossible to determine what actually had been done. The review of various saccharin studies suggests that bladder tumors resulted following in utero exposure. In utero exposure with sodium nitrite did not appear to cause reticuloendothelial changes. The numerous variations in protocol elements in the nitrite studies precluded identification of a prime element responsible for the variation in reticuloendothelial changes observed. It can be concluded from this review that achievement of reproducibility in long term studies requires minimal variation of protocol elements for the new study.     

Collaborative study comparing the spiral plate and aerobic plate count methods.

The spiral plate count method is a semiautomated plating technique that greatly reduces manpower and material costs normally associated with the pour plating technique. In this collaborative study, 8 laboratories compared the spiral and pour plating techniques, using 4 samples each of 3 products: frozen pumpkin pie, frozen chicken pot pie, and shampoo. The results show that 10 of the 12 comparisons of means of the pour and spiral methods were not significantly different; 2 values were significant at alpha = 0.01. Overall, the components of variance were less than that of the current milk standard, and the replicate per cent coefficient of variation was satisfactory. This study indicates that the spiral plate method is an acceptable alternative to the pour plate method; the spiral plate method has been adopted as official first action.     

Colorimetric and volumetric assays of colchicine in galenicals and in pharmaceutical preparations.

In the method described the amide group in the colchicine molecule is reduced with lithium aluminum hydride to the corresponding secondary amine. The latter is extracted with chloroform and then determined either colorimetrically by the copper dithiocarbamate reaction or volumetrically by dissolving in acid and titrating with sodium hydroxide or perchloric acid. The results were comparable with those obtained by the Egyptian Pharmacopoeia spectrophotometric method.     

Microbiological assays for antibiotics and vitamins: considerations for assuring accuracy.

Factors that may influence the accuracy and precision of microbiological manual and semiautomated turbidimetric methods as well as diffusion assays are discussed. Influence of kind of equipment, media, test bacteria, sample preparation, form of dose response lines, operations, and personnel on quality of assays is examined with the objective of reducing to insignificance those factors under control of the analyst that are responsible for low quality assays.     

Liquid chromatographic determination of N-methylcarbamate insecticides and metabolites in crops. I. Collaborative study

A liquid chromatographic (LC) multiresidue method for determining residues of N-methylcarbamate insecticides in crops was collaboratively studied in 6 laboratories. Methanol and a mechanical ultrasonic homogenizer are used to extract the carbamates. Water-soluble plant coextractives and nonpolar plant lipid materials are removed from the carbamate residues by liquid-liquid partitioning. Additional crop coextractives (e.g., carotenes, chlorophylls) are removed with a Nuchar SN-silanized Celite column. The carbamate residues are then separated on a reverse phase LC column, using an acetonitrile-water gradient mobile phase. Eluted residues are detected by an in-line post-column fluorometric detection technique. Seven carbamates and 2 carbamate metabolites were included in the collaborative study. Each collaborator determined all the carbamates at 2 levels (approximately 0.05 ppm and United States tolerance) in blind duplicate samples of grapes and potatoes. Fortified and control samples were analyzed. Repeatability coefficients of variation for all the carbamates on the 2 crops averaged 4.7% and ranged from 2.4 to 7.1%. Reproducibility coefficients of variation for all the carbamates on the 2 crops averaged 8.7% and ranged from 5.3 to 12.4%. Accuracy, measured by comparison with fortification values, averaged 95% and ranged from 79 to 103%. The estimated limit of quantitation is 0.01 ppm. The method has been adopted official first action.     

Collaborative study of speaker identiication by the voiceprint method.

