Bacterial and Fungal Numbers in Ruminal and Cecal Contents of the Blue Duiker (Cephalophus monticola)

Total and cellulolytic bacterial and fungal numbers were determined in ruminal and cecal contents of 20 blue duikers (Cephalophus monticola). The animals were equally divided by sex and fed two diets, either high roughage or high concentrate. The mean concentration for total bacterial numbers in the rumen was 26.0 × 10⁸/g of contents, with values ranging from 2 × 10⁸/g to 93 × 10⁸/g. Cellulolytic numbers averaged 6.0 × 10⁸/g with a range of 1.5 × 10⁸/g to 24.0 × 10⁸/g. No differences related to sex or diet were found. In contrast, total bacterial numbers in the cecum differed between diets (P < 0.02), i.e., 1,046 × 10⁶ bacteria per g for animals fed the high-forage diet compared with 166 × 10⁶/g for those fed the high-concentrate diet. Cellulolytic bacterial counts in the cecal contents averaged 3.1 and 7.0% of the total counts for the high-forage and high-concentrate diets, respectively. Low concentrations of fungi were found in both ruminal and cecal contents of some, but not all, animals. Unexpectedly, concentrations of bacteria and fungi in the rumen and cecum were highly correlated with their total numbers (concentration multiplied by total weight of contents).     

Comparison of treatments for the in vitro detachment of cellulolytic bacteria from ruminal digesta samples

Four treatments were applied to ruminal digesta samples in a randomized complete block design experiment to evaluate their effect on the extent of detachment of ruminal cellulolytic bacteria. The treatments were: blending with anaerobic dilution solution (ADS), blending with ADS after a 6-h cold incubation, blending with ADS+Tween 80, and blending with ADS +Tween 80 after a 6-h cold incubation. Compared with blending in ADs alone, significant increases (p<0.2) of 10% and 12% in the number of cellulolytic bacteria were observed with the 6-h cold and the 6-h cold plus Tween 80 treatments, respectively. Tween 80 had no effect on the number of cellulolytic bacteria released from ruminal digesta samples irrespective of chilling. It was concluded that these additional treatments afforded little value over blending in buffer alone in detaching cellulolytic bacteria from ruminal digesta.     

Studies on the Inoculation and Competitiveness of a Rhizobium leguminosarum Strain in Soils Containing Indigenous Rhizobia

The competitiveness of a Rhizobium leguminosarum strain was investigated at two separate locations in field inoculation studies on commercially grown peas. The soil at each location (sites I and II) contained an indigenous R. leguminosarum population of ca. 3 × 10⁴ rhizobia per g of soil. At site I it was necessary to use an inoculum concentration as large as 4 × 10⁷ CFU ml⁻¹ (2 × 10⁶ bacteria seed⁻¹) to establish the inoculum strain in the majority of nodules (73%). However, at site II the inoculum strain formed only 33% of nodules when applied at this (10⁷ CFU ml⁻¹) level. Establishment could not be further improved by increasing the inoculum concentration even as high as 10⁹ CFU ml⁻¹ (9.6 × 10⁷ bacteria seed⁻¹). The inoculum strain could be detected at both sites 19 months after inoculation. Analysis by intrinsic antibiotic resistance patterns and plasmid DNA profiles indicated that a dominant strain(s) and plasmid pool existed among the indigenous population at site II. Competition experiments were carried out under laboratory conditions between a dominant indigenous isolate and the inoculum strain. Both strains were shown to be equally competitive.     

Structure and Composition of Biological Slimes on Paper and Board Machines

Biological slimes (biofilms) collected from the wet end of paper and board machines were examined by electron microscopy and analyzed for fatty acid composition, neutral sugar composition, and ATP. Electron microscopy revealed minuscule prokaryotic organisms (diameter, 0.2 to 0.4 μm). Larger cells morphologically resembling Sphaerotilus and Leptothrix spp. were found in slimes from machines using recycled fiber or unbleached pulp. The bacteria were embedded in a slimy matrix and often contained reserve materials microscopically resembling poly-β-hydroxybutyrate and glycogen. Fatty acid analysis of the slimes revealed bacterial signature fatty acids in concentrations equivalent to the presence of 2 × 10¹⁰ to 2.6 × 10¹² (average, 7 × 10¹¹) bacterial cells (live and dead) per g (dry weight) of slime. The slimes contained several known components of bacterial polysaccharides in addition to glucose, indicating that the slime body consisted of bacterial polysaccharides. The slimes contained uronic acids equivalent to a binding capacity of 12.5 to 50 μmol of divalent cations per g (dry weight) of slime. The uronic acid-containing polysaccharides may be responsible for the accumulation of heavy metals in the slime. Calculation of the ATP contents of the slimes resulted in an estimate of 5 × 10¹² cells per g (dry weight) of slime when calibrated with pure bacterial cultures isolated from the slimes. From electron micrographs, an estimate ranging from 1 × 10¹⁰ to 1.5 × 10¹² (average, 4 × 10¹¹) cells per g (dry weight) of slime was obtained.     

