A rapid method for the determination of the anti-interferon activity of bacteria]

Methods for the rapid and immediate determination of the anti-interferon activity of bacteria have been developed. The use of these methods makes it possible to reduce the time of determination from 48 hours to 24 and 7 hours. The proposed methods require no additional production costs, while their sensitivity and specificity are not inferior to those ensured by the classical method. These methods are recommended for the etiological diagnosis of diseases caused by opportunistic enterobacteria.     

New rapid and simple methods for detection of bacteria and determination of their antibiotic susceptibility by using phage mutants

Three new methods applying a novel approach for rapid and simple detection of specific bacteria, based on plaque formation as the end point of the phage lytic cycle, are described. Different procedures were designed to ensure that the resulting plaques were derived only from infected target bacteria ("infectious centers"). (i) A pair of amber mutants that cannot form plaques at concentrations lower than their reversion rate underwent complementation in the tested bacteria; the number of plaques formed was proportional to the concentration of the bacteria that were coinfected by these phage mutants. (ii) UV-irradiated phages were recovered by photoreactivation and/or SOS repair mediated by target bacteria and plated on a recA uvrA bacterial lawn in the dark to avoid recovery of noninfecting phages. (iii) Pairs of temperature-sensitive mutants were allowed to coinfect their target bacteria at the permissive temperature, followed by incubation of the plates at the restrictive temperature to avoid phage infection of the host cells. This method allowed the omission of centrifuging and washing the infected cells. Only phages that recovered by recombination or complementation were able to form plaques. The detection limit was 1 to 10 living Salmonella or Escherichia coli O157 cells after 3 to 5 h. The antibiotic susceptibility of the target bacteria could also be determined in each of these procedures by preincubating the target bacteria with antibiotic prior to phage infection. Bacteria sensitive to the antibiotic lost the ability to form infectious centers.     

A rapid, guantitative, and selective estimation of radioactively labeled peptidoglycan in gram-positive bacteria

The Park and Hancock procedure for the isolation of bacterial cell wall peptidoglycans has been modified. The method developed permits rapid and accurate determination of small quantities of radioactively labeled material. The principal modifications consist of carrying out the procedure on glass-fiber filters after a vigorous hydrolysis of the cells with TCA (96°C, 30 min) and using Pronase in place of trypsin.     

Evaluation of a commercial fluorochromic system for the rapid detection and estimation of wine lactic acid bacteria by DEFT

A commercial fluorochromic system was evaluated for the rapid detection of lactic acid bacteria in fortified wines by the epifluorescent filter technique (DEFT). The viability test used, employing the fluorescence dyes SYTO 9 and propidium iodide, was able to detect and clearly differentiate viable from non-viable cells (killed with a 50% v/v ethanol solution). A good overall agreement ( r ₌ 0·92) was obtained between the DEFT count and the plate count in the range studied (5 × 10²–4 × 10⁹ cells ml⁻¹). Wine components which might otherwise interfere with the method could be removed by including simple wash steps in the protocol. This measure proved critical to the success of the procedure. For practical purposes, the rapid method studied seems to be a good alternative to the traditional cultural methods as part of quality control programmes in wine making. It may also be useful when studying the efficacy of certain treatments in the elimination of wine bacterial contaminants.     

Modified microdilution antimicrobial susceptibility test for anaerobic bacteria

A modified microdilution antimicrobial susceptibility test is described for anaerobic bacteria. The microdilution procedure at 24 and 48 h of incubation was compared with agar dilution using Wilkins-Chalgren (WC) broth and agar respectively. Results showed a total of 12.2% minor and major discrepancies with the microdilution test at 24 h incubation and 7.8% at 48 h. If anaerobic isolates are to be tested for antimicrobial susceptibility, then the 24-h microdilution procedure is an acceptable alternative to agar dilution.     

Interval estimation of genetic susceptibility for retrospective case-control studies

Background

This article describes classical and Bayesian interval estimation of genetic susceptibility based on random samples with pre-specified numbers of unrelated cases and controls.

Results

Frequencies of genotypes in cases and controls can be estimated directly from retrospective case-control data. On the other hand, genetic susceptibility defined as the expected proportion of cases among individuals with a particular genotype depends on the population proportion of cases (prevalence). Given this design, prevalence is an external parameter and hence the susceptibility cannot be estimated based on only the observed data. Interval estimation of susceptibility that can incorporate uncertainty in prevalence values is explored from both classical and Bayesian perspective. Similarity between classical and Bayesian interval estimates in terms of frequentist coverage probabilities for this problem allows an appealing interpretation of classical intervals as bounds for genetic susceptibility. In addition, it is observed that both the asymptotic classical and Bayesian interval estimates have comparable average length. These interval estimates serve as a very good approximation to the "exact" (finite sample) Bayesian interval estimates. Extension from genotypic to allelic susceptibility intervals shows dependency on phenotype-induced deviations from Hardy-Weinberg equilibrium.

