Nonselective Agar Media Research Articles

Impact of preculture temperature on peracetic acid-induced inactivation and sublethal injury of L. monocytogenes and subsequent growth potential of single cells

The disinfectant peracetic acid (PAA) that is used in the food industry can cause sublethal injury in L. monocytogenes. The effect of preculture temperature on the inactivation and sublethal injury of L. monocytogenes cells due to PAA was evaluated by plating on non-selective and selective agar medium supplemented with 5 % (w/v) NaCl. L. monocytogenes cells were precultured at 30 °C, 20 °C or 4 °C, and the former was used as reference temperature. Preculture of cells at 20 °C or 4 °C and subsequent exposure to PAA at the respective growth temperatures caused higher injury compared to cells grown at 30 °C and exposed to PAA 20 °C and PAA 4 °C, respectively. Survival was also affected by the preculture temperature; 20 °C-grown cultures resulted in lower survival at PAA 20 °C. Nevertheless, preculture at 4 °C resulted in a similar number of surviving cells when exposed to PAA 4 °C compared to cells precultured at 30 °C and exposed to PAA at 4 °C. Flow cytometry was subsequently used to quantify outgrowth capacity of stressed and sublethal damaged populations following sorting of single cells in nutrient rich medium (Tryptone soy broth supplemented with yeast extract [TSBY]). PAA treatment affected the outgrowth of L. monocytogenes at single-cell level resulting in increased outgrowth-times reflecting higher single cell heterogeneity. To conclude, the response of L. monocytogenes when exposed to PAA depended on the preculture conditions, and the highly heterogeneous outgrowth potential of PAA-injured cells may affect their detection accuracy and pose a food safety risk.

Prevalence and antibiotic susceptibility of neisseria gonorrhoeae in HIV positive and hiv negative cases at a tertiary care hospital

Introduction: Gonorrhea is one of the most common sexually transmitted infections (STIs) in developing countries, and the emergence of resistance to antimicrobial agents in Neisseria gonorrhoeae is a major hindrance in the control of gonorrhea. Over the past decade, Neisseria gonorrhoeae has developed resistance against commonly used antibiotics. Method: This was a prospective study done for a period of 3 years, both in men and women with the age group of (15 – 50 years) with any signs and symptoms for STIs were included. Discharge specimens like urethral, vaginal and endocervical were collected under strict aseptic conditions and smears were prepared for Gram staining and culture was done on selective and non-selective agar media. Antibiotic susceptibility was done according to Clinical and Laboratory Standards Institute guidelines. Results: Antibiotic resistance was higher in gonorrheal patients co-infected with HIV. The overall prevalence of gonorrhoea and its co-infection with HIV was 9% and 23.6% respectively. Highest resistance was seen with Penicillin, Tetracycline, Nalidixic acid, Ciprofloxine, Cefotaxime and Cefoxitin in both positive and negative cases of HIV. Conclusion: Periodic monitoring of antimicrobial sensitivity profile will help in detecting change in susceptibility pattern and the emergence of drug-resistant strains. Key words: Neisseria gonorrhoeae, Sexually Transmitted Infections (STI), Human Immune Deficiency Virus (HIV), Antibiotics, Resistance

Growth of Stressed Strains of Four Non-O157 Shiga Toxin-Producing Escherichia coli Serogroups in Five Enrichment Broths

The purpose of this study was to evaluate (i) the behavior of several strains of non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O103, O111, and O145) exposed to different stress conditions and (ii) the growth dynamics of stressed and nonstressed non-O157 STEC cells in five enrichment media. STEC strains were exposed to acid, cold, and freeze stresses. Lethal and sublethal injuries were determined by plating in parallel on selective and nonselective agar media. Freeze stress (8 days, -20°C) caused the most lethal (95.3% ± 2.5%) injury, as well as the most sublethal (89.1% ± 8.8%) injury in the surviving population. Growth of stressed and nonstressed pure cultures of non-O157 STEC on modified tryptic soy broth, buffered peptone water (BPW), BPW with sodium pyruvate, Brila, and STEC enrichment broth (SEB) was determined using total viable counts. To compare growth capacities, growth after 7 and 24 h of enrichment was measured; lag phases and maximum growth rates were also calculated. In general, growth on BPW resulted in a short lag phase followed by a high maximum growth rate during the enrichment of all tested strains when using all three stress types. Furthermore, BPW ensured the highest STEC count after 7 h of growth. Supplementing the medium with sodium pyruvate did not improve the growth dynamics. The two selective media, Brila and SEB, were less efficient than BPW, but Brila’s enrichment performance was remarkably better than that of SEB. This study shows that irrespective of the effect of background flora, BPW is still recommended for resuscitation of non-O157 STEC.

