Cell surface hydrophobicity and slime production of Staphylococcus epidermidis Brazilian isolates

The cell surface hydrophobicity of 60 isolates and three reference strains of Staphylococcus epidermidis was assayed by means of bacterial aggregation in liquid broth, phosphate-buffered saline, and in ammonium sulfate, as well as by affinity of the bacteria to n-hexadecane and polystyrene surfaces. In order to better characterize the isolates, the influence of bacterial growth time and enzyme treatment on cell hydrophobicity and the analysis of the slime production were also investigated. The strains presented the following profiles when assayed by the ammonium sulfate aggregation test (SAT): SAT < 1M, SAT 1M - <2M, SAT 2M - <4M, and SAT >or=4M. When SAT < 1M, the strains showed positive results for most of the cell surface hydrophobicity tests. None of the strains belonging to the groups with SAT >or= 1M showed spontaneous aggregation (SA), auto-aggregation (AA), or glass adherence, albeit 32 (62.7%) strains were polystyrene adherent and 42 (82.3%) presented weak adherence to n-hexadecane (>20%). The best correlation of the results was found among the AA and glass adherence tests (100%), followed by SA/ glass adherence (98%) and SA/ AA test (98%). The polystyrene adherence test and microbial adherence to n-hexadecane test (MATH) showed 78% correlation. Proteinase K treatment reduced bacterial adherence to polystyrene, but did not influence the SAT values. Three distinct groups of strains were distinguished by the polystyrene micromethod and glass tube adherence assay: 0.0-0.4 O.D. group, including non-glass adherent isolates; 0.5-0.7 O.D. group, including strains with variable profiles (adherent or non-adherent); and 0.8-1.3 O.D. group, composed of glass-adherent strains. Evaluation by a single method seemed not to reliably determine the surface hydrophobicity characteristics of S. epidermidis clinical isolates. Auto-aggregation properties of the strains that adhered to glass seemed related to slime expression, rather than cell surface hydrophobicity. Data also suggested involvement of protein components in adherence to polystyrene, but not in auto-aggregation properties assayed by SAT.     

Power Analysis for Real-Time PCR Quantification of Genes in Activated Sludge and Analysis of the Variability Introduced by DNA Extraction

The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.     

Cell Surface Hydrophobicity and Slime Production of <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> Brazilian Isolates

The cell surface hydrophobicity of 60 isolates and three reference strains of Staphylococcus epidermidis was assayed by means of bacterial aggregation in liquid broth, phosphate-buffered saline, and in ammonium sulfate, as well as by affinity of the bacteria to n-hexadecane and polystyrene surfaces. In order to better characterize the isolates, the influence of bacterial growth time and enzyme treatment on cell hydrophobicity and the analysis of the slime production were also investigated. The strains presented the following profiles when assayed by the ammonium sulfate aggregation test (SAT): SAT < 1M, SAT 1M − <2M, SAT 2M − <4M, and SAT ≥4M. When SAT < 1M, the strains showed positive results for most of the cell surface hydrophobicity tests. None of the strains belonging to the groups with SAT ≥ 1M showed spontaneous aggregation (SA), auto-aggregation (AA), or glass adherence, albeit 32 (62.7%) strains were polystyrene adherent and 42 (82.3%) presented weak adherence to n-hexadecane (>20%). The best correlation of the results was found among the AA and glass adherence tests (100%), followed by SA/ glass adherence (98%) and SA/ AA test (98%). The polystyrene adherence test and microbial adherence to n-hexadecane test (MATH) showed 78% correlation. Proteinase K treatment reduced bacterial adherence to polystyrene, but did not influence the SAT values. Three distinct groups of strains were distinguished by the polystyrene micromethod and glass tube adherence assay: 0.0–0.4 O.D. group, including non-glass adherent isolates; 0.5–0.7 O.D. group, including strains with variable profiles (adherent or non-adherent); and 0.8–1.3 O.D. group, composed of glass-adherent strains. Evaluation by a single method seemed not to reliably determine the surface hydrophobicity characteristics of S. epidermidis clinical isolates. Auto-aggregation properties of the strains that adhered to glass seemed related to slime expression, rather than cell surface hydrophobicity. Data also suggested involvement of protein components in adherence to polystyrene, but not in auto-aggregation properties assayed by SAT. Received: 13 May 2002 / Accepted: 5 July 2002     

Comparison of Fluorescence Microscopy and Solid-Phase Cytometry Methods for Counting Bacteria in Water

Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 10⁶ cells filter⁻¹. In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 10⁵, the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.     

