Assessment of media used for selective isolation of Aeromonas spp.

A. GAVRIEL AND A.J. LAMB. 1995. A group of selective media were evaluated for their ability to support growth and recovery of type species of the mesophilic, motile group from the genus Aeromonas . With both low and high inoculum densities Aer. hydrophila and Aer. caviae grew well on ampicillin dextrin agar, glutamate starch penicillin agar and starch glutamate ampicillin penicillin agar whereas Aer. veronii only grew with a high level of inoculum. Aeromonas sobria and Aer. schubertii displayed little growth on any of these media regardless of the inoculum size. These results indicate that environmental studies attempting to evaluate the relative distribution and abundance of the members of the motile Aeromonas group would not recover all the strains with equal effectiveness using these particular media.     

Comparison of different media for the isolation and enumeration of Aeromonas spp. in foods

Starch ampicillin agar (SA), starch glutamate ampicillin penicillin agar (SGAP) and Aeromonas medium (AM) were evaluated for enumeration of Aeromonas spp. from foods. Recovery from pure cultures of Aer. hydrophila and Aer. caviae was excellent on all media. Recovery of Aer. sobria was best on AM agar, where 95.5% were recovered, compared with 31.9% on SA agar and 33.3% on SGAP medium.     

Evaluation of 2 culture media for the isolation and enumeration of motile Aeromonas in different kinds of water

In recent years an increasing incidence of Aeromonas-related cutaneous infections and gastroenteritis has raised a serious public health problem. It appeared therefore timely to define a specific method allowing the rapid isolation and enumeration of the bacteria in their various aquatic habitats. In this line of research we have compared the growth of Aeromonas originating from different aquatic sources and raised on two media, i.e. RS-agar and PXA-agar. Whatever the aquatic system we observed that the PXA-agar medium clearly was better adapted for a quick enumeration of Aeromonas.     

The preparation of ampicillin dextrin agar for the enumeration of Aeromonas in water

Introduction of ampicillin dextrin agar (ADA) has revealed problems in details of the preparation. The final pH of the medium varied substantially between different laboratories. Measuring temperature has a pronounced effect on the pH (0·7 units lower at 50°C than at 6°C). Addition of agar during medium preparation resulted in a fall in pH of 0·5 units. If poured plates were stored in the refrigerator, the pH was reduced by 0·1–0·4 units, in particular during the first day. Recovery of Aeromonas from pure cultures and naturally polluted samples was unaffected by variation in pH between 7·1 and 8·3 but colony differentiation was optimal at a higher pH. The use of ADA at a final pH of 7·8 ± 0·2 (at 25°C) is recommended. Different types of dextrin differed in respect of solubility, fermentability and colony differentiation. Optimal results were obtained with Difco 161 and Merck 3006.     

Metal ion-catalysed hydrolysis of ampicillin in microbiological growth media

Anodic stripping voltammetry of bacterial growth medium containing copper(II) and ampicillin shows that Cu(II) is complexed by the antibiotic and that this complex decomposes to give Cu(II) complexes with ligands derived from ampicillin. At pH 7, substantial decomposition of ampicillin occurs over a few minutes, and even the very low levels of Cu(II) in Chelex-extracted medium are able effectively to catalyse the decomposition. The significance of this observation was shown during the screening of an Escherichia coli cosmid library for clones exhibiting increased resistance to Zn(II), Co(II) or Cd(II); the unexpected growth of the ampicillin-sensitive host E. coli strain on Luria-Bertani plates containing ampicillin and any of these metals was attributed to metal ion-catalysed decomposition of ampicillin. The instability of ampicillin (and other beta-lactam antibiotics) to metal ion-catalysed hydrolysis means that great care must be taken to ensure that such reactions do not occur in growth media. Furthermore, it is clear that double selection for resistance to ampicillin and metals such as Cu(II), Zn(II), Co(II) and Cd(II) is impossible.     

Sampling bias in snow leopard population estimation studies

Accurate assessments of the status of threatened species and their conservation planning require reliable estimation of their global populations and robust monitoring of local population trends. We assessed the adequacy and suitability of studies in reliably estimating the global snow leopard (Panthera uncia) population. We compiled a dataset of all the peer-reviewed published literature on snow leopard population estimation. Metadata analysis showed estimates of snow leopard density to be a negative exponential function of area, suggesting that study areas have generally been too small for accurate density estimation, and sampling has often been biased towards the best habitats. Published studies are restricted to six of the 12 range countries, covering only 0.3–0.9% of the presumed global range of the species. Re-sampling of camera trap data from a relatively large study site (c.1684 km²) showed that small-sized study areas together with a bias towards good quality habitats in existing studies may have overestimated densities by up to five times. We conclude that current information is biased and inadequate for generating a reliable global population estimate of snow leopards. To develop a rigorous and useful baseline and to avoid pitfalls, there is an urgent need for (a) refinement of sampling and analytical protocols for population estimation of snow leopards (b) agreement and coordinated use of standardized sampling protocols amongst researchers and governments across the range, and (c) sampling larger and under-represented areas of the snow leopard's global range.     