S104 The culture microbiome in the lungs of patients with COPD

Introduction and AimsPrevious studies have shown that the lungs of Cystic Fibrosis (CF) and bronchiectasis (BE, not caused by CF) patients are colonised by a range of aerobic and anaerobic...

Characterization of bacterial isolates from rotting potato tuber tissue showing antagonism to Dickeya sp. biovar 3 in vitro and in planta

Possibilities for biocontrol of biovar 3 Dickeya sp. in potato were investigated, using bacteria from rotting potato tissue isolated by dilution plating on nonselective agar media. In a plate assay, 649 isolates were screened for antibiosis against Dickeya sp. IPO2222 and for the production of siderophores. Forty‐one isolates (6·4%) produced antibiotics and 112 isolates (17·3%) produced siderophores. A selection of 41 antibiotic‐producing isolates and 41 siderophore‐producing isolates were tested in a potato slice assay for control of the Dickeya sp. Isolates able to reduce rotting of potato tuber tissue by at least 50% of the control were selected. Isolates were characterized by 16S rDNA analysis as Bacillus, Pseudomonas, Rhodococcus, Serratia, Obesumbacterium and Lysinibacillus genera. Twenty‐three isolates belonging to different species and genera, 13 producing antibiotics and 10 producing siderophores, were further characterized by testing acyl‐homoserine lactone (AHL) production, quorum quenching, motility, biosurfactant production, growth at low (4·0) and high (10·0) pH, growth at 10°C under aerobic and anaerobic conditions and auxin production. In replicated greenhouse experiments, four selected antagonists based on the in vitro tests were tested in planta using wounded or intact minitubers of cv. Kondor subsequently inoculated by vacuum infiltration with an antagonist and a GFP (green fluorescent protein)‐tagged biovar 3 Dickeya sp. strain. A potato endophyte A30, characterized as S. plymuthica, protected potato plants by reducing blackleg development by 100% and colonization of stems by Dickeya sp. by 97%. The potential use of S. plymuthica A30 for the biocontrol of Dickeya sp. is discussed.

Development of a semiselective medium for detection of Mycosphaerella pinodes in soil, plant debris and seed

Garden pea leaf blight, caused by Mycosphaerella pinodes, is a severe disease of peas worldwide. Detection of this pathogen from seed, plant debris and soil is difficult using non-selective agar media. This study was conducted to develop a semiselective medium for the isolation of this pathogen. Of the 14 carbon sources and 18 nitrogen sources tested, maltose and soluble starch were the most suitable carbon sources and casein was the most important nitrogen source for mycelial growth of three isolates of M. pinodes. Among 13 pesticides and two antibiotics tested, atrazine, mepronil, paraquat, propamocarb and TMTD showed no adverse effect on growth of M. pinodes. Based on these results, a starch–casein semiselective medium (designated as the SC-semiselective medium) was developed by amendment of 1 L Czapek–Dox agar with 30 g soluble starch, 2 g casein, 25 μg mL-1 propamocarb, 10 μg mL-1 benomyl, 50 μg mL-1 mepronil, 300 μg mL-1 atrazine, 100 μg mL-1 streptomycin sulfate and 200 μg mL-1 neomycin sulfate. This SC-semiselective medium was effective for detection of M. pinodes in artificially and naturally infested field soils, diseased seeds, leaves, and stems of peas. It should have potential for applications in research on biology, ecology and control of M. pinodes in peas.

