Cylinder Plate Assay Research Articles

DERIVATIZATION OF NEOMYCIN SULPHATE AND AREA UNDER CURVE METHOD FOR ESTIMATION OF NEOMYCIN SULPHATE AND CLOBETASOL PROPIONATE IN CREAM

Neomycin sulphate is official in IP 2018, USP 2004 and BP 2003; which includes Microbial assay (Cylinder Plate assay) for the estimation therefore chemical derivatization of Neomycin Sulphate is done using 1-Fluoro-2, 4-Dinitrobenzene (DNFB); also known as Sanger’s reagent, to make it detectable by UV with good accuracy and precision. A fixed dose combination of Neomycin Sulphate, Clobetasol Propionate and Chlorocresol (preservative) in cream formulation is used to treat different types of Skin infections. The literature review reveals that methods like UV, HPLC, HPTLC, GC-MS etc. have been reported for Neomycin Sulphate and Clobetasol Propionate individually and along with other drugs. But no reported method found in combination for Neomycin sulphate and Clobetasol propionate. A simple, accurate, precise and economical UV spectrophotometric i.e. Area Under Curve Method (AUC) was developed. All the dilutions of drugs were prepared in Methanol: Acetonitrile (50:50). Area under curve was integrated in the wavelength range of 235.0- 245.0 nm, 252.0-262.0 nm and 223.0-233.0 nm for CSP, NMS and CCS respectively. The AUC method for Clobetasol Propionate (CSP), Neomycin Sulphate (NMS) and Chlorocresol (CCS) was found to be linear over the range of 0.8-1.2 μg/ml, 8.0-12.0 μg/ml and 1.6-2.4 μg/ml respectively. The result of analysis was analysed and validated statistically and recovery study was found within range of 98-102%. The % RSD was not more than 2.0 % which indicates good precision. All the validation parameters were carried out according to ICH Q2 (R1) guidelines.

MICROBIOLOGICAL ASSAY FOR QUANTITATIVE DETERMINATION OF IMIPENEM IN POWDER FOR INJECTION

This work describes the development and validation of a microbiological method using the cylinder-plate assay for quantitative determination of imipenem in powder for injection. The aim was to obtain a low-cost and suitable methodology that can be alternative to physicochemical techniques already described, contributing for the quality control of this antibiotic. Firstly, the analytical conditions were optimized, testing the microorganism, inoculum concentration and best range of sample and standard concentrations, in a way that provides the adequate measurement of the inhibition halos. Staphylococcu s epidermidis ATCC 12228 was selected as test microorganism, as well as 2.0 % of inoculum concentration. The validation protocol followed the official guidelines, and the parameters evaluated were linearity, precision (intermediate precision and repeatability) and accuracy. All standard curves ranging 0.5-2.0 µg mL-1 showed r values higher than 0.999, and ANOVA confirmed that were no deviation from linearity (p-value < 0.05). The method also proved to be precise with RSD (relative standard deviation) values ranging 0.28-0.64 for repeatability and 2.49 for intermediate precision. It was performed three days of experiments, being three assays of eight plates a day. The drug mean content was 101.05%. Accuracy was assessed by recovery test, with standard recovery percentage of 101.70-107.90% (mean recovery = 104.86%), which was considered satisfactory. Therefore, the proposed microbiological method was considered validated and suitable for application in quantitative determination of this drug, being useful for quality control routine.

&lt;i&gt;In Vitro&lt;/i&gt; Comparison of Probiotic Properties of &lt;i&gt;Lactobacillus fermentum&lt;/i&gt; SK54 Isolated from New Born Baby with &lt;i&gt;Lactobacillus rhamnosus&lt;/i&gt; GG ATCC 53103