Differential media for quantitative recovery of waterborne Aeromonas hydrophila.

Because of the ubiquity of Aeromonas spp., their prevalence in drinking water, and the increasing number of reports on Aeromonas sp.-related infections, a standard method for routine and quantitative recovery had to be defined. On the basis of a survey of 10 media for recovery analysis and subsequent differentiation assays in mixed cultures, we conclude that ampicillin-dextrin agar performed the best for the recovery of Aeromonas spp. in drinking water and the differentiation by simple criteria of that genus from other common waterborne bacteria.     

Comparison of transport media for recovery of Aeromonas from clinical specimens

The efficacy of Amies, Stuart, and Cary and Blair transport media for the survival of the three Aeromonas species over 21 d was determined utilizing simulated clinical specimens of aeromonad-contaminated pus and faeces. Aeromonas sobria had the worst survival rate of the three Aeromonas species. Cary and Blair medium performed the best, as colony counts of all three Aeromonas species in simulated pus and faeces stored for 7 d were comparable to the colony counts at initial inoculation.     

Sampling bias in the determination of first and last occurrences

Background: Past studies of first and last occurrence dates of phenological events have revealed close associations with climatic parameters. Consequently, it is widely acknowledged that recent shifts in the beginning, duration or ending of such events are a response to present climate change. In addition, in recent times, there have tended to be many more observers than in earlier times, especially in urban areas. Furthermore, the number of individuals (plants or animals) observed has often changed markedly. In many situations it is not possible to obtain the average first or last occurrence date of a group of individuals, and only the most extreme occurrence is recorded. This common observational difficulty leads to sampling bias that needs to be taken into account.

Aim: Our aim is to use statistical models to quantify the sampling bias and its dependence on sample size and the variability and correlation amongst the individuals under consideration.

Methods: n-dimensional multivariate normal distribution and two-way fixed-effects analysis of variance models were developed to examine the dependence of the sampling bias on the above factors. Our results are compared with real data.

Results: For first and last occurrence observations, which are the most common index in many phenological studies, we found that changes in observational practice and sample size can, in certain circumstances, easily produce changes in bias that can swamp (or indeed reverse) any climatic change effects.

Conclusions: Our new, realistic statistical models allow the sampling bias to be quantified and calculated in terms of the number of individuals under observation, their variability and the degree of correlation between individuals.     

Comparison of different media for the enumeration of Aeromonas sp. in freshwaters

I. KERSTERS, N. SMEYERS AND W. VERSTRAETE. 1996. Ampicillin-dextrin agar (ADA), m-Aeromonas agar (mA), starch glutamate ampicillin penicillin C-glucose agar (SGAP-10C), trehalose agar (TRE) and ampicillin bile salts inositol xylose agar (MIX) were evaluated for the isolation of Aeromonas sp. from aquatic environments. Recovery of pure cultures was excellent on all media, except for Aer. sobria on SGAP-10C and MIX agars. Recovery of Aeromonas sp. from freshwaters was comparable on ADA, mA, SGAP-10C and TRE. The most selective media were SGAP-10C and ADA, which yielded an average reduction factor of more than 2.9 log. The ability to differentiate Aeromonas sp. from the background microbiota present in freshwaters was best on ADA. The present findings indicate that ADA is the most adequate medium for the selective isolation of Aeromonas sp. from freshwaters.     

Sampling bias in studies of dentoalveolar pathology in past human populations

Two subsamples of 30 subjects drawn from the same skeletal population (Monte d'Argento, Italy, Medieval age) were examined in a parallel blind study of dentoalveolar pathology, tooth wear and calculus, in order to assess the influence of sampling bias on dietary reconstruction. The effect of interobserver error was controlled and random subdivision was performed on a sample homogeneous for age and social condition. In spite of this, appreciable differences in the frequencies of some pathologies and, therefore, in dietary reconstruction were found, thus pointing to potential effectiveness of sampling bias in archaeological partial samples.     

SGAP-10C agar for the isolation and quantification of Aeromonas from water

Glutamate starch penicillin (GSP) medium was used for the simultaneous isolation of Pseudomonas and Aeromonas. Modifications to reduce the number of Pseudomonas and background flora and to improve the recovery of Aeromonas from water samples are described. The original medium was modified by adding glucose and ampicillin. The addition of 10 micrograms/l of C-glucose to the medium (SGAP-10C) permitted better recuperation of stressed cells of aeromonads and the ampicillin reduced the numbers of Pseudomonas. The best temperature for the recovery of aquatic aeromonads was 28 degrees C. The recovery of different species of Aeromonas on SGAP-10C was 93%. The selectivity of the medium was validated because 95.5% of 28 colonies tested with an Aeromonas-like morphology belonged to the genus Aeromonas. Moreover, when 45 strains of different genera were cultured on the medium, only Vibrio alginolyticus presented a confusing morphology. When the SGAP-10C was compared with GSP with 45 river samples, the new medium gave a significantly better recovery of Aeromonas spp., especially when large numbers of Pseudomonas spp. were present. SGAP-10C used at 28 degrees C and 48 h was an efficient selective medium for the isolation of Aeromonas from fresh waters.     

