Acid Agar Research Articles

Occurrence of the vancomycin-resistant genes vanA, vanB, vanC1, vanC2 and vanC3 in Enterococcus strains isolated from poultry and pork

It is suspected that the use of avoparcin as a feeding antibiotic for the fat stock contributes to development of cross-resistance against vancomycin and teicoplanin. After isolating enterococci strains from poultry and pork meat by cultivation on citrate azide Tween carbonate agar (CATC) and screening the vancomycin resistance on Columbia colistin nalidixic acid agar (CNA, supplemented with 5% sheepblood and 5 mg vancomycin/l) the polymerase chain reaction (PCR) was used for the detection of the vancomycin resistance genes vanA (‘high level’), vanB (‘moderate high level’), vanC1, vanC2 and vanC3 (‘low level’). Out of 1643 E.-isolates from 115 poultry and 50 pork samples, 420 isolates could be identified as vancomycin resistant, 202 isolates of which carry the vanA, one isolate both the vanA and the vanC1, 38 isolates the vanC1, 14 isolates the vanC2, nine isolates both the vanC1 and the vanC3 gene and 156 isolates carry no gene. The vanB gene was not found in these isolates. Comparing vanA-positive food isolates with those from different human sources by means of the pulsed field gel electrophoresis (PFGE) it could clearly be demonstrated that they do not show homological fingerprints according to the source of origin. It is therefore unlikely that there is a close genetic relationship between isolates from animal foodstuff and humans.

Isolation and Identification of Tetracycline Resistant Lactic Acid Bacteria from Pre-packed Sliced Meat Products

In recent years, the food chain has been recognised as one of the main routes for transmission of antibiotic resistant bacteria between the animal and human population. In this regard, the current study aimed to investigate if tetracycline resistant (tetR) lactic acid bacteria (LAB) are present in ready-to-eat modified atmosphere packed (MAP) sliced meat products including fermented dry sausage, cooked chicken breast meat and cooked ham. From population graphs based on doubling tetracycline concentrations between 0 and 256 microg ml(-1), only fermented dry sausage was shown to contain a high-level retR LAB population (5.10(1) - 2,23.10(4) CFU/g), and this in four out of ten examined sausages. From these four positive sausages, a total of 100 strains were isolated on de Man, Rogosa and Sharpe-sorbic acid (MRS-S) agar without tetracycline (n = 45) and on MRS-S agar supplemented with a tetracycline breakpoint concentration of 64 microg ml(-1) (n = 55). Using resistance histograms derived from the disc diffusion method, all these strains were grouped as sensitive to rifampicin, erythromycin and ampicillin. All strains from the tetracycline-containing MRS-S plates were resistant to tetracycline. Identification with whole-cell protein profiling revealed that the total strain set represented four different species: Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus sakei subsp. carnosus and Lactobacillus curvatus. All species are commonly associated with fermented dry sausage, either as starter culture or as natural contaminants. The latter three species were found to comprise all tetracycline resistant strains. To our knowledge, this is the first report providing evidence for the presence of tetR LAB in final ready-to-eat pre-packed fermented dry sausages.

Screening Fungi Isolated from Woody Compost for Lignin-Degrading Potential

Thirty-four fungi isolated from woody compost were screened for lignin-degrading ability using a kraft pine lignin medium, and for the production of extracellular phenoloxidases associated with lignin degradation using gallic acid agar, tannic acid agar, and syringaldazine tests. Nine isolates tested gave positive results in all four assessments, with an additional five showing positive results on three tests. Twelve isolates modified kraft pine lignin, eleven of which also tested positive for laccase using syringaldazine as the substrate. From these results it is concluded fungi with lignin-degradative potential are common in and easily isolated from woody compost. The ten isolates positive on gallic and/or tannic acid agars, but negative on lignin agar and syringaldazine, may be involved in humus formation.