A lactobacillus strain isolated from the feces of a healthy baby was identified as Lactobacillus fermentum SK54. The probiotic properties of this strain including antimicrobial activity, survival in acid, bile, simulated gastric fluid [SGF] and simulated intestinal fluid [SIF], antibiotic susceptibility and adherence to Caco-2 cells were examined and compared them with a well known commercial probiotic strain Lactobacillus rhamnosus GG ATCC 53103 isolated from human intestine. L. fermentum SK54 and L. rhamnosus GG showed inhibitory properties toward gastrointestinal pathogens i.e., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella Typhimurium, and Shigella sonnei by cylinder-plate assay. The inhibitory substances from L. fermentum SK54 composed of acid substances naturally produced by lactobacilli and high molecular weight proteinaceous compound [MW &gt; 3.5 kDa] which sensitive to proteolytic enzymes, pH stability [pH 2.0-5.0] and heat stability [60, 80, 100°C for 10 min and 121°C for15 min]. L. rhamnosus GG produced only acid inhibitory substances. Both strains survived well at low pH [pH 2.0-4.0], bile salts [0.1-0.3% w/v], SGF and SIF. They also had the same 14 tested antibiotic sensitivity pattern and expressed high adherence to Caco-2 cells. This study provides a rationale to support L. fermentum SK54 as a potential probiotic to prevent or act as a therapeutic agent against pathogenic strains.

Evaluation of in-vitro antibacterial activity and anti-inflammatory activity for different extracts of Rauvolfia tetraphylla L. root bark

ObjectiveTo assess the in-vitro antibacterial activity and anti-inflammatory activity of orally administered different extracts (Hydro-alcoholic, methanolic, ethyl acetate and hexane) of Rauvolfia tetraphylla (R. tetraphylla) root bark in Carrageenan induced acute inflammation in rats. MethodsIn-vitro antibacterial activity was evaluated for extracts against four Gram positive and four Gram negative bacteria by using cylinder plate assay. Hydro-alcoholic extract (70% v/v ethanol) at 200, 400 and 800 mg/kg doses and methanolic, ethyl acetate and hexane extracts at doses 100, 200 and 400 mg/kg were tested for anti-inflammatory activity in Carrageenan induced rat paw oedema model and paw thickness was measured every one hour up to 6 hrs. ResultsAll extracts of R. tetraphylla root bark showed good zone of inhibition against tested bacterial strains. In Carrageenan induced inflammation model, hydro-alcoholic and methanolic extract of R. tetraphylla root bark at three different doses produced significant (P<0.001) reduction when compared to vehicle treated control group and hexane, ethyl acetate extracts. ConclusionIn the present study extracts of R. tetraphylla root bark shows good in-vitro antibacterial activity and in-vivo anti-inflammatory activity in rats.

Bovine Intestinal Bacteria Inactivate and Degrade Ceftiofur and Ceftriaxone with Multiple β-Lactamases

The veterinary cephalosporin drug ceftiofur is rapidly degraded in the bovine intestinal tract. A cylinder-plate assay was used to detect microbiologically active ceftiofur, and high-performance liquid chromatography-mass spectrometry analysis was used to quantify the amount of ceftiofur remaining after incubation with bovine intestinal anaerobic bacteria, which were isolated from colon contents or feces from 8 cattle. Ninety-six percent of the isolates were able to inactivate ceftiofur to some degree, and 54% actually degraded the drug. None of 9 fungal isolates inactivated or degraded ceftiofur. Facultative and obligate anaerobic bacterial species that inactivated or degraded ceftiofur were identified with Vitek and Biolog systems, respectively. A subset of ceftiofur degraders also degraded the chemically similar drug ceftriaxone. Most of the species of bacteria that degraded ceftiofur belonged to the genera Bacillus and Bacteroides. PCR analysis of bacterial DNA detected specific β-lactamase genes. Bacillus cereus and B. mycoides isolates produced extended-spectrum β-lactamases and metallo-β-lactamases. Seven isolates of Bacteroides spp. produced multiple β-lactamases, including possibly CepA, and metallo-β-lactamases. Isolates of Eubacterium biforme, Bifidobacterium breve, and several Clostridium spp. also produced ceftiofur-degrading β-lactamases. An agar gel overlay technique on isoelectric focusing separations of bacterial lysates showed that β-lactamase enzymes were sufficient to degrade ceftiofur. These results suggest that ceftiofur is inactivated nonenzymatically and degraded enzymatically by multiple β-lactamases from bacteria in the large intestines of cattle.