Conclusions

The suggested classical and Bayesian interval estimates appear to perform reasonably well. Generally, the use of exact Bayesian interval estimation method is recommended for genetic susceptibility, however the asymptotic classical and approximate Bayesian methods are adequate for sample sizes of at least 50 cases and controls.     

A method for rapid abundance estimation of semiplanktonic meiofauna

Many meiofaunal copepods and plathelminths enter the tidal waters at night thus exhibiting a life-style intermediate between benthic and planktonic. At the same time, ostracods may leave their interstitial dwelling and move across the sediment surface. In laboratory experiments, the percentage of plathelminth populations emerging from the sediment varied with the species, temperature, light conditions, and the dimensions of the sediment cores studied, but not with tidal level, season, ambient density of conspecifics, or the sediment composition. Therefore, the swimming activity may be utilised for extraction of semiplanktonic meiofauna provided that the extraction procedure is standardised with respect to temperature, light and core size. For free-living plathelminths from the Wadden Sea intertidal a robust standard procedure is as follows: sediment cores 1.6 cm in diameter (2 cm² surface area) and 3 cm deep are fitted into cylindrical containers and submerged into aquaria containing filtered seawater (ambient salinity, room temperature, darkness) for 24 h. The sediment containers are then removed and the aquarian water filtered through appropriate meshes; the residue contains the emergent faunal component. For plathelminths, this procedure reduces sorting time by some 90% compared with the standard shaking–decantation method and thus makes it possible to process a high number of samples in a short time. Similar procedures may be developed for copepods and epibenthic ostracods. Received in revised form: 28 August 2000 Electronic Publication     

Riboprints as a tool for rapid preliminary identification of sphingomonads

Fourtythree strains of the genus Sphingomonas and close relatives were subjected to riboprint analyses generated after digestion of genomic DNA with the restriction enzyme EcoRI and hybridization with E. coli rrnB operon. The majority of strains were characterized by a complex banding pattern in the riboprints. High degrees of similarities in the riboprints were only observed among strains of the same species such as S. yanoikuyae, S. aromaticivorans, S. subarctica and S. chlorophenolica. Strains of different species including close phylogenetic relatives such as S. asaccharolytica, S. mali and S. pruni were easily distinguished by the differences in the riboprints even after visual evaluation. Thus, our data demonstrate that riboprint analysis is useful for preliminary identification of new sphingomonad isolates at the species level.     

Standardization of antimicrobial disc susceptibility test for anaerobic bacteria.

Both agar diluiton and agar diffusion tests with 8 clinically useful or potentially useful antimicrobial agents were performed with 74 strains of Bacteroides fragilis. Correlation of results obtained by the two methods and applicability of the single disc test to the measurement of antimicrobial susceptibility of anaerobes were analyzed. Prediction of susceptibility, intermediate susceptibility, and resistance of anaerobic bacteria to antimicrobial agents, based on the measurement of inhibition zone diameter, appeared to be satisfactory generally.     

Simple and rapid method for disruption of bacteria for protein studies

A simple and rapid method was developed for the extraction of proteins from both pathogenic and nonpathogenic bacteria. The method involves the treatment of cells with acetone followed by sodium dodecyl sulfate extraction of cellular proteins. Polyacrylamide gel electrophoresis revealed that the protein composition of extracts made by this method was comparable to that of extracts made by established methods, namely, sonication and agitation with beads. This technique has been successfully applied to the extraction of proteins from a wide variety of bacteria, including pathogens.     

A simple and rapid method for screening amylolytic bacteria

A laboratory class was designed for the study of the ecology of amylolytic bacteria in soil, although other sources may be equally suitable for this purpose. Groups of three students carried out the following: (a) preparation and sterilization of medium and plates, (b) collection and preparation of soil samples, spreading the samples on the plates, (c) incubation of the plates at 37 degrees C overnight, a further 1 h incubation at 60 degrees C to observe amylolytic activity due to thermophilic bacteria, and (d) interpretation and discussion of the results. These tasks are accomplished in two periods of 4h on consecutive days. No sophisticated instruments are required for these experiments, which can be carried out in three classes of 4h each. On the first day the students prepare culture media, buffers and reagents, as well as collect and grow soil samples. The second day is spent for both taxonomic identification of colonies and the HAI determination.