Multiple bacterial samples

Occasionally, analytical studies such as western blot analysis of LPS or LOS will require preparation of same quantities of these glycolipids from multiple bacterial samples. Two methods have been described which can be used for such isolations (18,19). One relies on a modified phenol-water extraction (20), and the second utilizes SDS solubility of bacteria followed by enzymatic degradation of bacterial proteins (19). Neither preparation gives a highly purified preparation, but both enrich for LPS or LOS and can serve as adequate substitutes in acrylamide gel and western blot studies. These two methods are presented below. 

Epothilones A, B and E (4,5 and 6) (Fig. 2) are representative members of a new class of bacterially derived natural products which exhibit potent biological activity. Isolated by Hofle and coworkers [6] from a soil sample collected near the Zambesi river, the compounds have provided a great deal of excitement in the scientific community due to their potent cytotoxicity against a number of multiple drug-resistant tumor cell lines and because of the mechanism by which they exert this effect. Like Taxol [7], the epothilones promote the combination of a- and 3-tubulin subunits and stabilize the resulting microtubule structures. This mode of action inhibits the cell division process and is, therefore, an attractive strategy for cancer chemotherapy [7,8]. 

Automated programmable instruments that can carry out the repeated thermal cycles necessary for PCR and that can accommodate multiple samples simultaneously are now widely available. The procedure is usually performed with thermostable DNA polymerases. PCR is widely used to facilitate detection of minute amounts of viral DNA. The technique can also be used to detect specific point mutations, provided the approximate site of mutation is known. One limiting feature of this approach arises from the fact that the bacterial polymerases frequently make errors when synthesizing new strands and so can introduce mutations that are not present in the original sample. 

In our further study we investigated effects of various numbers of virulent (strain C) and avirulent (strain A) E. coli on serum microbiostasis. Bovine serum was distributed in 1-ml quantities into small screw-topped tubes and was infected with various numbers of strain A or strain C bacteria. After 12- and 24-hr incubation at 37 °C, samples of infected sera were plated on IPAM. Numbers of bacteria were determined by counting bacterial colonies on plates after 12-hr incubation. Results showed that bovine serum inhibited bacilli of avirulent strain A if the inoculated serum contained less than 12,000 bacilli per 1 ml of serum. Larger inocula of strain A multiplied in serum but at a slower rate than in broth medium (Table III). Bacteria of virulent strain C overcame the iron starvation in bovine serum even when serum was inoculated with as few as 100 cells/ml of serum. The results of this study showed that the rate of bacterial multiplication in bovine serum is determined partly by bacterial numbers in the inoculum. Small inocula of virulent and avirulent bacteria were more inhibited than large inocula. The growth of even minute inocula of virulent bacteria in bovine serum... 

Bioluminescence-based analytical assays were used to measure various analytes in nanoliter sample volumes. Nanoliter volumes of multiple bioluminescent analytical assays were deposited in an array format and lyophilized. ATP-firefly luciferase (FFL) and NADH-bacterial luciferase (BL) platform reactions were compared. We achieved parallel sample delivery via sample-hydrated membranes. A CCD camera measured the luminescent kinetics for each assay. These miniaturized assays and instruments can he prepared as micro-analytical systems to operate in point-of-care (POC) diagnostic devices. 

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