Factors Influencing the Induction of Freezing Tolerance by Abscisic Acid in Cell Suspension Cultures of Bromus inermis Leyss and Medicago sativa L

A 2-gram fresh weight inoculum of bromegrass (Bromus inermis Leyss. culture BG970) cell suspension culture treated with 7.5 × 10⁻⁵ molar abscisic acid (ABA) for 7 days at 25°C survived slow cooling to −60°C. Over 80% of the cells in ABA treated cultures survived immersion in liquid N₂ after slow cooling to −40 or −60°C. In contrast, a 6-gram fresh weight inoculum only attained a hardiness level of −28°C after 5 days of ABA treatment. Ethanol (2 × 10⁻² molar) added to the culture medium at the time of ABA addition, inhibited the freezing tolerance of bromegrass cells by 25°C. A 6-gram inoculum of both control and ABA treated bromegrass cells altered the pH of the medium more than a 2-gram inoculum. ABA inhibited the increase in fresh weight of bromegrass by 20% after 4 days. Both control and ABA (10⁻⁴ molar) treated alfalfa cells (Medicago sativa L.) grown at 25°C hardened from an initial LT₅₀ of −5°C to an LT₅₀ of −23°C by the third to fifth day after subculture. Thereafter, the cells dehardened but the ABA treated cells did not deharden to the same level as the control cells. ABA inhibited the increase in fresh weight of alfalfa by 50% after 5 days.     

Decomposition Studies in Two Central Ontario Lakes Having Surficial pHs of 4.6 and 6.6

The rates of cellulose breakdown, composition of detrital microflora, and density of bacterial populations were determined in the epilimnetic sediments and water columns of two poorly buffered, oligotrophic, Canadian Shield lakes having mean surficial pHs of 4.6 (Bat Lake) and 6.6 (Harp Lake). The decomposition rate was significantly lower in oxic sediment of the acidified lake than of the circumneutral lake, but water column rates were almost identical in the two lakes. These results are explained in terms of the groups of cellulolytic microorganisms which were observed by phase-contrast microscopy as being active at the different sites: fungi in Bat Lake water and Cytophaga-like bacteria in the water and sediment of Harp Lake. Cytophaga-like bacteria were also the main decomposers in Bat Lake sediment, but their activity was restricted at porewater pHs of <5.0. Acridine orange direct counts of bacteria in the top centimeter of sediment ranged from 3.7 × 10⁸ to 1.0 × 10⁹ per g, and counts in planktonic water samples ranged from 4.9 × 10⁵ to 1.2 × 10⁶ per ml. Bacterial densities at most sites decreased significantly (P < 0.001) from August to late October, but did not show a consistent pattern of differences related to pH.     

Production and Characterization of Amylase from Calvatia gigantea

α-Amylase (EC 3.2.1.1) was excreted by Calvatia gigantea in liquid growth media containing different sources of starch. Among the factors affecting enzyme production in shake flasks were the type and the concentration of starch and the nitrogen source supplied. Optimum cultural conditions for maximum enzyme production were: soluble starch concentration, 5%; inoculum size, 3.75 × 10⁵ conidia per ml; 5-day cultivation time at 28 to 30°C. The observed maximum yield of 81.3 U of saccharifying enzyme activity per ml of growth medium was the highest ever reported in the literature for submerged cultures. Partially purified enzyme functioned optimally at pH 4.5 to 5.5 and 53 to 58°C. The activation energy of enzymic hydrolysis of starch in the range of 20 to 40°C was 8,125 cal/mol (ca. 3.41 × 10⁴ J). The apparent K_m value of the enzyme at 25°C was 7.68 × 10⁻⁴ g/ml. Some of the properties of the enzyme under investigation were similar to those of α-amylases excreted from molds producing large amounts of the enzyme.     