EUCAST Definitive Document EDef 7.1: method for the determination of broth dilution MICs of antifungal agents for fermentative yeasts: Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST)∗

INTRODUCTIONAntifungal susceptibility tests are performed onfungi that cause disease, especially if they belongto a species exhibiting resistance to commonlyused antifungal agents. Antifungal susceptibilitytesting is also important for resistance surveil-lance, for epidemiological studies and forcomparing the in-vitro activity of new and exist-ing agents.Dilution methods are used to establish theMICs of antimicrobial agents. These are thereference methods for antimicrobial susceptibilitytesting, and are used mainly to establish theactivity of a new antifungal agent, to confirm thesusceptibility of organisms that give equivocalresults in routine tests, and to determine thesusceptibility of fungi where routine dilution testsmay be unreliable. Fungi are tested for theirability to produce visible growth in microdilutionplate wells containing broth culture media andserial dilutions of the antifungal agents (brothmicrodilution). The MIC is defined as the lowestconcentration (in mg⁄L) of an antifungal agentthat inhibits the growth of a fungus. The MICprovides information concerning the susceptibil-ity or resistance of an organism to the antifungalagent and can help in making correct treatmentdecisions.The method described in this document isintended for testing the susceptibility ofyeasts that cause clinically significant infections(primarily Candida spp.). The method encom-passes only those yeasts that are able to fermentglucose. Thus, the susceptibility of non-fermenta-tive yeasts, e.g., Cryptococcus neoformans, cannotbe determined by the current procedure, and themethod is not suitable for testing the yeast formsof dimorphic fungi.SCOPEThe standard method described in this documentprovides a valid method for testing the suscepti-bility of glucose-fermenting yeasts to antifungalagents by determination of the MIC. MICs indi-cate the activity of a given antifungal drug underthe described test conditions, and can be used inmaking decisions concerning patient manage-ment after taking into account other factors, e.g.,pharmacokinetics, pharmacodynamics and resis-tance mechanisms. The MIC also allows fungi tobe categorised as ‘susceptible’ (S), ‘intermediate’(I) or ‘resistant’ (R) to a drug. In addition, MICdistributions can be used to define wild-type ornon-wild-type fungal populations.This method is intended primarily to facilitatean acceptable degree of conformity, i.e., agree-ment within specified ranges among laboratories,in measuring the susceptibility of yeasts to anti-fungal agents. The method is designed to be easyto perform, rapid, economical, and suitable forreading by microdilution plate readers in order toallow direct transfer, storage and manipulation ofthe data by computer. The method is alsointended to yield results that are concordantwith those obtained using the procedure

Activity and Community Composition of Denitrifying Bacteria in Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-Using Solid-phase Denitrification Processes

Two laboratory-scale solid-phase denitrification (SPD) reactors, designated reactors A and B, for nitrogen removal were constructed by acclimating activated sludge with pellets and flakes of poly(3-hydoxybutyrate-co-3hydroxyvalerate) (PHBV) as the sole added substrate under denitrifying conditions, respectively. The average denitrification rate in both reactors was 60 mg NO3 � -N g �1 (dry wt) h �1 under steady-state conditions, whereas washed sludge taken from the reactors showed an average denitrification rate of 20 mg NO3-N g �1 (dry wt) h �1 with fresh PHBV as the sole substrate. The difference in the denitrification rate between the two might be due to the bioavailability of intermediate metabolites as the substrate for denitrification, because acetate and 3-hydroxybutyrate were detected in the reactors. Most of the predominant denitrifiers isolated quantitatively by the platecounting method using non-selective agar medium were unable to degrade PHBV and were identified as members of genera of the class Betaproteobacteria by studying 16S rRNA gene sequence information. nirS and nosZ gene clone library-based analyses of the microbial community from SPD reactor A showed that most of the nirS and nosZ clones proved to be derived from members of the family Comamonadaceae and other phylogenetic groups of the Betaproteobacteria. These results suggest that the efficiency of denitrification in the PHBV-SPD process is affected by the availability of intermediate metabolites as possible reducing-power sources as well as of the solid substrate, and that particular species of the Betaproteobacteria play the primary role in denitrification in this process.