Power analysis for real-time PCR quantification of genes in activated sludge and analysis of the variability introduced by DNA extraction

The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.     

Quantitative and specific detection of the biocontrol agent, Serratia plymuthica, in plant extracts using a real-time TaqMan? assay

A Serratia plymuthica-specific TaqMan? assay was designed based on the consensus nucleotide sequence from the 3??- end of the luxS gene present in all S. plymuthica strains tested. The specificity of the assay was demonstrated by testing 21 Serratia spp. strains and 30 isolates belonging to various species that can potentially coexist with S. plymuthica in the same environment. Positive reactions in the TaqMan? assay were observed only for S. plymuthica isolates and not for other bacteria. The TaqMan? assay could detect down to 1.95 ng of S. plymuthica DNA, down to 5 bacterial cells per reaction (100?cfu ml^?1) in vitro, down to 50 bacterial cells per reaction (1,000?cfu ml^?1) in spiked potato root extracts and down to 5 bacterial cells per reaction (100?cfu ml^?1) in spiked potato haulm extracts. We used this assay to quantify S. plymuthica A30 cells in potato and tomato haulms and roots grown from S. plymuthica A30-inoculated potato seed tubers and tomato seeds. The results were comparable with the spread-plating of plant extracts on a newly developed S. plymuthica A30 selective medium (CVTR2Arif). The TaqMan? assay can be used to quantify S. plymuthica isolates in different ecosystems and in complex substrates.     

The effects of natural biofilms on the reattachment of young adult zebra mussels to artificial substrata

This laboratory study examined the effects of natural biofilms on the reattachment of young adult zebra mussels, Dreissena polymorpha, in Petri dishes. Natural biofilms were developed in glass and polystyrene Petri dishes using water samples collected at various times of the year. Biofilms were developed over 1, 3, 8, and 14 d. Controls were clean glass and polystyrene Petri dishes. Zebra mussels collected from the field (< or = 10 mm, ventral length) were placed in the dishes and their reattachment by byssal threads was recorded after 1 d. Zebra mussels reattached to the dish surface or the shells of other mussels in the dish, or remained unattached. The data indicate that reattachment to clean glass was greater than to clean polystyrene (p < or = 0.05, ANOVA), but there were no consistent differences between reattachment to filmed polystyrene and filmed glass dish surfaces. Zebra mussels in control and filmed glass dishes reattached in higher percentages to the dish surface compared to the shells of other mussels (p < or = 0.05, ANOVA). There was no difference in mussel of reattachment between the dish surface and the shells of other mussels in most control polystyrene dishes (p > 0.05, ANOVA), whereas in filmed polystyrene the percentage of reattachment to the dish surface was greater than to the shells of other mussels (p < or = 0.05, ANOVA). These results indicate that substratum wettability and the presence of biofilms on some types of substrata can be factors in the reattachment of young adult zebra mussels.     

Bacterial biomass and productivity in sediments,stromatolites, and water of hamelin pool,shark bay,Western Australia

Heterotrophic bacterial biomass and growth rates were examined in stromatolites formed from four different types of benthic cyanobacterial mats. Bacteria in algal mats were counted using direct microscopy and biomass was estimated from the numbers of bacteria. Heterotrophic bacterial growth rates were estimated from the rate of incorporation of tritiated thy‐midine into DNA. Pustular mat, which occurs in the upper in‐tertidal zone, contained relatively few bacteria in the surface layers (0–5 mm), having about 0.2 x 10⁶ cells mm^‐3, or 20 mgC m^‐2 per millimetre depth. Other mats in the lower intertidal and subtidal zones had from 1 x 10⁶ cells mm^‐3 to 8 x 10⁶ cells mm^‐3. Heterotrophic bacterial productivities were 2.1 to 5.0 mgC m^‐2 h^‐1. Turnover times were an average of 1 day in the sandy sediment and 5 days in the colloform mat. Although these results are minimum estimates, they indicate that heterotrophic bacteria contribute substantially to the carbon cycle in stromatolites, by utilizing about 20 to 30% of primary production.     