SGAP-10C agar for the isolation and quantification of Aeromonas from water

Glutamate starch penicillin (GSP) medium was used for the simultaneous isolation of Pseudomonas and Aeromonas . Modifications to reduce the number of Pseudomonas and background flora and to improve the recovery of Aeromonas from water samples are described. The original medium was modified by adding glucose and ampicillin. The addition of 10 μg/l of C-glucose to the medium (SGAP-10C) permitted better recuperation of stressed cells of aeromonads and the ampicillin reduced the numbers of Pseudomonas . The best temperature for the recovery of aquatic aeromonads was 28°C. The recovery of different species of Aeromonas on SGAP-10C was 93%. The selectivity of the medium was validated because 95·5% of 28 colonies tested with an Aeromonas -like morphology belonged to the genus Aeromonas . Moreover, when 45 strains of different genera were cultured on the medium, only Vibrio alginolyticus presented a confusing morphology. When the SGAP-10C was compared with GSP with 45 river samples, the new medium gave a significantly better recovery of Aeromonas spp., especially when large numbers of Pseudomonas spp. were present. SGAP-10C used at 28°C and 48 h was an efficient selective medium for the isolation of Aeromonas from fresh waters.     

Comparison of media for isolation of poultry intestinal bacteria

The effects of medium composition, incubation temperature, and length of incubation were determined for recovery of the predominant intestinal bacteria from turkey poults. Incubation of recovery media at 41 degrees C resulted in significantly higher counts than at 37 degrees C. In 2- and 3-week-old turkey poults. RGCAP-30, RGCAP-10, and RGCA-30 gave the highest recoveries of cecal bacteria. M98-5 was less effective and brain heart infusion agar was definitely inadequate. However, there was no significant difference between RGCAP-30 and brain heart infusion agar for recovery of duodenal bacteria. In older birds (6 weeks of age), M98-5 was equal or superior to the RGCA-based media. The choice of a primary isolation medium is thus dependent on the site to be sampled and the age of the bird.     

Comparison of media for the isolation of Enterobacter sakazakii

Enterobacter sakazakii is associated with neonatal infections and is occasionally present at low levels (<1 CFU/g) in powdered infant formula milk (IFM). It has been previously reported that some E. sakazakii strains do not grow in standard media for Enterobacteriaceae and coliform bacteria; therefore, a reliable method is needed for recovery of the organism. Three E. sakazakii enrichment broths-Enterobacteriaceae enrichment broth (EE), E. sakazakii selective broth (ESSB), and modified lauryl sulfate broth (mLST)-were compared with a novel broth designed for maximum recovery of E. sakazakii, E. sakazakii enrichment broth (ESE). One hundred seventy-seven strains (100%) grew in ESE, whereas between 2 and 6% of strains did not grow in EE, mLST, or ESSB. E. sakazakii possesses alpha-glucosidase activity, and a number of selective, chromogenic agars for E. sakazakii isolation based on this enzyme have been developed. E. sakazakii isolation agar produced fewer false-positive colonies than did Druggan-Forsythe-Iversen agar. However, the latter supported the growth of more E. sakazakii strains. It was also determined that 2% of E. sakazakii strains did not produce yellow pigmentation on tryptone soya agar at 25 degrees C, a characteristic frequently cited in the identification of E. sakazakii. The recovery of desiccated E. sakazakii (0.2 to 2000 CFU/25 g) from powdered IFM in the presence of a competing flora was determined with various enrichment broths and differential selective media. Current media designed for the isolation and presumptive identification of E. sakazakii do not support the growth of all currently known E. sakazakii phenotypes; therefore, improvements in the proposed methods are desirable.     

Nutrient media for the isolation and accumulation of Listeria

Dried culture media for the isolation and accumulation of Listeria from pathological material and foodstuffs have been developed. The media are suitable for use in bacteriological and sanitary-hygienic practice. The optimum nutrient base has been selected: dried broth (from sprat hydrolysate), produced by the Research and Manufacturing Amalgamation "Culture Media". The optimum concentrations of ingredients, stimulating the growth of Listeria and inhibiting the growth of associated microbes, have been experimentally established. The samples of died accumulation and isolation culture media ensuring the growth of L. monocytogenes, diluted 10(-6), after 24-hour incubation at 37 degrees C have been obtained. The possibility of using these media for the bacteriological diagnosis of listeriosis in pregnant women has been demonstrated.