Characterization of the Microbial Community and Culturable Denitrifying Bacteria in a Solid-phase Denitrification Process Using Poly(ε-caprolactone) as the Carbon and Energy Source

A laboratory-scale solid-phase denitrification process for nitrogen removal was constructed by acclimating sewage activated sludge with poly(ε-caprolactone) (PCL) as the sole substrate under denitrifying conditions. The sludge thus acclimated exhibited a nitrogen removal rate of 6-7 mg NO3-N g-1 h-1 with PCL as the sole source of reducing power. The microbial community and the distribution of denitrifying bacteria in this process were studied by rRNA-targeted fluorescence in situ hybridization, quinone profiling and standard cultivation methods. The culture-independent molecular and biomarker approaches demonstrated that members of the class Alphaproteobacteria predominated and those of Betaproteobacteria were the second most abundant group of bacteria in the process. The plate counts of denitrifying bacteria with a non-selective agar medium accounted for 6% of the total count and 10% of the direct viable count on average. The most probable number (MPN) obtained with PCL-containing medium under denitrifying conditions was one order of magnitude lower than the plate count. Most of the denitrifying isolates from the MPN enrichment tubes and 10% of the predominant denitrifying bacteria isolated by the plate-counting method were capable of degrading PCL. 16S rRNA gene sequence comparisons showed that the greater majority of the predominant denitrifiers were members of the genera Comamonas, Diaphorobacter and Paracoccus. All of the PCL-degrading denitrifying strains isolated were assigned to a previously unknown species of the genus Comamonas. The results of this study suggest that, apart from their PCL-degrading capacity, members of Alphaproteobacteria and Betaproteobacteria are mainly responsible for nitrogen removal in the PCL-acclimated denitrification process.

The Acid-Tolerant Microbiota Associated with Plaque from Initial Caries and Healthy Tooth Surfaces

The intent of this study was to compare the inherent acid tolerance of bacteria in samples of dental plaque from tooth sites in subjects with and without initial caries. Plaque was collected from approximal surfaces showing early enamel caries and from healthy tooth surfaces in the same subjects, as well as from enamel surfaces of caries-free individuals. In addition to plating on blood agar, the plaque samples were plated directly on non-selective solid agar medium buffered to pH 7.0, 6.0, 5.5, 5.0, 4.5 and 4.0 to avoid any loss of adaptation to acid during primary isolation of plaque bacteria. The results showed that approximately 50% of the total cultivable plaque microbiota from caries, as well as healthy tooth sites, was able to grow at pH 5.5 and 1% at pH 5.0, pH values regarded as critical for the demineralization of tooth enamel. At pH 5.0, members of the genus Streptococcus were the dominant group, but mutans streptococci accounted for less than half of the streptococcal viable count. The other acid-tolerant streptococcal isolates included Streptococcus anginosus, Streptococcus constellatus, Streptococcus gordonii, Streptococcus intermedius, Streptococcus mitis, Streptococcus oralis,Streptococcus salivarius and Streptococcus sanguis. Analysis of the results indicated that the mutans streptococci in dental plaque were highly variable with respect to acid tolerance, and that both caries and healthy sites harboured significant numbers of mutans streptococci that were not acid-tolerant.

Pretreatment effect on inactivation of Escherichia coli O157:H7 inoculated beef jerky