Detection of Alicyclobacillus spp. in Fruit Juice by Combination of Immunomagnetic Separation and a SYBR Green I Real-Time PCR Assay

An approach based on immunomagnetic separation (IMS) and SYBR Green I real-time PCR (real-time PCR) with species-specific primers and melting curve analysis was proposed as a rapid and effective method for detecting Alicyclobacillus spp. in fruit juices. Specific primers targeting the 16S rDNA sequences of Alicyclobacillus spp. were designed and then confirmed by the amplification of DNA extracted from standard strains and isolates. Spiked samples containing known amounts of target bacteria were used to obtain standard curves; the correlation coefficient was greater than 0.986 and the real-time PCR amplification efficiencies were 98.9%- 101.8%. The detection limit of the testing system was 2.8×10¹ CFU/mL. The coefficient of variation for intra-assay and inter-assay variability were all within the acceptable limit of 5%. Besides, the performance of the IMS-real-time PCR assay was further investigated by detecting naturally contaminated kiwi fruit juice; the sensitivity, specificity and accuracy were 91.7%, 95.9% and 95.3%, respectively. The established IMS-real-time PCR procedure provides a new method for identification and quantitative detection of Alicyclobacillus spp. in fruit juice.     

A Widely Used In Vitro Biofilm Assay Has Questionable Clinical Significance for Enterococcal Endocarditis

Biofilm formation may play an important role in the pathogenesis of infections caused by Enterococcus faecalis, including endocarditis. Most biofilm studies use a polystyrene dish assay to quantify biofilm biomass. However, recent studies of E. faecalis strains in tissue and animal models suggest that polystyrene dish results need to be interpreted with caution. We evaluated 158 clinical E. faecalis isolates using a polystyrene dish assay and found variation in biofilm formation, with many isolates forming little biofilm even when different types of media were used. However, all tested clinical isolates were able to form biofilms on porcine heart valve explants. Dextrose-enhanced biofilm formation in the polystyrene dish assay was found in 6/12 (50%) of clinical isolates tested and may explain some, but not all of the differences between the polystyrene dish assay and the heart valve assay. These findings suggest that in studies assessing the clinical relevance of enterococcal biofilm-forming ability, ex vivo biofilm formation on a relevant tissue surface may be warranted to validate results of in vitro assays.     

A bioassay technique for Entomophthora sphaerosperma on the spruce budworm,Choristoneura fumiferana

A technique for conducting bioassays of Entomophthora sphaerosperma on sixth-instar larvae of the spruce budworm, Choristoneura fumiferana, was developed. Four assays were conducted by showering conidia on 10 larvae for each of 10 to 20 doses per assay. Dose was estimated by averaging estimates of the concentration of spores falling on water agar dishes before and after insect exposure. Maximum-likelihood probit analysis indicated significant regressions between log dose and probit mortality for all four assays. LC₅₀ values ranged from 11.21 to 18.77 spores/mm² with a weighted mean of 16.13 spores/mm². Slope estimates ranged from 0.92 to 1.87 with a weighted mean of 1.13. These low slope values may have been indicative of a highly variable test insect population, but also suggested a nontoxic infection process by the pathogen.     