Screening Pregnant Women for Group B Streptococcal Colonization

The recovery rates of group B streptococcus (GBS) from anorectal swabs (RS) and vaginal swabs (VS) that were enriched were compared to the routine method to determine the optimal procedure. Separate RS and VS were collected from women attending antenatal clinics. RS and VS were placed in 2 ml enrichment and selective broth. Swabs were inoculated onto colistin/nalidixic acid agar (CNA) upon arrival in the laboratory and onto 5% sheep blood agar (SBA) and CNA after 24 h enrichment. The routine method consisted of a VS sent in transport medium and inoculated in the laboratory onto SBA (no enrichment). The overall GBS colonization rate was 24% (64/264). Of the 64 GBS carriers, 77% were colonized in the vagina and 89% were colonized in the anorectum. The anorectum was the only site of colonization in 24% of the women, whereas the vagina was the only site of colonization in 11% of cases. Enrichment increased the detection of GBS from both RS (55 versus 42; P < 0.025) and from VS (49 versus 27; P < 0.001). Of the 64 cases, enriched RS detected 86%, enriched VS detected 77% and the standard VS detected only 41%. Enriched RS and enriched VS collectively detected 99% of cases. SBA was better than CNA for subculture of the enrichment broth because of a higher recovery rate (98–100% versus 80–82%; P < 0.01) and the fact that the hemolysis on SBA made it easier to differentiate GBS from enterococci. The data confirm that optimal screening of pregnant women for GBS should include a combined RS/VS swab placed in enrichment broth that is then subcultured onto SBA after 24 h incubation.

Evaluation of the Strep B OIA Test Compared to Standard Culture Methods for Detection of Group B Streptococci

Objective:This study evaluated the accuracy of the commercial product Strep B OIA (optical immunoassay) compared to the standard agar and broth culture methods for detecting vaginal colonization with group B streptococcus (GBS).Methods:Preoperative vaginal cultures were obtained from 141 nonpregnant gynecological patients undergoing major gynecologic surgery. Major gynecologic surgery was defined as benign gynecologic, gyne-oncology, and urogynecologic procedures. The results of the Strep B OIA test were compared to the results obtained from SXT agar (selective for GBS), colistin-nalidixic acid (CNA) agar, and Todd-Hewitt broth cultures.Results:The prevalence of vaginal GBS colonization in this population was 20.6%. The sample sensitivity and specificity of the OIA method were 58.6% and 85.7%, respectively. These values are lower than the sensitivity and specificity of 85.4% and 91.5%, respectively, given in the OIA package insert. Although the sample negative predictive value was fairly high (88.9%), the positive predictive value was low (51.5%).Conclusion:Although a previous study stated that the product Strep B OIA reduces the time required to obtain results (30 minutes versus days) and can, therefore, function as a useful diagnostic tool in the management of early-onset GBS disease, the present study's finding of low sensitivity and low positive predictive value indicates that this test may have very limited clinical value. Infect. Dis. Obstet. Gynecol. 7:202–205, 1999.

Screening pregnant women for group B streptococcal colonization.

The recovery rates of group B streptococcus (GBS) from anorectal swabs (RS) and vaginal swabs (VS) that were enriched were compared to the routine method to determine the optimal procedure. Separate RS and VS were collected from women attending antenatal clinics. RS and VS were placed in 2 ml enrichment and selective broth. Swabs were inoculated onto colistin/nalidixic acid agar (CNA) upon arrival in the laboratory and onto 5% sheep blood agar (SBA) and CNA after 24 h enrichment. The routine method consisted of a VS sent in transport medium and inoculated in the laboratory onto SBA (no enrichment). The overall GBS colonization rate was 24% (64/264). Of the 64 GBS carriers, 77% were colonized in the vagina and 89% were colonized in the anorectum. The anorectum was the only site of colonization in 24% of the women, whereas the vagina was the only site of colonization in 11% of cases. Enrichment increased the detection of GBS from both RS (55 versus 42; P < 0.025) and from VS (49 versus 27; P < 0.001). Of the 64 cases, enriched RS detected 86%, enriched VS detected 77% and the standard VS detected only 41%. Enriched RS and enriched VS collectively detected 99% of cases. SBA was better than CNA for subculture of the enrichment broth because of a higher recovery rate (98-100% versus 80-82%; P < 0.01) and the fact that the hemolysis on SBA made it easier to differentiate GBS from enterococci. The data confirm that optimal screening of pregnant women for GBS should include a combined RS/VS swab placed in enrichment broth that is then subcultured onto SBA after 24 h incubation.