Changes in inoculated Escherichia coli O157:H7 populations were determined during drying (62.5°C, 10 h) of whole muscle beef jerky slices pretreated by: (1) immersing in boiling water (94°C, 15 s), then marinating (4°C, 24 h); (2) seasoning (4°C, 24 h), then immersing in a pickling brine (78°C, 90 s); (3) immersing in a vinegar/water (750/750 mL) solution (57.5°C, 20 s), then marinating (4°C, 24 h); and (4) marinating (4°C, 24 h), then immersing in a vinegar/water (750/750 mL) solution (57.5°C, 20 s). Samples were analysed (bacterial enumeration with selective and nonselective agar media, pH, and a w) following inoculation, each preparation step, and at 0, 4, 6, 8 and 10 h of drying. While all pre-drying treatments resulted in significant ( P<0.05) bacterial reductions, treatment 2 resulted in the greatest pre-drying reduction (3.1–4.1 log cfu/cm 2) and the highest overall reduction at 10 h drying (5.7–5.8 log cfu/cm 2). Total reductions for treatments 1, 3 and 4 after 10 h drying were 4.3–4.5, 4.9–5.2 and 4.7–4.8 log cfu/cm 2, respectively. Bacterial populations declined to <1.0 log cfu/cm 2 after 30 d storage and remained at this level throughout 90 d storage. These results should be useful in developing guidelines for jerky preparation by consumers and processors.

EFFECT OF MARINADE AND DRYING TEMPERATURE ON INACTIVATION OF ESCHERICHIA COLI O157:H7 ON INOCULATED HOME DRIED BEEF JERKY

ABSTRACT Beef slices were inoculated (5.7–7.5 log CFU/cm2) with a 4‐strain composite of E. coli O157:H7, stored (4C, 24 h), marinated (4C, 24 h), dried for 10 h at 62.5C or 68.3C, and stored for 90 days at 21C. Unmarinated beef slices dried for 10 h at 62.5C were used to determine the relative contribution of the marinate versus temperature treatment in the 62.5C trials. Samples were analyzed (bacterial enumeration with selective and nonselective agar media, pH, and aw) following inoculation, marinating, at 4, 6, 8 and 10 h of drying, and after 30, 60 and 90 days of storage. Marination resulted in slight changes in bacterial populations (−0.3 to + 0.6 log CFU/cm2), but did not enhance bacterial reduction during drying. For all treatments, most bacterial reductions occurred in the first 4 h of drying, with little reduction thereafter. After 10 h of drying, bacterial reductions were 3.2–3.4 log CFU/cm2 for unmarinated beef slices dried at 62.5C. Reductions of 2.2 and 3.0–4.6 log CFU/cm2 were achieved in marinated jerky slices dried at 62.5C and 68.3C, respectively. No treatment resulted in the recommended 5‐log reduction at the end of 10 h drying. However, bacteria did become undetectable by direct plating (&lt;10 CFU/cm2) following 30 days of storage in all treatments except the unmarinated beef slices plated on tryptic soy agar (TSA). Additional work is needed to develop procedures for adequate destruction of E. coli O157:H7 during drying of beef jerky.

Culturability as an indicator of succession in microbial communities.

Successional theory predicts that opportunistic species with high investment of energy in reproduction and wide niche width will be replaced by equilibrium species with relatively higher investment of energy in maintenance and narrower niche width as communities develop. Since the ability to rapidly grow into a detectable colony on nonselective agar medium could be considered as characteristic of opportunistic types of bacteria, the percentage of culturable cells may be an indicator of successional state in microbial communities. The ratios of culturable cells (colony forming units on R2A agar) to total cells (acridine orange direct microscopic counts) and culturable cells to active cells (reduction of 5-cyano-2,3-ditolyl tetrazolium chloride) were measured over time in two types of laboratory microcosms (the rhizosphere of hydroponically grown wheat and aerobic, continuously stirred tank reactors containing plant biomass) to determine the effectiveness of culturabilty as an index of successional state. The culturable cell:total cell ratio in the rhizosphere decreased from approximately 0.25 to less than 0.05 during the first 30-50 days of plant growth, and from 0.65 to 0.14 during the first 7 days of operation of the bioreactor. The culturable cell:active cell ratio followed similar trends, but the values were consistently greater than the culturable cell:total cell ratio, and even exceeded I in early samples. Follow-up studies used a cultivation-independent method, terminal restriction fragment length polymorphisms (TRFLP) from whole community DNA, to assess community structure. The number of TRFLP peaks increased with time, while the number of culturable types did not, indicating that the general decrease in culturability is associated with a shift in community structure. The ratio of respired to assimilated C-14-labeled amino acids increased with the age of rhizosphere communities, supporting the hypothesis that a shift in resource allocation from growth to maintenance occurs with time. Results from this work indicate that the percentage of culturable cells may be a useful method for assessing the successional state of microbial communities.