Highly sensitive detection and quantification of the pathogen Yersinia ruckeri in fish tissues by using real-time PCR

Yersinia ruckeri is the causative agent of enteric redmouth diseases (ERM) and one of the major bacterial pathogens causing losses in salmonid aquaculture. Since recent ERM vaccine breakdowns have been described mostly attributed to emergence of Y. ruckeri biotype 2 strains, rapid, reproducible, and sensitive methods for detection are needed. In this study, a real-time polymerase chain reaction (PCR) primer/probe set based on recombination protein A (recA) gene was designed and optimized to improve the detection of Y. ruckeri. The primer/probe set proved to have a 100 % analytical specificity and a sensitivity of 1.8 ag μl^?1, equivalent to 1.7 colony-forming units (CFU)?ml^?1, for purified DNA, 3.4 CFU g^?1 for seeded liver, kidney, and spleen tissues, and 0.34 CFU/100 μl^?1 for seeded blood, respectively. The assay was highly reproducible with low variation coefficient values for intra- and inter-run experiments (2.9 % and 9.5 %, respectively). Following optimization, the assay was used to detect changes in the bacterial load during experimental infection. Rainbow trout (Onchorhynchus mykiss) were exposed to two strains of Y. ruckeri (biotype 1 and biotype 2) by intraperitoneal inoculation. Internal organs (liver, kidney, spleen) and blood were biopsied from dead fish daily for 15 days to quantify copies of pathogen DNA per gram of tissue. The findings showed the efficacy of this real-time PCR assay to quantify Y. ruckeri cells in the fish tissues and also confirmed this assay as a non-lethal method for the detection of this pathogen in blood samples.     

Real-Time PCR Quantification of the Plant Growth Promoting Bacteria Herbaspirillum seropedicae Strain SmR1 in Maize Roots

The plant growth promoting bacteria Herbaspirillum seropedicae SmR1 is an endophytic diazotroph found in several economically important crops. Considering that methods to monitor the plant–bacteria interaction are required, our objective was to develop a real-time PCR method for quantification of PGPB H. seropedicae in the rhizosphere of maize seedlings. Primer pairs were designed, and their specificity was verified using DNA from 12 different bacterial species. Ten standard curves of qPCR assay using HERBAS1 primers and tenfold serial dilutions of H. seropedicae SmR1 DNA were performed, and PCR efficiency of 91 % and correlation coefficient of 0.99 were obtained. H. seropedicae SmR1 limit of detection was 10¹ copies (corresponding to 60.3 fg of bacterial DNA). qPCR assay using HERBAS1 was used to detect and quantify H. seropedicae strain SmR1 in inoculated maize roots, cultivated in vitro and in pots, harvested 1, 4, 7, and 10 days after inoculation. The estimated bacterial DNA copy number per gram of root was in the range 10⁷–10⁹ for plants grown in vitro and it was around 10⁶ for plants grown in pots. Primer pair HERBAS1 was able to quantify H. seropedicae SmR1, and this assay can be useful for monitoring plant–bacteria interaction.     

Morpho‐pathological and Molecular Characterization of Bipolaris oryzae in Rice (Oryzae sativa)

Fifty isolates of Bipolaris oryzae from rice were characterized morpho‐pathologically and molecularly. Based on colony morphology and growth pattern on PDA, these isolates were grouped into four categories: black with suppressed growth (21 isolates), black with cottony growth (16 isolates), black with fluffy growth (12 isolates) and white with cottony growth (1 isolate). The frequency of the black and suppressed type was the highest (42%) with maximum aggressiveness (mean spore count of 1854/cm²), whereas the white and cottony growth isolate had lowest frequency (2%) and aggressiveness (548/cm²). Thirteen B. oryzae isolates (four isolates from Groups I, II and III and one isolate from Group IV) were further tested for their variability with random amplified polymorphic DNA (RAPD) primers. Twenty RAPD primers were screened, of which 10 gave amplification; however, only six primers gave reproducible results. Based on the molecular similarity of the RAPD profiles, the isolates were grouped in to three major clusters and maximum linkage distance between them was determined as 0.29 units. This study establishes the variability among B. oryzae isolates.     