Hormonal Control of Parthenocarpic Ovary Growth by the Apical Shoot in Pea

The role of the apical shoot as a source of inhibitors preventing fruit growth in the absence of a stimulus (e.g. pollination or application of gibberellic acid) has been investigated in pea (Pisum sativum L.). Plant decapitation stimulated parthenocarpic growth, even in derooted plants, and this effect was counteracted by the application of indole acetic acid (IAA) or abscisic acid (ABA) in agar blocks to the severed stump. The treatment of unpollinated ovaries with gibberellic acid blocked the effect of IAA or ABA applied to the stump. [3H]IAA and [3H]ABA applied to the stump were transported basipetally, and [3H]ABA but not [3H]IAA was also detected in unpollinated ovaries. The concentration of ABA in unpollinated ovaries increased significantly in the absence of a promotive stimulus. The application of IAA to the stump enhanced by 2- to 5-fold the concentration of ABA in the inhibited ovary, whereas the inhibition of IAA transport from the apical shoot by triiodobenzoic acid decreased the ovary content of ABA (to approximately one-half). Triiodobenzoic acid alone, however, was unable to stimulate ovary growth. Thus, in addition to removing IAA transport from the apical shoot, the accumulation of a promotive factor is also necessary to induce parthenocarpic growth in decapitated plants.

Oral staphylococcus in older subjects with rheumatoid arthritis.

To determine if Staphylococcus aureus and Staphylococcus epidermidis, etiologic bacterial agents to late prosthetic joint infections (LPJI), are more prevalent in the oral flora of older individuals with rheumatoid arthritis (RA) than in an age and gender-matched nonarthritic control population (NA). Cultures were obtained from the nares, oropharynx, saliva, tongue, and gingival crevice, and the results were compared between older patients with RA and controls. University of Michigan Medical Center, Ann Arbor, VA Medical Center, and University of Michigan School of Dentistry. A total of 111 community-dwelling subjects with a diagnosis of RA and 83 gender-matched control subjects. Colistin nalidixic acid agar plates with 5% sheep's blood were inoculated and incubated. Isolates were speciated using the API Staph Trac micro method and catalase and coagulase tests. Individuals with RA had a higher prevalence of S. aureus isolated from the oral cavity. However, only the oropharynx and tongue revealed higher rates; all other sites were insignificant. The presence of oral S. aureus was associated with xerostomia. Staphylococcus epidermidis was not detected from any of the oral sites sampled. Sixty-two percent (10/16) of the S. aureus isolates from the RA subjects were resistant to penicillin and ampicillin, whereas none were resistant to a cephalosporin. These findings suggest that rheumatoid arthritis may be a risk factor for LPJI in older prosthetic joint patients undergoing invasive dental procedure in the posterior oral cavity. This increased risk is caused, in part, by a higher prevalence of S. aureus in the posterior oral cavity. The prevalence and the antibiotic resistance of S. aureus must be considered when determining the need for chemoprophylaxis.

Investigation of persistent colonization by Pseudomonas aeruginosa-like strains in a spring water bottling plant.