Detection of Xanthomonas sp., the causal agent of onion bacterial blight, in onion seeds using a newly developed semi-selective isolation medium

Onion bacterial blight, caused by Xanthomonas sp., is a potentially severe disease in several tropical and subtropical areas. Although little research has been undertaken on this pathosystem, seed transmission of the pathogen has been hypothesized. Because of an important bacterial microflora naturally associated with onion seeds, detection of the pathogen is difficult using non-selective agar media. A new semi-selective medium, whose selectivity was obtained by a combination of four antibiotics, was developed. The new NCTM1 medium contained (per liter) yeast extract 7 g, peptone 7 g, glucose 7 g, agar 15 g, neomycin 10 mg, cephalexin 30 mg, trimethoprime 3 mg, pivmecillinam 100 mg and propiconazole 20 mg. Plating efficiencies, using 16 pure cultures of the pathogen, ranged from 79% to 142%, with an average of 110% compared to the basal medium. All onion Xanthomonas sp. strains from several countries grew on NCTM1 medium. The pathogen was repeatedly isolated using this medium from seed samples containing approximately 106 saprophytic bacteria per gram, as well as from symptomless plant material. Xanthomonas sp. was detected only in seeds originating from one infected seed production site. This is the first report of selective isolation of Xanthomonas sp. from onion seeds. NCTM1 medium should be a valuable tool to study the ecology and epidemiology of Xanthomonas sp. causing onion bacterial blight.

Fungi in therapy-resistant apical periodontitis.

The occurrence of yeasts in 967 microbiological endodontic samples taken from root canals in persistent endodontic infections was studied. The sampling was done by general practitioners in various parts of Finland from root canal infections which did not respond favourably to standard conservative therapy. The samples were cultivated aerobically on a non-selective enriched horse blood agar medium, on TSBV agar medium in 5% CO2 and anaerobically on horse blood agar medium. Micro-organisms were found in 692 of the samples while 275 showed no growth. Forty-eight fungi were isolated from 47 samples which is 7% of the culture-positive samples. Twenty yeast strains were identified further by their colony morphology, growth and cellular characteristics and patterns of carbohydrate assimilation. All isolates except one belonged to the genus Candida. Candida albicans was the most common species. C. glabrata was found together with C. albicans in one sample. C. guilliermondii, C. inconspicua and Geotrichum candidum were each isolated once. Yeasts were found in pure culture in six samples and together with bacteria in 41 samples. In all the samples except two, the accompanying facultative bacteria were Gram positive. The most frequent of them were alpha- and non-haemolytic Streptococcus species which were found in 31 samples. Anaerobic bacteria were isolated together with yeasts from 12 root canals. They included both Gram positive species such as Peptostreptococcus micros and Gram negative species such as Fusobacterium nucleatum. The regular isolation of yeasts, also in pure culture, indicates that yeasts may have an important role in cases of apical periodontitis persisting after conventional treatment.

Variations in the predominant cultivable microflora of dental plaque at defined subsites on approximal tooth surfaces in children

The distribution and composition of the resident microflora were determined in approximal gingival margin plaque from 21 premolars extracted from schoolchildren (mean age 12.0 ± 1.8 yr). Indigo carmine (5% w/v) was used to visualize plaque to facilitate sampling. About 1 mm 2 of plaque was removed from sites away from (A), to the side of (S), and below (B) the contact area. Plaque samples were dispersed, serially diluted, and cultured on selective and non-selective agar media. An average of seven to nine species was found at each subsite. Streptococcus and Actinomyces were subdivided on the basis of a range of biochemical tests. The predominant Actinomyces and streptococcal species at most subsites were A. naeslundii and Strep. mitis biovar I. A. naeslundii and A. odontolyticus were isolated more often at subsite B (90.5 and 57.1%, respectively), and A. israelii at subsite S (66.7%). Strep. mitis I and Strep. sanguis were found more frequently at subsite S (76.2 and 66.7%, respectively), whereas Strep. mutans, Strep. sobrinus, Strep. gordonii and Veillonella spp. were recovered most commonly from subsite B (85.7, 33.3, 38.1 and 76.2%, respectively). The isolation frequencies of Strep. mutans and Strep. sobrinus were significantly higher at subsite B (A < B, p < 0.01 and p > 0.05, respectively), Veillonella spp. were significantly higher at subsites B and S (A < B, p > 0.01; B > S, p < 0.05), while Neisseria spp. were most common at subsite A (A > B, p < 0.03). IgA1 protease-producing species were found at each subsite, but they formed only a small proportion of the total Streptococcus population. This study has shown that local variations were evident at different subsites, both with respect to species prevalence and to proportions of each species within each subsite. The population shifts in gingival margin plaque appear to relate to the location of plaque in relation to the most caries-prone site below the contact area B.