Assimilatory Sulfur Metabolism in Marine Microorganisms: Sulfur Metabolism, Protein Synthesis, and Growth of Alteromonas luteo-violaceus and Pseudomonas halodurans During Perturbed Batch Growth

The antibiotic protein synthesis inhibitor chloramphenicol specifically blocked the incorporation of [³⁵S]sulfate into the residue protein of two marine bacteria, Pseudomonas halodurans and Alteromonas luteo-violaceus. Simultaneous inhibition of total protein synthesis occurred, but incorporation of ³⁵S into low-molecular-weight organic compounds continued. A. luteo-violaceus rapidly autolyzed, with similar reduction in cell counts, total culture protein and cellular sulfur, whereas P. halodurans remained viable. Treatment with chloramphenicol, growth during nitrogen and carbon limitation, and the carbon and energy sources used for growth did not alter the sulfur content of P. halodurans protein. The mean value (1.09%, by weight), representing a wide variety of environmentally relevant growth conditions, was in agreement with model protein composition. The variability of cellular composition of P. halodurans and A. luteo-violaceus is discussed with respect to the measurement of bacterial growth in natural environments. Total carbon and nitrogen per cell varied greatly (coefficient of variation, ca. 100%) depending on growth conditions. Variation in total sulfur and protein per cell was much less (coefficient of variation, <50%), but the least variation was found for sulfate incorporation into residue protein (coefficient of variation, ca. 15%). Thus, sulfate incorporation into residue protein can be used as an accurate measurement of de novo protein synthesis in these bacteria.     

Evaluating Total Lymphocyte Count as a Surrogate Marker for CD4 Cell Count in the Management of HIV-Infected Patients in Resource-Limited Settings: A Study from China

Objective

To evaluate the correlation of total lymphocyte count (TLC) and CD4 cell count and the suitability of TLC as a surrogate marker for CD4 cell count of HIV-infected patients in China.

Methods

Usefulness of TLC as a surrogate marker for a CD4 cell count <350 cells/mm³ for HIV-positive patients in China was evaluated by 977 pairs of TLC and CD4 cell count from 977 outpatients. The result was then validated by a literature review which was conducted on 9 relevant articles. Further investigation using the 977 pairs of TLC and CD4 cell count data was done to determine a TLC threshold for predicting a CD4 cell count <500 cells/mm³. Correlation and receiver operating characteristic (ROC) analysis were performed for both CD4 cell counts, and the sensitivity and specificity were computed.

Results

Good correlation was noted between TLC and CD4 count (r = 0.60, 95% CI, 0.56–0.64). TLC obtained a relatively high diagnostic performance (area under ROC curve, 0.80) for predicting a CD4 cell count <350 cells/mm³, with a sensitivity of 0.65 (95% CI, 0.61–0.68) and a specificity of 0.80 (95% CI, 0.75–0.85) at the TLC threshold of 1570 cells/mm³. The literature review suggested that for a CD4 cell count <350 cells/mm³, the optimal TLC threshold was 1500 cells/mm³, which was similar to the figure presented in this observational study. As for predicting a CD4 cell count <500 cells/mm³, TLC obtained a high diagnostic performance (area under ROC curve, 0.82) as well with a sensitivity of 0.70 (95% CI, 0.67–0.73) and a specificity of 0.80 (95% CI, 0.73–0.87).

Conclusions

When considering the antiretroviral therapy for HIV-infected Chinese individuals, total lymphocyte count can be considered as an inexpensive and easily available surrogate marker for predicting two clinically important thresholds of CD4 count of 350 cells/mm³ and 500 cells/mm³.     