Ninety-seven strains, producing a fluorescent pigment under UV light and/or a green diffusive pigment on cetrimide-naladixic acid agar, were isolated from a spring water bottling plant. These strains were presumptively identified as Pseudomonas aeruginosa, but they could not be confirmed as strains of this species nor identified by the API 20NE identification system. The isolates and reference strains were clustered by computer-assisted whole-cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The numerical analysis of the protein electrophoregrams resulted in the formation of four clusters at a similarity level of 80% and two unclustered type strains. One cluster included strains isolated during a 4-month period and reference strains of several biotypes of P. fluorescens. The remaining isolates formed another cluster with a very high similarity of level, which included two groups of strains based on biochemical characterization by the API 20NE Test System. Strains were typed by random amplified polymorphic DNA (RAPD)-PCR and two different RAPD patterns were obtained, corresponding to each biochemical profile. This persistent colonization seems to be caused by a single species present in the bottling system, with two clonal origins, not related to P. aeruginosa or to any of the other type strains tested. Partial 16S rDNA sequence of a representative strain of one cluster of isolates had a level of similarity of 99.3% with P. alcaligenes. This study shows that characteristics similar to P. aeruginosa on cetrimide-naladixic acid agar can be exhibited by several groups of fluorescent pseudomonads that do not belong to this species, clearly showing that confirmation tests must be performed before a decision regarding the water quality is made.

Stress co-tolerance and trehalose content in baking strains of Saccharomyces cerevisiae.

Fourteen wild-type baking strains of Saccharomyces cerevisiae were grown in batch culture to true stationary phase (exogenous carbon source exhausted) and tested for their trehalose content and their tolerance to heat (52 degrees C for 4.5 min), ethanol (20% v/v for 30 min), H2O2 (0.3 M for 60 min), rapid freezing (-196 degrees C for 20 min, cooling rate 200 degrees C min-1), slow freezing (-20 degrees C for 24 h, cooling rate 3 degrees C min(-1)), salt (growth in 1.5 M NaCl agar) or acetic acid (growth in 0.4% w/v acetic acid agar) stresses. Stress tolerance among the strains was highly variable and up to 1000-fold differences existed between strains for some types of stress. Compared with previously published reports, all strains were tolerant to H2O2 stress. Correlation analysis of stress tolerance results demonstrated relationships between tolerance to H2O2 and tolerance to all stresses except ethanol. This may imply that oxidative processes are associated with a wide variety of cellular stresses and also indicate that the general robustness associated with industrial yeast may be a result of their oxidative stress tolerance. In addition, H2O2 tolerance might be a suitable marker for the general assessment of stress tolerance in yeast strains. Trehalose content failed to correlate with tolerance to any stress except acetic acid. This may indicate that the contribution of trehalose to tolerance to other stresses is either small or inconsistent and that trehalose may not be used as a general predictor of stress tolerance in true stationary phase yeast.

Evaluation of Media for Selective Enumeration of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, and Bifidobacteria

Fifteen media were evaluated to determine their suitability for selective enumeration of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, and bifidobacteria using 5 to 6 strains of each of the four groups of organisms. Streptococcus thermophilus agar was found to be suitable for selective enumeration of S. thermophilus under aerobic incubation at 37°C for 24h. The MRS agar at pH 5.2 or reinforced clostridial agar at pH 5.3 could be used for the selective enumeration of L. delbrueckii ssp. bulgaricus when the incubation was carried out at 45°C for ≥72h. However, the recovery of this organism was lower on MRS agar at pH 5.2 or reinforced clostridial agar at pH 5.3 than that obtained on MRS agar. The recovery of L. acidophilus and bifidobacteria on MRS agar and MRS-maltose agar was similar; MRS-maltose agar could be used to enumerate total counts of L. acidophilus and bifidobacteria. For selective enumeration of L. acidophilus, MRS-salicin agar or MRS-sorbitol agar could be used. For selective enumeration of bifidobacteria, MRS NNLP (nalidixic acid, neomycin sulfate, lithium chloride, and paromomycin sulfate) agar was suitable; however, determination of bifidobacteria by differential counts between L. acidophilus enumerated on MRS-salicin agar or MRS-sorbitol agar and the total counts of L. acidophilus and bifidobacteria obtained from MRS-maltose agar resulted in higher recovery of some strains of bifidobacteria. Other media that were evaluated in this study were not suitable for selective enumeration.