Chemical composition and antimicrobial activity of the essential oil of Sagittaria trifolia

The essential oil of Sagittaria trifolia, a well-known famous medicinal foodstuff in China, was analyzed for the first time using GC-MS. Twenty-eight constituents were identified. The major components of the oil were hexahydrofarnesyl acetone (62.3%), tetramethylhexadecenone (5.8%), myristaldehyde (4.7%), n-pentadecane (2.9%), and 2-hexyldecanol (2.9%).The antimicrobial activity of the essential oil was evaluated against seven microorganisms, including two clinically isolated strains and five reference strains, using microbiological cylinder plate assay and broth microdilution methods. The results showed that the oil had a significant antimicrobial effect on four of them. This antimicrobial activity can partly explain why the oil is used medicinally during childbirth and for skin diseases in Chinese traditional medicine.

Microbiological assay of ketoconazole in shampoo

Ketoconazole, an anti-fungal agent, is often incorporated in several pharmaceutical forms and in shampoo formulation it is known to be effective against fungal infection on the scalp. This paper describes a method to quantify ketoconazole in shampoo by comparing the cylinder plate assay and the HPLC method. The test organism used for the agar diffusion assay was Candida albicans ATCC 10231. Three different concentrations of ketoconazole were used for the diffusion assay. A mean zone diameter was obtained for each concentration. A standard curve was obtained by plotting the three values derived from the zone diameters. A prospective validation of the method showed that the method was linear ( r = 0.9982), precise (R.S.D. = 2.57%) and accurate. The results obtained by the two methods were statistically evaluated by analysis of variance (ANOVA) and the results obtained indicate that there is no significant difference between these two methods.

Screening of Iranian Plants for Antimicrobial Activity

The methanol extracts of 306 plants of 52 families obtained from Northeast of Iran (Khorasan Province), were tested for antimicrobial activity (in vitro) using the cylinder plate assay method. Activity against Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Pseudomonas aeruginosa, Morganella morganii (Gram negative), Bacillus subtilis and Staphylococcus aureus (Gram positive) and Candida albicans is discussed.

Validation Study of Assay for Neomycin in Feed

Abstract The neomycin microbiological cylinder plate assay using an ionic extraction method was validated for 2 swine feeds with neomycin base concentrations of 140.0 and 280.0 g/ton, 2 cattle feeds with neomycin base concentrations of 140.0 and 280.0 g/ton, and 2 premixes with neomycin base concentrations of 1.4 and 7.0 g/lb. The accuracy, precision, and reproducibility were evaluated by assaying the 6 feed samples in duplicate on 6 different days by an Upjohn laboratory (Laboratory 1) and 2 outside laboratories (Laboratories 2 and 3) for a total of 72 data points per laboratory. Three plates were used for each sample assayed. The recovery over all complete (type C) feed samples and (type B) premixes tested, ranged from 82 to 109% for Laboratory 1,85 to 114% for Laboratory 2, and 78 to 134% for Laboratory 3. Mean percent recoveries for all samples tested were 98.0,98.9, and 103.7% for Laboratories 1,2, and 3, respectively. Day-to-day performance yielded 95% confidence limits of ±11.9, ±16.9, and ±25% for Laboratories 1,2, and 3, respectively. Of 215 assay values, 206 (96%) fell within 20% of the actual targeted recovery (100%). Plate-to-plate variation of each of the 3 laboratories also was assessed. Statistical estimates for the use of 1,5, and an infinite number of plates showed that when 2 plates were used by Laboratories 1,2, and 3, than 99,99, and 95%, respectively, of the assay results fell within the acceptable assay compliance limits (70–125% recovery) for complete feeds and premixes. Slight improvement was found only for Laboratory 3, when more than 2 plates were used.

