FLUORESCENT DNA BINDING DYE-BASED APPROACH FOR MEASURING THE GROWTH OF AN ESCHERICHIA COLI LYSINE AUXOTROPH AND QUANTIFYING LYSINE

Microbiological assays for determination of bioavailable lysine appear to have many advantages. However, since the developed assay is based on bacterial growth and considerable optical density (OD) is required to detect distinguishable differences in extent of growth, it can be time consuming. The purpose of this study was to explore the fluorescence as an alternative method to measure bacterial growth instead of OD and examine the possibility to shorten the time required for the lysine assay. An assay based on SYTO 9 green fluorescent DNA binding dye (Live/Dead BacLight Protocol, Molecular Probes) was used to stain all bacteria in a population. Additional experiments were carried out to determine the ability of fluorescence based on SYTO 9 to overcome problems associated with high nonbacterial background that contributes to OD. From this study it appears that using fluorescence based on SYTO 9 green fluorescent staining, the E. coli lysine auxotroph growth assay can be completed in 9 h instead of 11 h and has the advantage of improved detection sensitivity. Problems associated with interference by high background nonbacterial OD can be partially resolved by fluorescence.     

ASSESSMENT OF AN ESCHERICHIA COLI METHIONINE AUXOTROPH GROWTH ASSAY FOR QUANTIFYING CRYSTALLINE METHIONINE SUPPLEMENTED IN POULTRY FEEDS

Estimating availability of methionine is relevant to feed formulation since diets can be supplemented with crystalline methionine to meet the minimum requirements of rapidly growing birds. Bacterial assays have been developed to measure the bioavailable levels of several essential amino acids in feeds, including methionine. The E. coli methionine auxotroph strain used in this study exhibited a linear extent of growth response to increasing concentrations of methionine added to the minimal test media, in the range of 0 to 4 μg/mL. In addition the growth rates of the E. coli auxotroph were significantly (P < 0.01) different when the methionine concentrations were varied (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 μg/mL) in minimal media. To assay feeds, feed grade methionine was added to poultry feed mixtures and samples were diluted with M9 media. Using this assay for estimating crystalline methionine added to feed, the extent of growth of the methionine auxotroph was correlated with the levels of crystalline methionine supplemented in the feed (R²= 0.9873). For all supplementation levels methionine recovery percentages ranged from 71 to 80% indicating that the bacterial assay response to crystalline methionine was relatively constant in the presence of the feed matrix. The overall results indicate that the rapid detection of crystalline methionine added to feeds is possible using this E. coli methionine auxotroph growth-based assay.     

ADAPTATION OF A METHIONINE AUXOTROPH ESCHERICHIA COLI GROWTH ASSAY TO MICROTITER PLATES FOR QUANTITATING METHIONINE

A rapid microtiter methionine assay was developed using a methionine auxotroph E. coli strain. The bacterial strain was first grown on rich media to promote extensive bacterial growth and the cells were depleted to exhaust all endogenous methionine. After depletion, cells were transferred to minimal media with increasing concentrations of methionine and microtiter plates were incubated at 37C for 6 h. Methionine microtiter standard curves yielded linear growth responses to increasing concentrations of methionine in the range of 0 to 26.8 μM. Addition of different antibiotic and antifungal agents to the media did not significantly alter the linear growth response observed in the microtiter assay. This microtiter plate E. coli methionine assay has potential as a rapid in vitro assay method for quantifying methionine.     

