An alternative approach to the yeast extract-nalidixic acid method for determining the proportion of metabolically active aquatic bacteria

When metabolizing aquatic bacteria were determined by the conventional approach to the yeast extract-nalidixic acid method the apparent percentage of viable bacteria depended on the critical length beyond which cells were scored as viable. An alternative approach allowed the identification of an optimum critical length and the fixing of a lower limit to the true percentage of viable bacteria.     

Epiphytic bacteria: development of a method for determining respiring bacteria on leaves

A method was developed for studying the total numbers and the proportion of active bacteria on leaf surfaces. It involves staining gelatin impressions of leaves treated with an electron transport system indicator, 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT). The method is rapid, inexpensive and allows simultaneous observation of numbers, ecology and respiratory activity of epiphytic bacteria. The total numbers of epiphytic bacteria for four species of aquatic plants varied between 0.6 to 10.2 times 10⁶/cm². The proportion of active bacteria on leaves of aquatic plants ranged from 2.2 to 42.9%. The method was also applied to a comparison of surface fouling of glass slides and aquatic leaf surfaces, indicating significant differences in numbers of bacteria but little difference in the proportion active on the two surfaces.     

A new high-throughput screening method for determining active and enantioselective hydrolases

A new high-throughput screening method using fluorescein sodium salt as an indicator to obtain hydrolases with high enantioselectivity is developed, which is demonstrated to be sensitive and reliable. The results determined by the method correlate well with those from GC analysis. This method can be applied to determine activity and enantioselectivity of not only lipase and esterase, but also other enzymes which catalyze hydrolysis reaction releasing proton, such as the protease or amidase. Because of the application of small amount of optically pure enantiomers, screening large libraries of enzymes is allowed at low cost and in short time.     

Isolation of bacteria producing polyhydroxyalkanoates (PHA) from municipal sewage sludge

Bacterial isolates from sludge samples collected at a local municipal sewage treatment plant were screened for bacteria producing polyhydroxyalkanoates (PHA). Initially Sudan black B staining was performed to detect lipid cellular inclusions. Lipid-positive isolates were then grown in a nitrogen limitation E2 medium containing 2% (w/v) glucose to promote accumulation of PHA before the subsequent staining with Nile blue A. The positive isolates were quantified initially with a u.v. spectrophotometer, for a very large number of isolates (105) and among them high PHA-producing isolates (15) were selected and were confirmed by gas chromatographic analysis. The GC analysis showed the polymers produced by 13 of the selected isolates to be polyhydroxybutyrate (PHB), and the remaining two isolates produced polyhydroxybutyrate-co-hydroxyvalerate (PHB-co-HV) copolymer. The proportion of the PHA-positive bacterial isolates showed variability in the number of PHA accumulators during various months. The correlation of PHB production with the cell dry weight (CDW) was found to be statistically significant. The metabolism of PHB in these selected 15 isolates was studied using the Nile blue A staining, which showed an initial increase in the fluorescence followed by a decline, on further incubation. All the selected 15 isolates were classified to genus level by studying their morphological and biochemical characteristics. There were seven Bacillus species, three Pseudomonas species, two Alcaligenes species, two Aeromonas species, and one Chromobacterium species.     

Nonstaining (KOH) method for determination of gram reactions of marine bacteria.

A rapid nonstaining (KOH) method for the determination of the Gram reactions of bacteria is described, and its application to marine isolates is discussed. All gram-positive and gram-negative results obtained by Gram staining were confirmed by the KOH method. Gram-variable bacteria produced equivocal results.     

Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae)

Nickel was found to be required for expression of urease activity in batch cultures of Thiocapsa roseopersicina strain 6311, Chromatium vinosum strain 1611 and Thiocystis violacea strain 2311, grown photolithotrophically with NH₄Cl as nitrogen source. In a growth medium originally free of added nickel and EDTA, the addition of 0.1–10 M nickel chloride caused an increase in urease activity, while addition of EDTA (0.01–2 mM) caused a strong reduction. Variation of the nitrogen source had no pronounced influence on the level of urease activity in T. roseopersicina grown with 0.1 M nickel in the absence of EDTA. Only nickel, of several heavy metal ions tested, could reverse suppression of urease activity by EDTA. Nickel, however, did not stimulate and EDTA did not inhibit the enzyme in vitro. When nickel was added to cultures already growing in a nickel-deficient, EDTA-containing medium, urease activity showed a rapid increase which was not inhibited by chloramphenicol. It is concluded that the (inactive) urease apoprotein may be synthesized in the absence of nickel and can be activated in vivo without de novo protein synthesis by insertion of nickel into the pre-formed enzyme protein.     

Evaluation of analytical methods for determining the distribution of biofilm and active bacteria in a commercial heating system

Danish district heating systems have good water quality, but continue to suffer from biofouling and biocorrosion. Localisation analyses of bacteria using microautoradiography were performed for one system in order to obtain detailed information for solving these problems. A mass balance showed that 77% of the bacteria were located at surfaces, with 23% in the bulk water, and 9% of the total carbon originated from biomass, while 91% was dissolved in the bulk water. The presence of active bacteria was determined with microautoradiography which showed that biofilms contained 99% and 1% were in the bulk water. A high bacterial functional diversity was observed, with active mesophilic and thermophilic bacteria under aerobic and anaerobic conditions and with potentially corrosive biofilm bacteria present. The study reveals that by applying the activity based approach, the ratio of living and dead bacteria in the biofilm and bulk water in this type of system could be accurately determined. Also, the results emphasise that to minimise biofilm growth and biocorrosion, monitoring should be established focusing on the surfaces, since bulk water parameters do not reflect bacterial activity.     

Enumeration of potentially pathogenic bacteria from sewage sludges.

To ascertain the health risks that may be posed by the land application of sewage sludges, a scheme was devised to determine the types and numbers of pathogenic and potentially pathogenic bacteria present in sludges. A processing treatment was adapted to sludge to give a homogenate which yielded the greatest numbers of viable bacteria. Conventional methods were successful in enumerating Klebsiella, Staphylococcus, gram-negative enteric bacteria, and commonly used indicator organisms. Modifications of conventional methods improved the enumeration of Salmonella, Mycobacterium sp., fluorescent Pseudomonas sp., and Clostridium perfringens. However, Shigella methodology yielded only one isolate. Utilizing the proposed scheme, the population densities of these organisms were estimated in three domestic wastewater sludges. In light of these results, the potential impact of land application of sewage sludges is discussed.