Culturable Populations of Sporomusa spp. and Desulfovibrio spp. in the Anoxic Bulk Soil of Flooded Rice Microcosms

Most-probable-number (MPN) counts were made of homoacetogenic and other bacteria present in the anoxic flooded bulk soil of laboratory microcosms containing 90- to 95-day-old rice plants. MPN counts with substrates known to be useful for the selective enrichment or the cultivation of homoacetogenic bacteria (betaine, ethylene glycol, 2,3-butanediol, and 3,4,5-trimethoxybenzoate) gave counts of 2.3 × 10³ to 2.8 × 10⁵ cells per g of dry soil. Homoacetogens isolated from the terminal positive steps of these dilution cultures belonged to the genus Sporomusa. Counts with succinate, ethanol, and lactate gave much higher MPNs of 5.9 × 10⁵ to 3.4 × 10⁷ cells per g of dry soil and led to the isolation of Desulfovibrio spp. Counting experiments on lactate and ethanol which included Methanospirillum hungatei in the medium gave MPNs of 2.3 × 10⁶ to 7.5 × 10⁸ cells per g of dry soil and led to the isolation of Sporomusa spp. The latter strains could grow with betaine, ethylene glycol, 2,3-butanediol, and/or 3,4,5-trimethoxybenzoate, but apparently most cells of Sporomusa spp. did not initiate growth in counting experiments with those substrates. Spores apparently accounted for 2.2% or less of the culturable bacteria. It appears that culturable Desulfovibrio spp. and Sporomusa spp. were present in approximately equal numbers in the bulk soil. Multiple, phylogenetically-distinct, phenotypically-different, strains of each genus were found in the same soil system.     

Broad Distribution of Diverse Anaerobic Ammonium-Oxidizing Bacteria in Chinese Agricultural Soils

Anaerobic ammonium-oxidizing (anammox) bacteria have been detected in many marine and freshwater ecosystems. However, little is known about the distribution, diversity, and abundance of anammox bacteria in terrestrial ecosystems. In this study, anammox bacteria were found to be present in various agricultural soils collected from 32 different locations in China. Phylogenetic analysis of the 16S rRNA genes showed “Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia” in the collected soils, with “Candidatus Brocadia” being the dominant genus. Quantitative PCR showed that the abundance of anammox bacteria ranged from 6.38 × 10⁴ ± 0.42 × 10⁴ to 3.69 × 10⁶ ± 0.25 × 10⁶ copies per gram of dry weight. Different levels of diversity, composition, and abundance of the anammox bacterial communities were observed, and redundancy analysis indicated that the soil organic content and the distribution of anammox communities were correlated in the soils examined. Furthermore, Pearson correlation analysis showed that the diversity of the anammox bacteria was positively correlated with the soil ammonium content and the organic content, while the anammox bacterial abundance was positively correlated with the soil ammonium content. These results demonstrate the broad distribution of diverse anammox bacteria and its correlation with the soil environmental conditions within an extensive range of Chinese agricultural soils.     

Inoculation Response of Legumes in Relation to the Number and Effectiveness of Indigenous Rhizobium Populations

The response of legumes to inoculation with rhizobia can be affected by many factors. Little work has been undertaken to examine how indigenous populations or rhizobia affect this response. We conducted a series of inoculation trials in four Hawaiian soils with six legume species (Glycine max, Vigna unguiculata, Phaseolus lunatus, Leucaena leucocephala, Arachis hypogaea, and Phaseolus vulgaris) and characterized the native rhizobial populations for each species in terms of the number and effectiveness of the population for a particular host. Inoculated plants had, on average, 76% of the nodules formed by the inoculum strain, which effectively eliminated competition from native strains as a variable between soils. Rhizobia populations ranged from less than 6 × 10⁰/g of soil to 1 × 10⁴/g of soil. The concentration of nitrogen in shoots of inoculated plants was not higher than that in uninoculated controls when the most probable number MPN counts of rhizobia were at or above 2 × 10¹/g of soil unless the native population was completely ineffective. Tests of random isolates from nodules of uninoculated plants revealed that within most soil populations there was a wide range of effectiveness for N₂ fixation. All populations had isolates that were ineffective in fixing N₂. The inoculum strains generally did not fix more N₂ than the average isolate from the soil population in single-isolate tests. Even when the inoculum strain proved to be a better symbiont than the soil rhizobia, there was no response to inoculation. Enhanced N₂ fixation after inoculation was related to increased nodule dry weights. Although inoculation generally increased nodule number when there were less than 1 × 10² rhizobia per g of soil, there was no corresponding increase in nodule dry weight when native populations were effective. Most species compensated for reduced nodulation in soils with few rhizobia by increasing the size of nodules and therefore maintaining a nodule dry weight similar to that of inoculated plants with more nodules. Even when competition by native soil strains was overcome with a selected inoculum strain, it was not always possible to enhance N₂ fixation when soil populations were above a threshold number and had some effective strains.     