Tissue concentrations of clindamycin after multiple oral doses in normal cats

Eighteen normal cats were randomly allocated into two blocks with three treatment groups and dosed orally with clindamycin aqueous solution for 10 days at a dosage rate of 5.5 mg/kg twice daily (Group 1), 11 mg/kg twice daily (Group 2), or 22 mg/kg once daily (Group 3). At the end of dosing, all cats were killed and tissues were taken for clindamycin concentration analysis. Clindamycin was extracted from tissues using solid-phase extraction columns followed by microbiological assay of clindamycin using a cylinder plate assay using M. luteus. Recovery from each tissue was determined by inoculating known concentrations of clindamycin into drug-naive tissues and comparing the observed concentration from the expected concentration. Confirmation that the bioassay detected clindamycin and not N-desmethylclindamycin, its active metabolite, was done using gas-chromatography-mass-spectrometry. Concentrations were highest in the lung, with tissue:serum ratios greater than 3 in all groups. Concentrations were higher in Group 3 than Group 1 (P less than 0.05). Only liver concentrations in Group 3 were statistically higher than in Group 2, although all tissues except bone marrow and CSF had numerically higher concentrations in Group 3 than Group 2. The tissue:serum ratio was greater than 1 in all tissues studied except bone, cerebrospinal fluid, brain, and skeletal muscle.

High performance liquid chromatographic determination of cefoperazone in human serum and urine

A gradient high-performance liquid chromatographic (HPLC) procedure has been developed for the determination of microgram amounts of cefoperazone in human serum and urine. The method employes a μ Bondapak C 18 column and gradient elution with two mobile phases. Excellent separation of the drug from potential degradation products as well as from representative penicillins (sodium ampicillin, sodium methicillin, potassium penicillin G) and aminoglycosides (tobramycin, gentamicin, kanamycin) has been demostrated. Coefficients of variation of 7.3% or less were obtained for 25–100 μg/ml cefoperazone in both serum and urine. Average recoveries of the drug from spiked serum and urine samples corresponded to 97.6% and 98.6%, respectively. Amounts as low as 1 μg cefoperazone per ml of sample can be estimated using sample volumes corresponding to 0.1 ml serum or 1 ml urine. Good correlation between the HPLC assay and microbiological cylinder—plate assay employing Micrococcus luteus ATCC 9341 has been demostrated for human serum and urine of patients treated with cefoperazone. While the microbiological method is less time-consuming, it lacks specificity in the presence of other antibiotics. The HPLC method can be used to analyze cefoperazone in the presence of penicillins and aminoglycosides which can potentially be co-administered with cefoperazone.

Cylinder Plate Assay for Determining Bacitracin in Premix Feeds and Finished Feeds: Collaborative Study

A cylinder plate assay procedure was studied by 10 laboratories. For premix feeds, 3 samples of bacitracin methylene disalicylic acid and 3 samples of bacitracin zinc premixes covering the range of 10 to 50 g/lb were used. The repeatability standard deviation was 2.11, and the reproducibility standard deviation was 2.13. The average recovery of bacitracin was 101.5%. The method has been adopted official first action. For finished feeds, 6 samples of bacitracin methylene disalicylic acid and 6 samples of bacitracin zinc covering the range of 10 to 800 g/ton were used in the study. The procedure included a sample cleanup step using disposable reverse phase columns. This step appears to be the cause of the poor results reported by most collaborators. Continued study is needed to develop an acceptable method for finished feeds.

Improved microbiological assay procedures for dihydrostreptomycin residues in milk and dairy products.

Because dihydrostreptomycin can remain as a slowly removed antibiotic residue in dairy animals and because of the need for more sensitive procedures with which to provide information concerning antibiotic residues in food products, procedures were developed in more sensitive assays of dihydrostreptomycin in milk and some representative dairy products. The cleanup procedures that aided these improvements were (i) precipitation of milk proteins by acidification and (ii) centrifugation to extract cheeses and to remove physical barriers to diffusion in the cylinder plate assay. In addition, a thin, single seeded assay layer was used to maximize diffusion. Levels of dihydrostreptomycin as low as 0.06 microgram/ml in milk and 0.2 to 0.4 microgram/g in cheeses were measurable; these levels were some fourfold more sensitive than those presently recommended by the Food and Drug Administration.