ASSESSMENT OF IMPEDANCE MICROBIOLOGICAL METHOD FOR THE DETECTION OF ESCHERICHIA COLI IN FOODS

This study was conducted to determine the sensitivity and specificity of the impedance-based microbiological method for the detection of Escherichia coli in foods within 24 h of testing. A Malthus Microbiological Analyzer system (Malthus System V, Malthus Instruments Ltd., Bury, United Kingdom), and a modified Malthus Coliform Broth Medium (MCBM), and an incubation temperature of 44C were used. The sensitivity of the impedance method was determined by testing E. coli-negative food samples spiked with different concentrations of E. coli. The specificity of the method was determined by testing E. coli -negative food samples spiked with Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa. The test results were compared with those obtained by the Most Probable Number (MPN) method. Milk, milk products, raw and ready-to-eat meats, and vegetables were tested for the presence of E. coli by both methods. The sensitivity of the impedance method and the MPN method for the detection of foods containing 10¹ CFU/g was 100% and 84.4%, respectively. Both methods had a specificity of 100% for food samples spiked with 10¹ CFU/g E. coli. The specificity of the impedance and the MPN methods for the detection of E. coli in naturally contaminated milk and meat samples was 100% and 95.7% respectively. E. coli was detected in foods by the impedance method within 4–24 h of testing at a detection limit of 1 CFU/mL. These results demonstrate that the impedance method can be used as a rapid and sensitive method for the detection of E. coli in foods.     

POTENTIAL RAPID BIOASSAY FOR ALIMET® USING A METHIONINE ESCHERICHIA COLI AUXOTROPH

A potential rapid bioassay for methionine hydroxy analog (MHA) feed additive (ALIMET®) was examined using a methionine auxotroph E. coli strain. Bacterial cells were grown in minimal media containing a concentration range of 0 to 26.8 μM of either L-methionine or MHA as ALIMET®. Increasing either methionine or MHA concentration increased the growth rate of the methionine auxotroph. The estimated substrate affinities for methionine compared to MHA were not significantly different (P > 0.13) and the maximum growth rate estimates were also similar (P > 0.34). Methionine and MHA standard curves yielded linear responses (R²= 0.96) to increasing concentrations of the respective substrate. Based on these results it appears that the E. coli methionine auxotroph would have potential utility for further development of a rapid bioassay of ALIMET®.     

CONSTRUCTION AND GROWTH KINETICS OF A BIOLUMINESCENT METHIONINE AUXOTROPH ESCHERICHIA COLI STRAIN FOR POTENTIAL USE IN A METHIONINE BIOASSAY

Methionine is one of the essential and first limiting amino acids in animal nutrition. In this study, an Escherichia coli methionine auxotroph bacterial strain that exhibits a linear growth response to methionine concentrations was transformed with a plasmid containing genes encoding ampicillin resistance and bioluminescence in order to develop a microbiological technique for methionine quantitation. Transformants were selected based on antibiotic resistance and plasmid containing candidates were confirmed by restriction enzyme digestion and gel electrophoresis. To confirm the bioluminescent phenotype, video imaging of the strain using long exposure photography yielded colonies exhibiting bioluminescence. The strain was also tested in the presence of ampicillin supplemented media with increasing methionine concentrations and growth response (measured as optical density, OD), growth rates and methionine affinities were compared before and after transformation. Although the transformed E. coli methionine auxotroph exhibited somewhat different growth kinetic responses than the nontransformed strain, the standard curves used for estimating methionine concentrations were not different. Based on the results in this study the transformed bioluminescent strain could be used as an OD-based assay if bioluminescence equipment and materials are not available.     

A NOTE ON THE CONTAMINATION OF MILK DURING INCUBATION AT 22° FOR THE CLOT-ON-BOILING TEST

SUMMARY: During the removal of samples from thick-walled vessels, either by pouring or pipetting, the remaining liquid frequently became contaminated, no matter whether the vessel was closed by a rubber bung or a glass Kali bottle top over a cotton wool plug. This was not the case with vessels of thin heat-resistant glass. It seems that the necks of thin- but-not thick-walled vessels can be sterilized in the bunsen flame.     