Evidence for the Cooccurrence of Nitrite-Dependent Anaerobic Ammonium and Methane Oxidation Processes in a Flooded Paddy Field

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) are two of the most recent discoveries in the microbial nitrogen cycle. In the present study, we provide direct evidence for the cooccurrence of the anammox and n-damo processes in a flooded paddy field in southeastern China. Stable isotope experiments showed that the potential anammox rates ranged from 5.6 to 22.7 nmol N₂ g⁻¹ (dry weight) day⁻¹ and the potential n-damo rates varied from 0.2 to 2.1 nmol CO₂ g⁻¹ (dry weight) day⁻¹ in different layers of soil cores. Quantitative PCR showed that the abundance of anammox bacteria ranged from 1.0 × 10⁵ to 2.0 × 10⁶ copies g⁻¹ (dry weight) in different layers of soil cores and the abundance of n-damo bacteria varied from 3.8 × 10⁵ to 6.1 × 10⁶ copies g⁻¹ (dry weight). Phylogenetic analyses of the recovered 16S rRNA gene sequences showed that anammox bacteria affiliated with “Candidatus Brocadia” and “Candidatus Kuenenia” and n-damo bacteria related to “Candidatus Methylomirabilis oxyfera” were present in the soil cores. It is estimated that a total loss of 50.7 g N m⁻² per year could be linked to the anammox process, which is at intermediate levels for the nitrogen flux ranges of aerobic ammonium oxidation and denitrification reported in wetland soils. In addition, it is estimated that a total of 0.14 g CH₄ m⁻² per year could be oxidized via the n-damo process, while this rate is at the lower end of the aerobic methane oxidation rates reported in wetland soils.     

Populations of Anaerobic Phototrophic Bacteria in a Spartina alterniflora Salt Marsh

Habitat-simulating media were used with the Hungate anaerobic roll tube technique to enumerate culturable anaerobic photosynthetic bacteria in sediment, tidal waters, and Spartina alterniflora plant samples collected from the salt marsh at Sapelo Island, Ga. No phototrophs were detected in samples of creekside (low marsh) sediment or in tidal waters in creekside regions. In the high marsh region, 90% of anaerobic phototrophic bacteria occurred in the top 5 mm of sediment and none were detected below 6 mm. There was a seasonal variation, with maximal populations occurring in summer and fall (mean, 4.4 × 10⁵ phototrophs g of dry sediment⁻¹) and minimal numbers occurring in winter (mean, 3.9 × 10³ phototrophs g of dry sediment⁻¹). During winter and late spring, phototrophs had a patchy distribution over the high marsh sediment surface. In contrast, during late summer they had a random uniform distribution. Tidal water collected over high marsh sediment contained an average of 8.7 × 10² phototrophs ml⁻¹, with no significant seasonal variation. Anaerobic phototrophic bacteria were also cultured from the lower stem tissue of S. alterniflora growing in both the high (4.3 × 10⁴ phototrophs g of dry tissue⁻¹) and creekside (4.9 × 10⁴ phototrophs g of dry tissue⁻¹) marsh regions. Chromatium buderi, Chromatium vinosum, Thiospirillum sanguineum, Rhodospirillum molischianum, and Chlorobium phaeobacteroides were the predominant anaerobic phototrophic species cultured from high marsh sediment. The two Chromatium species were dominant.     