Collaborative Study of a Microbiological Assay for Lasalocid Sodium in Feeds

The microbiological cylinder plate assay method for lasalocid sodium reported earlier was studied in a slightly modified form by 7 laboratories. Assay values paralleled recovery values and all data were corrected by the corresponding daily recovery factor for the feed type. Agreement between laboratories for mashes and pellets containing 0.0064, 0.0080, and 0.0096% lasalocid sodium was satisfactory. Coefficients of variation for mashes were 14.6, 9.8, and 10.7% and for pellets, 15,2, 10.9, and 15.6%. The average of the values found for the 3 mashes was 99.4% of the calculated value and for the 3 pellets, 86.5%. The method has been adopted as official first action.

Agar Well Technique for Antibiotics in Animal Feeds

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.

Practical quantitative assay for 5- fluorocytosine in serum and other body fluids.

A method is described for assaying 5-fluorocytosine levels in serum or other body fluids. It is simpler and less expensive than the standard cylinder plate assay and is therefore more practical for routine diagnostic laboratory testing.

A practical cylinder plate assay method for determination of nitrofuran derivatives in tissues

A practical cylinder plate assay method for determination of nitrofuran derivatives in tissues

Sensitive Microbial Assay Procedure for Chlortetracycline in Milk and Related Products

Abstract A microbiological assay procedure with increased sensitivity was developed for milk and some of its products. The procedure is based on the classical cylinder-plate assay procedures with the following modifications: closer adherence to a consistent pH of 4.5, centrifugation of samples to remove the physical barriers to diffusion of the antibiotic, and use of a thin, single layer of seeded agar. Recoveries in milk by this procedure averaged 65.3% versus 37.9% for those obtained using FDA methods. Improvements in recoveries were noted with the milk products studied. Of the chlortetracycline added to milk, 25% was converted to the epimer form which has negligible microbiological inhibitory properties. Binding and/or the inability to account for the antibiotic was found to be negligible.

Microbiological Assay of Streptomycin in Feeds

Abstract Modifications of the official AOAC method for the determination of streptomycin in feeds were collaboratively studied. The cylinder plate assay uses Bacillus subtilis ATCC 6633 as the test organism; low levels of streptomycin are determined with sulfadiazine-sensitized assay agar and high levels are determined with nonsensitized assay agar. The modifications include a single extraction and either mechanical shaker or high-speed blender as alternative methods of extraction. Four feed levels at 6 to 75 g streptomycin/ton were analyzed by the sensitized method. The average recoveries ranged from 81 to 98% and the coefficients of variation ranged from 4.5 to 9.7%. Also, four feed levels containing 37.5 to 300 g streptomycin/ton were analyzed by the non-sensitized assay. The spread of recoveries among 18 collaborators was too wide for this method to be acceptable at the levels studied. It is recommended that the sulfadiazine-sensitized assay be adopted as official first action at 6 g/ton and that the non-sensitized assay be approved at the level of 2.8 g/lb and study continued at lower levels.

Placental transfer of dicloxacillin at term

Following a single dose of 500 mg. of dicloxacillin the serum concentration of the antibiotic in women in active labor followed the relationship y = 25.8 e−0.0129t, where y = concentration in microgram per milliliter and t = minutes after injection; the corresponding relationship within the cord serum, y = 3.0 e−0.0032t, demonstrated transport across the placenta to the fetus. When 250 mg. of the antibiotic was injected intramuscularly, insufficient material transferred to be detectable by the cylinder plate assay method (0.20 μg per milliliter lower limit of sensitivity). When compared to other penicillin analogs, the amount of transfer, as judged by the initial total fetal serum concentration, was best related to the initial maternal concentration of antibiotic unbound to serum protein. This finding is consistent with a proposed limitation of available transferable forms of penicillin type antibiotics by serum protein binding. Dicloxacillin appeared in the amniotic fluid approximately 2 hours after intravenous administration to the mothers.