Performance of a new DNA probe for the detection of group B streptococcal colonization of the genital tract

To test the performance characteristics of a new DNA probe designed for the rapid identification of heavy colonization of the genital tract with group B streptococci. Vaginal and combined vaginal-perianal samples were collected from 193 pregnant women and cultured on colistin-nalidixic acid agar plates. Bacterial growth was classified semiquantitatively. Specimens were also tested by a new DNA probe in two formats: a direct assay performed on the swabs soon after collection and an assay performed after the swabs were incubated for 24 hours in an enriched culture medium. The agar cultures were positive in 36 of 193 patients (18.6%, 95% confidence interval 13.2–24). Nineteen women were lightly colonized, and 17 were heavily colonized (at least 10 4 colonies/mL). The combined vaginal-perianal swabs yielded positive results more often than the vaginal swabs alone (26 versus 16, χ 2 = 24, P < .01). In its direct form, the assay had only 8.3% sensitivity in identifying colonized women. In heavily colonized women, the sensitivity of the assay increased slightly to 12%. After a 16–24-hour incubation, the sensitivity of the assay was 81%. The direct assay is insufficiently sensitive for clinical use. The delayed assay offers no advantage over standard cultures.

Efficacy of selected media for recovery of Escherichia coli O157:H7 from frozen chicken meat containing sodium chloride, sodium lactate or polyphosphate

The viability of Escherichia coli O157:H7 in frozen chicken meat (CM) containing sodium chloride (NaCl), sodium lactate, or polyphosphate over an 18 month period was determined. Additionally, several recovery media were evaluated. Ground CM (10 g) was inoculated (10 6 cfu g -1) with E. coli O157:H7, then held at -20°C. Surviving cells were enumerated monthly using a nonselective medium, tryptic soy agar (TSA). After 11 months, seven additional recovery media were also used to evaluate the ability to recover freeze/NaCl-injured cells. In all treatments, the initial population decreased (P < 0·05) during storage. In CM with no additives, E. coli O157:H7 decreased by 0·6 log 10 cfu g -1 in 18 months. Addition of NaCl and sodium lactate resulted in reductions (P < 0·05) ranging from 1·4-2·4 log 10 cfu g -1 over the same period. Addition of polyphosphate did not affect recovery (P < 0·05). Recovery of E. coli O157:H7 was least (P < 0·05) from CM containing 8% NaCl, and recovery was least efficient (P < 0·05) on selective media (i.e. MacConkey sorbitol agar, MSA). The addition of Mg 2+ to MSA did not enhance recovery. Highest recoveries (P < 0·05) occurred on TSA, TSA plus 1% pyruvic acid and phenol red sorbitol agar (PRSA). E. coli O157:H7 survived (≥18 months) well in frozen poultry, and selective media such as MSA were inadequate for recovering freeze/NaCl-injured cells.

Anhydrides as antimycotic agents added to polyethylene films for food packaging

AbstractIncorporation of free acids or salts of antimycotic food additives, including propionic acid, benzoic acid or sorbic acid, into low‐density polyethylene (LDPE) films failed to inhibit mould growth when the films contacted inoculated media. However, LDPE films into which benzoic anhydride was incorporated exhibited antimycotic activity when in contact with media and cheese. Benzoic anhydride, which had been added to LDPE film, was hydrolysed within 5h and detected as benzoic acid in potato dextrose agar (PDA) and cheese after contact with the film. LDPE films, into which 1% benzoic anhydride was incorporated, completely inhibited Rhizopus stolonifer. Penicillium spp., and Aspergillus toxicarius growth on PDA. Lower amounts of anhydride partially inhibited growth by increasing the lag phase and reducing the rate of growth in most cases. LDPE films incorporated with 0.5–2% benzoic anhydride delayed mould growth on cheese. These data suggest that addition of antimycotic agents to LDPE during film manufacture may be a feasible way of controlling surface mould growth in foods such as cheese.