In the voiceprint method known and unknown voices are compared to determine if they are the same speaker. An unknown voice is recorded over the telephone onto a quality recorder. The known voice is similarly recorded, repeating the questioned message verbatim. An examiner determines, by means of acoustic spectrography and aural analysis, the similarities or differences existing between the known and unknown speakers. A positive conclusion is based on at least 10 pairs of like sounds. Five decisions are available for the examiner to make: positive identification, positive elimination, probable identification, probable elimination, and unable to make a decision with the sample submitted. Seven collaborators, chosen from various parts of the United States, were given recordings of 4 unknown speakers, 2 male and 2 female, to compare with similar recordings of 6 known speakers, 3 male and 3 female. A total of 21 positive identifications and 63 positive eliminations was possible. Twenty correct identifications and 47 correct positive eliminations were made. In 16 tasks, all involving the same unknown sample, collaborators reported they could not make a determination due to the poor quality of the sample. No false identifications were made; however, a trainee examiner, with less than 2 years experience, eliminated a known speaker when in fact a match did exist, thereby falsely eliminating the speaker. Examiners with more than 2 years of experience correctly identified all existing matches. This study indicates that a trained examiner can make very reliable decisions, using the aural and visual methods of comparing known and unknown voices. The voiceprint method has been adopted as official first action.     

Collaborative study of the determination of available lysine in proteins and feeds.

In the proposed method 1-fluoro-2,4-dinitrobenzene (DNFB) is reacted with the free epsilon-amino groups in protein of form DNFB-epsilon-amino lysine which is stable to acid hydrolysis. The sample is acid hydrolyzed and unavailable lysine is determined with an amino acid analyzer; total lysine is determined on the untreated sample. The available lysine, which was bound by DNFB, is determined by difference. The available lysine has been determined in 3 samples of 44% protein soybean meal by 5 collaborators, following the method outlined. The range for available lysine in reference standard 1 was 2.02-2.14%, in reference standard 2, 2.59-2.73% and in reference standard 3, 0.55-0.91%. The method has been adopted as official first action.     

Microbiological plate and turbidimetric assays of chlortetracycline in feeds: collaborative study.

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.     

Morphological and molecular cytogenetic characterization of partial octoploid Tritileymus

This study used cytological observation, genomic in situ hybridization (GISH), and seed storage protein electrophoresis to characterize a partial octoploid Tritileymus M842-1 derived from Leymus mollis (NsNsXmXm, 2n = 28) and common wheat cv. 7182 (Triticum aestivum L.), and its relationship with alien chromosomes and those from wheat. Mitotic observations showed that the chromosome configuration of M842-1 was 2n = 56 = 28II. Mitotic and meiotic GISH using two different probes for L. mollis and Psathyrostachys huashanica indicated that M842-1 contained 14 Ns genome chromosomes from L. mollis, which formed seven pairs of uniform bivalents, and confirmed the genomic composition as AABBDDNsNs. The separation of seed storage protein by polyacrylamide gel electrophoresis indicated that M842-1 expressed some L. mollis-specific glutenin and gliadin bands. The seeds of M842-1 were as large as those of wheat cultivars, while they also had a long spike and were covered with wax on the leaves and stems. Disease screening demonstrated that M842-1 was immune to stripe rust, highly resistant to powdery mildew, and moderately susceptible to wheat scab during the adult plant stage. This study demonstrated that the partial octoploid Tritileymus M842-1 could serve as a donor source in wheat breeding programs for the introduction of novel variation to promote high yield and disease resistance.     

Free radical generation assays: new methodology for accelerated oxidation studies at low temperature in complex food matrices

A novel method for the rapid screening of antioxidant efficacy and oxidative stability in food and feed matrices has been developed. The analyses are described as free radical generation (FRG) assays. The new procedure combines the use of azo-initiators with analytical equipment that is widely used for antioxidant research such as the oxidative stability instrument and the oxygen bomb. The use of initiators instead of high temperatures as a driving force to increase the rate of oxidation improves the correlation between the accelerated screening of foodstuffs and real shelf life. The improved correlation can be mainly explained by the fact that food products are analyzed in their original status, maintaining all interfacial phenomena of the food matrix. Furthermore, the lower temperature of analysis reduces differences between the reaction kinetics of the assay and those of the oxidation during actual shelf life. Consequently, the correlation between the accelerated analysis and shelf life is improved, particularly when compared to accelerated oxidation at high temperatures. The FRG assays could be used successfully to evaluate the efficacy of natural antioxidants in heat-sensitive food products such as emulsions and meat products. A good correlation was observed between the accelerated tests and the oxidation parameters obtained from standard shelf-life evaluation. It was possible to successfully compare the efficacy of several antioxidants and to predict shelf life for these heat-sensitive food matrices.     