Meiotic and mitotic stability of transforming DNA in the phytopathogenic fungus Magnaporthe grisea

Magnaporthe grisea was transformed with cosmid pAN7-2 encoding hygromycin B resistance but containing no homology with the M. grisea genome. Rearrangement of the integrated DNA was detected in several hygromycin B-resistant progeny from cross Guy11-T10-1 (a single-copy integration site transformant)x2539 (sensitive wild-type parent), but not in hygromycin B-resistant progeny from four other crosses. Transformants produced typical lesions when inoculated onto host plants. Southern hybridization revealed rearrangements of integrated DNA in single conidial isolates of highcopy transformant 2539-T1-1 re-isolated from host plants, characterized by excision of one or more copies of the transforming plasmid. Plasmid loss and rearrangement were also observed within single conidial isolates derived from transformant 2539-T1-1 following ten asexual generations on non-selective agar medium. These examples of instability of integrated DNA in M. grisea transformants suggest that caution should be exercised in the use of transformation for assessing the phenotypic effects of specific introduced genes.

Faecal microflora of capt ive koalas, Phascolarctos cinereus (Marsupialia: Phascolarctidae).

The faecal microflora of four Phascolarctos cinereus kept at the Saitama Children&amp;apos;s Zoo in Japan was investigated using 10 selective and 4 nonselec tive agar media. Total viable counts (in log form) of faecal bacteria (8.2 g of wet faeces) were significantly lower than the direct microscopic counts (11.2 g of wet faeces). Four groups of obligate anaerobes (bacteroidaceae, peptococcaceae, bifidobacteria , and clostridia) and three groups of facultative anaerobes (enterobacteria, streptococci and lactobacilli) were isolated. Dominant components of the flora were obligate anaerobes, in particular bacteroidaceae and peptococcaceae.

Maintenance of the recombinant plasmid pIJ2 in chemostat cultures of Streptomyces lividans 66 (pIJ2)

AbstractThe maintenance of the recombinant plasmid pIJ2 in chemostat cultures of Streptomyces lividans 66 (pIJ2) was investigated. The presence of the plasmid coding for a neomycin phosphotransferase was detected by plating samples from the continuous cultures on nonselective and selective agar medium containing neomycin.The plasmid was lost from the host strain under all conditions tested. However, the kinetics of the plasmid segregation from the chemostat populations were dependent on the growth‐limiting substrate of the medium, the dilution rate of the continuous culture and the cultivation temperature.Size differences between the original plasmid and plasmid DNA isolated from neomycin‐resistant clones after long‐term chemostat cultivation were not observed. In neomycin‐sensitive clones no extrachromosomal DNA was found.

Relative recovery of anaerobes on different isolation media

The recovery of clinical anaerobic isolates on selective and nonselective agar media, as well as the time required to detect the isolates, was examined. Of a total of 235 isolates, 77, 46, and 40% were detected on Schaedler blood agar, colistinnalidixic acid blood agar, and kanamycin-vancomycin-lysed blood agar, respectively, and 94% were detected on the combination of Schaedler blood agar with kanamycin-vancomycin-lysed blood agar. A total of 19% of the anaerobes were detected after incubation for 1 day, and 70% were detected after 2 days.