Recent IDF publications

This chapter discusses the preparation of Lactobacillus sorbic acid (LaS) agar according to the formulation of Reuter. The combination of anaerobic incubation and reading of the results after 2 days enables selective enumeration of “lactobacilli.” Acetate, citrate, sorbic acid, and low pH suppress other bacterial flora, including enterococci and Streptococcus salivarius ssp. thermophilus. Productivity for Lactobacillus delbrueckii ssp. bulgaricus may be slightly reduced. This medium is good for isolating lactobacilli from a mixed flora, but can be inhibitory for some yoghurt cultures and for sublethally damaged bacteria. The chapter describes the preparation of LaS. The chapter reviews that all well-grown colonies after 48 h incubation at 30 or 37∼ are considered as lactobacilli.

Selective isolation of fluorescent and nonfluorescent Pseudomonas species from Sohag Governorate (Upper Egypt).

Three hundred and fourty nine isolates belonging to ten different species of Pseudomonas were isolated and identified from soil and water samples on four selective isolation media: Modified Trypticase soy agar (TSA), ACC-agar containing ampicillin-chloramphenicol-cycloheximide, Nitrate plus organic acid agar and Tryptophan substrate agar. The fluorescent species were P. putida (21.2% of the total Pseudomonas species), P. fluorescens (15.8%), P. aeruginosa (9.7%) and P. aureobaciens (2.6%), while the nonfluorescent species were P. stutzeri (12.4%), P. alcaligenes (11.2%), P. pseudoalcaligenes (9.5%), P. mendocina (8.3%), P. lemoignei (5.7%) and P. ruhlandii (3.4%). Modified TSA and ACC media were efficient for the isolation of both fluorescent and nonfluorescent species, whereas Nitrate and Tryptophan media were more selective for the isolation of nonfluorescent species. The optimum temperature for the isolation of different species ranged between 20 degrees and 37 degrees C.

Cultural studies of four North American species of Perenniporia (Aphyllophorales: Polyporaceae)

Perenniporia phloiophila colonizes the dead, outer bark of live oak ( Quercus virginiana ) and is known in the southeastern United States on this host only. On 2% malt extract agar, P. phloiophila has an average growth rate of 5–6 mm day −1 at its optimum temperature range of 30–35 °C. P. phloiophila gives strong oxidase reactions on gallic and tannic acid agar media and does not produce chlamydospores in cultures of single dikaryons or homokaryons. P. medulla-panis has an average growth rate of 6–8 mm day −1 at its optimum temperature range of 30–35°. It has strong reactions on gallic and tannic acid agar media and produces chlamydospores in culture. Vegetative incompatibility between different dikaryotic isolates of P. phloiophila resulted in antagonistic hyphal interactions and dark pigmented zones between opposing isolates. Chlamydospores developed in the interaction zones of incompatible dikaryotic isolates. Self crosses of dikaryons resulted in complete intermingling of isolates with no interaction zone or pigmentation. Incompatible matings of homokaryotic isolates of P. phloiophila have abundant chlamydospores in the interaction zone while chlamydospores are absent in compatible matings of single spore isolates. Chlamydospores occur in all homokaryotic isolates of P. medulla-panis . Both species are heterothallic and tetrapolar. In vitro wood decay abilities of P. phloiophila, P. medulla-panis, P. ohiensis and P. fraxinophila were studied using wood of silverleaf oak, white fir and velvet ash. Percent weight loss (PWL) was greatest on silverleaf oak wood for three of the four species tested. P. medulla-panis caused the greatest PWL on white fir while P. fraxinophila caused the greatest PWL on silverleaf oak and velvet ash wood. Significant ( P = 0·05) differences in PWL occurred with P. phloiophila isolates tested at 25 and 30°. Mycelium of P. phloiophila readily colonized Emory oak bark blocks but only one isolate caused significant PWL.