Collaborative study of a spectrofluorometric assay for Rauwolfia serpentina tablets and powdered root.

Reserpine-rescinnamine group alkaloids are extracted from Rauwolfia serpentina preparations into a dimethylsulfoxide (DMSO)-methanol mixture and diluted with 0.5N H2SO4. The chloroform extract of this solution is passed through a 0.1N NaOH-Celite column and then through a silica gel column. The weakly basic alkaloids trapped on the latter column are eluted with a methanol mixture; a portion of the eluate is treated with nitrous acid and the reserpine-rescinnamine content is determined by measuring the intensity of fluorescence of the oxidation product. The following means and standard deviations (11 collaborators) were obtained for the determination of reserpine-rescinnamine group alkaloids in 4 samples of Rauwolfia serpentina (NF reference powder, 100 mg and 50 mg commercial tablets, and a 45 mg synthetic tablet formulation) : 0.174% +/- 0.0112, 0.131% +/- 0.0047, 0.160% +/- 0.0100, and 0.153% +/- 0.0083, respectively.     

Physiological studies on salinity and nitrogen interaction in alfalfa. II. Photosynthesis and transpiration

The interaction between salinity and nitrogen (N) forms and concentration was studied with alfalfa (Medicago sativa L.) grown in pots with fine sand under greenhouse conditions. Salinity (0–100 mM NaCl) caused a substantial reduction in carbon assimilation rate, stomatal conductance, water use efficiency, and leaf area, while transpiration rate was least affected. Salinity effects were considerably moderated by additional N supply, varied with form, concentration, and stage of plant growth. The photosynthesis was reduced more in ammonium‐ than in nitrate‐fed plants, while the transpiration rate was relatively lower in nitrate‐fed plants grown either with or without NaCl. The plants also responded differently to salinity and N levels at two harvests. This indicated a change in plant behaviour with age. The promotive effect of N on photosynthesis and other parameters in saline as well as in non‐saline conditions may be attributed to the enhanced synthesis and availability of carbon assimilatory enzymes and cofactors required for optimal photosynthesis.     

Comparison of high pressure liquid chromatographic and chemical methods for vitamin D3 concentrates. II. Collaborative study.

A collaborative study was carried out which compared the official chemical method, 43.B14-43.B24, the official rat bioassay, 43.165, and the high pressure liquid chromatographic method for vitamin D3 resin, vitamin D3 resin in oil, and dry concentrate. A total of 340 samples were distributed to 17 collaborators for analysis. Five laboratories performed both the chemical and HPLC methods on 5 sets of blind duplicates. A 2-way analysis of variance comparing both methods for each sample showed a significant (P less than 0.01) difference between methods only for Sample 5. When the 2 methods were compared over all the samples, no significant (P less than 0.05) difference was found. Except for Sample 5, there were no differences in the repeatability of the methods. Per cent recoveries on Sample 3, which contained exactly 0.200 X 10(6) IU/g, showed 98.2% for the chemical method and 100.6% for the HPLC method for the 5 laboratories that performed both methods. The assay results of the HPLC and chemical methods are in good agreement with those found by the biological assay on Samples 1-4, but not for Sample 5. Evidence indicates that Sample 5 degraded partially to isotachysterol, and while the HPLC method yielded a reasonable value on this material, the chemical method erroneously showed full potency. An amendment is included for the collaboratively studied HPLC method which detects and eliminates 5,6-trans-vitamin D3, a possible interferant.