REGIONAL DISTRIBUTION AND PROPERTIES OF THE GLYCINE CLEAVAGE SYSTEM WITHIN THE CENTRAL NERVOUS SYSTEM OF THE RAT: EVIDENCE FOR AN ENDOGENOUS INHIBITOR DURING IN VITRO ASSAY

—The activity of the glycine cleavage system (GCS) was determined in homogenates from five specific regions of the rat CNS (telencephalon, midbrain, cerebellum, medulla-pons, and spinal cord). An inverse trend was noted between the glycine content and the specific activity of the GCS in the regions. A 25-fold range in the enzyme activities was found between the telencephalon (highest) and the spinal cord (lowest). The properties of the GCS activity in CNS homogenates agreed with those properties previously described for this system in partially purified preparations of liver and brain mitochondria (Kikuchi , 1973; Bruin et al., 1973). Within the CNS homogenates, the liberation of CO₂ from the carboxyl carbon of glycine was quantitatively coupled to the formation of serine. The presence of an endogenous inhibitor(s) within neural tissues was suggested by the non-additivity of the activities when homogenates from the various regions were combined. Moreover, homogenates of CNS tissue inhibited the GCS activity of liver homogenates, and an inverse relationship was found between the level of GCS activity in a given region of the CNS and its ability to inhibit the GCS activity of liver homogenates. This inhibition of liver activity was greatest when liver was incubated with homogenates of spinal cord (86%) and lowest when incubated with homogenates of telencephalon (20%). Because of this endogenous inhibition, the apparent activity of the GCS measured in vitro may not reflect the contribution of this enzyme system in the metabolism of glycine in vivo. Although the significance of this inhibition is not known, a possible role is discussed for the regulation of the levels in glycine and one-carbon pools within the CNS.     

草鱼生长激素非竞争式酶联免疫吸附测定法的建立及鉴定

应用草鱼生长激素（ｇｃＧＨ）单克隆抗体及多价兔抗血清建立了草鱼ＧＨ非竞争式酶’联免疫吸附测定ＥＬＩＳＡ系统。用正辛酸法对腹水单抗进行了分离纯化,获得了高纯度的单抗制备物。聚丙烯酸胺凝胶电泳表明纯化的单抗由分子量分别为５５ｋＤ和２５ｋＤ的两条蛋白带组成。用纯化单抗铺底,用兔抗血清作后续抗体建立了一种测定草鱼ＧＨ的非竞争式双抗夹心ＥＬＩＳＡ方法。交叉试验表明该测定系统只与草鱼ＧＨ和基因重组鲤生长激素（ｒｃＧＨ）具有剂量依存的结合反应,而与大马哈鱼生长激素（ｓＧＨ）、牛生长激素（ｂＧＨ）、大马哈鱼促性腺激素（ｓＧｔＨ）、及黑鲢促性腺激素（ｂｓｃＧｔＨ）等均无交叉反应。该 ＥＬＩＳＡ方法的灵敏度可达０．８ｎｇ／ｍｌ,组内变异系数为 ５．９ ％,组间变异系数为７．６％,回收率达９０％以上。初步应用表明,鲤和团头鲂垂体抽提液、草鱼血清、鲤血清及鲫血清在该测定系统中有剂量依存的反应曲线,而大口鲶、黄颡鱼、中华鲟及黄鳝鱼垂体抽提液及大口鲶、胡子鲶和罗非鱼血清在该测定系统中没有交叉反应。     

DEVELOPMENT AND EVALUATION OF A RAPID ENZYME-LINKED IMMUNOFILTRATION ASSAY (ELIFA) AND AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B

An enzyme-linked immunofiltration assay (ELIFA) and a microtitre plate enzyme-linked immunosorbent assay (ELISA) were developed and compared for their ability to detect staphylococcal enterotoxin B (SEB). The double antibody capture format was used for both assays. Factors which improved the sensitivity of the ELIFA system were (1) addition of casein and thimerosal to the antigen dilution buffer; (2) addition of polyethylene glycol (MW 6000) to the detection and conjugate antibody dilution buffers; and (3) washing with diethanolamine buffer prior to addition of the substrate/chromogen. The ELIFA system had a turnaround time of approximately 1 h and a detection limit of 1 ng/mL of purified SEB. The ELISA had a total turnaround time of 21 h, or 3 h using plates pre-coated overnight with the capture antibody. The detection limit of the ELISA for purified SEB was 0.05 ng/mL. The detection limit of SEB in cheese samples spiked with purified enterotoxin and subjected to a simple extraction procedure was 1 ng/mL and 0.1 ng/mL of extract, with the ELIFA and the ELISA, respectively.