CD4 and Viral Load Dynamics in Antiretroviral-Na?ve HIV-Infected Adults from Soweto,South Africa: A Prospective Cohort

BackgroundCD4 count is a proxy for the extent of immune deficiency and declines in CD4 count are a measure of disease progression. Decline in CD4 count is an important component: for estimating benefits of ARV treatment; for individual level counselling on the rapidity of untreated disease progression and prognosis; and can be used in planning demand for health services. Our objective is to report CD4 decline and changes in viral load (VL) in a group of HIV-infected adults enrolled in a randomized trial of preventive treatment for TB in South Africa where clade C infection predominates.MethodsHIV-infected, tuberculin skin test positive adults who were not eligible for antiretroviral (ARV) treatment were randomized to a trial of preventive treatment from 2003–2005. VL and CD4 count were assessed at enrollment and CD4 counts repeated at least annually. During follow-up, individuals whose CD4 counts decreased to <200 cells/mm³ were referred for antiretroviral therapy (ART) and were analytically censored.Results1106 ARV naïve adults were enrolled. Their median age was 30 years and male to female ratio was 1∶5. Median baseline CD4 count was 490 cells/mm³ (IQR 351–675). The overall mean decline in CD4 count was 61 cells/mm³ per annum. Adjusting for age, gender, baseline hemoglobin, smoking and alcohol use had little impact on the estimate of CD4 decline. However, VL at baseline had a major impact on CD4 decline. The percent decline in CD4 count was 13.3% (95% CI 12.0%, 14.7%), 10.6% (95% CI 8.8%, 12.4%), and 13.8% (95% CI 12.1%, 15.5%) per annum for baseline VLs of <10,000 (N = 314), 10,001–100,000 (N = 338), >100,000 (N = 122) copies/ml.ConclusionsOur data suggests that six and a half years will elapse for an individual''s CD4 count to decline from 750 to 350 cells/mm³ in the absence of ART.     

DNA content of spermatozoa in different genetic lines of turkeys

The variability of DNA content in turkey spermatozoa of four different lines and its correlation with body weight and sperm concentration were studied. In lines selected for lower body weight the DNA content was 2.034 and 2.036 pg per spermatozoon. In lines selected for higher body weight the DNA content was 2.267 and 2.370 pg per spermatozoon. Sperm concentration in 1 mm³ of semen, however, was higher in lines with a lower body weight (6.08–6.21 million) in comparison with lines selected for higher body weight (5.46–5.67 million). The correlations between the DNA content and sperm concentration were negative (r ranged from ?.457 to ?.860).     

Morphological, Biochemical and Molecular Characterization of Trichoderma harzianum Isolates for their Efficacy as Biocontrol Agents

The random amplified polymorphic DNA (RAPD) procedure was used to examine the genetic variability among 8 isolates of Trichoderma harzianum , and their ability to antagonize Sclerotium rolfsii using a dual culture assay was correlated with RAPD profiles. Eight oligodeoxynucleotide primers were selected for the RAPD assays, which resulted in 86 bands for 8 isolates of T . harzianum . The data were entered into a binary matrix and a similarity matrix was constructed using the DICE similarity (SD) index. An unweighted pair grouping mathematical averaging (UPGMA) cluster based on SD values was generated using the NTSYS computer program. A mean coefficient of similarity obtained for pairwise comparisons was c. 30% and it showed that the variability among the isolates of T. harzianum was very high. Using the dual culture method in antagonism experiments, the T. harzianum isolates were classified in to antagonism classes. Further, T. harzianum isolates were screened for chitinase and β-1,3-glucanase activity. RAPD was efficient in demonstrating the high intraspecific genetic variation among isolates. The dendrogram did not show the grouping of isolates by their level of antagonism. Relationship among polymorphism existent, the aggressiveness and the origin of isolates were not found.     

Filamentous growth ofPseudomonas aeruginosa

Summary The growth of two strains ofPseudomonas aeruginosa in stirred batch cultures was monitored by optical density, DNA concentration, and acridine orange direct cell count measurements. Growth of adherent bacteria in pure culture was also observed on suspended glass discs by light and scanning electron microscopy. Strain MUCOID produced significant numbers of filamentous cells in broth culture and in the adherent population, while strain PAO 381 did not produce elongated cells. Filamentous growth of MUCOID could be prevented by the addition of 5 × 10^–2 M Mg²⁺. However, the addition of 0.66 mM EDTA caused an increased proportion of the population (>50%) of MUCOID cells to become filamentous in broth culture. The results are discussed and related to theories regarding bacterial plasticity, and filamentation of normally bacillary cells.