Comparison of Three Selective Isolation Media for the Detection of L. monocytogenes in Foods

391 different foods were examined in order to compare three selective isolation media for Listeria: blood agar with nalidixic acid, Palcam agar, Listeria selective Oxford medium (Oxford agar). The percentage of positive samples (presence of Listeria in 25 g) obtained on the three media after an enrichment procedure is identical: 15.8%. The species L. monocytogenes is found in respectively 8.43%, 8.43% and 8.18% of the foods examined on Palcam agar, Oxford agar and nalidixic acid agar; this represents more than 50% of the isolates of the genus Listeria. However, Palcam agar and Oxford agar offer the advantages of a great reduction of the development of the contaminating microflora and a clearly less fastidious reading. The technique used for the direct counting of L. monocytogenes on these two selective media does not allow the detection of a low contamination of foods (theoretical positivity threshold = 100 Listeria/g): 65.2% of the positive samples would not have been detected without prior enrichment. 41.1% of the isolated L. monocytogenes belong to serovar 1/2a, 5.8% to serovar 1/2b, 20.58% to serovar 1/2c, 5.8% to serovar 3b and 24.47% to serovar 4b.

New methods for the highly selective isolation of Streptosporangium and Dactylosporangium from soil

Two methods for the isolation of the rare actinomycetes Streptosporangium and Dactylosporangium in soil, are described. The methods use the ability of both the sporangiospores of Streptosporangium and the globose bodies (aleuriospore) of Dactylosporangium to withstand dry heating and treatment with benzethonium chloride (BC). In addition, the differential antibiotic resistance of these actinomycetes is also utilized. (i) To isolate streptosporangia, an air-dried soil sample is first subjected to dry heat treatment (120°C, 1 h). A water suspension of the heated sample is then treated with 0.01% BC, diluted and cultured on HV agar supplemented with nalidixic acid and leucomycin. (ii) To isolate dactylosporangia, a water suspension of a soil sample given dry heat and 0.03% BC treatment is cultured on HV agar supplemented with nalidixic acid and tunicamycin. The dry heat and BC treatment drastically eliminated bacteria and unwanted actinomycetes contaminants, including streptomycetes, from the isolation plates, thereby facilitating the selective isolation of streptosporangia and dactylosporangia. Nalidixic acid in HV agar was useful to suppress the growth of dry heat- and BC-resistant bacteria. The use of leucomycin and tunicamycin increased the selectivity of HV agar for streptosporangia and dactylosporangia, respectively, through elimination of the growth of unwanted actinomycetes which remained after the employment of pretreatments. From various field soils (vegetable, orchard, corn, and rice paddy), the methods described above consistently achieved the highly selective isolation of streplosporangia and dactylosporangia, respectively, as each group accounted for more than 20% of the total number of colonies recovered.

Anomalies in the enumeration of starved bacteria on culture media containing nalidic acid and tetracycline

Culturable counts of antibiotic resistant, genetically engineeredPseudomonas fluorescens were determined on antibiotic-containing plate count agar during starvation in water. Prior to starvation, colony counts obtained on all media separated into two groups. The mean of the colony counts on plate count agar with or without tetracycline (4.9 × 10(6) ml(-1)) was significantly higher than the mean colony counts on plate count agar containing either nalidixic acid or nalidixic acid plus tetraclycline (2.5×10(6) ml(-1)). After 20 days of starvation the highest mean colony counts continued to be obtained on plate count agar (7.2 × 10(6) ml(-1)) with slightly, but significantly, lower counts obtained on plate count agar containing either nalidixic acid (5.6 × 10(6) ml(-1)) or tetraclycline (1.5×10(6) ml(-1)). A combination of nalidixic acid and tetracycline in plate count agar, however, dramatically reduced colony counts (8.3 × 10(2) ml(-1)) after this starvation period. The addition of catalase to plate count agar containing nalidixic acid and tetracycline negated the effect caused by this combination of antibiotics. When colony counts obtained over the entire 20 day incubation were considered, the addition of MgSO4 to plate count agar containing nalidixic acid and tetracycline resulted in a significant increase in colony counts. Other combinations of antibiotics, nalidixic acid+carbenicillin, nalidixic acid+kanamycin, streptomycin+tetracycline, streptomycin+carbenicillin, rifampicin+tetracycline, rifampicin+carbenicillin, and rifampicin+kanamycin, did not inhibit colony formation of starved cells. Antibiotic resistant strains ofP. putida andEscherichia coli also displayed sensitivity to the combination of nalidixic acid and tetracycline in plate count agar after starvation.