Application of Real-Time PCR To Study Effects of Ammonium on Population Size of Ammonia-Oxidizing Bacteria in Soil

Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in agricultural and natural ecosystems and has a large global impact. In the past, the ecology and physiology of AOB were not well understood because these organisms are notoriously difficult to culture. Recent applications of molecular techniques have advanced our knowledge of AOB, but the necessity of using PCR-based techniques has made quantitative measurements difficult. A quantitative real-time PCR assay targeting part of the ammonia-monooxygenase gene (amoA) was developed to estimate AOB population size in soil. This assay has a detection limit of 1.3 × 10⁵ cells/g of dry soil. The effect of the ammonium concentration on AOB population density was measured in soil microcosms by applying 0, 1.5, or 7.5 mM ammonium sulfate. AOB population size and ammonium and nitrate concentrations were monitored for 28 days after (NH₄)₂SO₄ application. AOB populations in amended treatments increased from an initial density of approximately 4 × 10⁶ cells/g of dry soil to peak values (day 7) of 35 × 10⁶ and 66 × 10⁶ cells/g of dry soil in the 1.5 and 7.5 mM treatments, respectively. The population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) remained between 0.7 × 10⁹ and 2.2 × 10⁹ cells/g of soil, regardless of the ammonia concentration. A fertilization experiment was conducted in a tomato field plot to test whether the changes in AOB density observed in microcosms could also be detected in the field. AOB population size increased from 8.9 × 10⁶ to 38.0 × 10⁶ cells/g of soil by day 39. Generation times were 28 and 52 h in the 1.5 and 7.5 mM treatments, respectively, in the microcosm experiment and 373 h in the ammonium treatment in the field study. Estimated oxidation rates per cell ranged initially from 0.5 to 25.0 fmol of NH₄⁺ h⁻¹ cell⁻¹ and decreased with time in both microcosms and the field. Growth yields were 5.6 × 10⁶, 17.5 × 10⁶, and 1.7 × 10⁶ cells/mol of NH₄⁺ in the 1.5 and 7.5 mM microcosm treatments and the field study, respectively. In a second field experiment, AOB population size was significantly greater in annually fertilized versus unfertilized soil, even though the last ammonium application occurred 8 months prior to measurement, suggesting a long-term effect of ammonium fertilization on AOB population size.     

Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes

Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the species Bacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and the Clostridium lituseburense groups. Another probe was designed for the genera Streptococcus and Lactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectale group. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 × 10¹⁰ cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectale group-specific probe detected a mean of 7.2 × 10¹⁰ cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, and Streptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 × 10⁷ to 7 × 10⁸ per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.     

Influence of Cyanobacterial Hyperscum on Heterotrophic Activity of Planktonic Bacteria in a Hypertrophic Lake

The response of the planktonic heterotrophic bacterial community to the buildup and breakdown of a semipermanent, crusted, floating cyanobacterial mat, or hyperscum, that covered 1 to 2 ha was studied in a hypertrophic lake (Hartbeespoort Dam, South Africa). The initial response of bacteria in the main basin to the release of dissolved organic carbon (DOC) from the hyperscum 1 km away was an increase in activity per cell from 35 × 10⁻¹² to 153 × 10⁻¹² μg of C cell⁻¹ h⁻¹ for total cell counts, while activity per cell for metabolically active cells increased from 19 × 10⁻¹¹ to 85 × 10⁻¹¹ μg of C cell⁻¹ h⁻¹. No major population growth occurred at this stage. Later, with the continuous supply of DOC from the hyperscum, total bacterial numbers increased from 6.6 × 10⁶ to 20 × 10⁶ cells ml⁻¹, while the activity per cell declined. Metabolically active bacteria followed the same trend. Shorter-term DOC increases caused only increases in bacterial activity per cell. The data from Hartbeespoort Dam demonstrate an interesting and little-documented mechanism by which aquatic bacteria respond to increased DOC concentration and which may be universal for aquatic systems.     

Evidence for a Terpene-Based Food Chain in the Gulf of Alaska

A mixture of ¹⁴C-terpenes was prepared from conifer seedlings and introduced into fresh seawater samples taken near Seward, Alaska. Initial rates of oxidation by the indigenous bacteria were linear and faster than the rates of toluene oxidation. Turnover times were 4 to 19 days. Autoradiographic measurements with ³H-terpenes indicated that at least 10% of the 0.6 × 10⁹ to 2.7 × 10⁹ bacteria per liter present could catabolize terpenes. The rate of terpene oxidation, 24 μg of terpenes per g of cells per h with 3 μg of terpenes added per liter, was a constant function of bacterial biomass. The specific affinity of the process was estimated to be between 8.1 and 81 liters/g of cells per h, indicating a high state of induction and the probable presence of terpenes. Terpene-oxidizing bacteria were grown on [¹⁴C]alanine and added to fresh seawater samples. Transfer of the bacterial radioactivity into larger particles at a rate of 146 pg/liter per h from the 2.3 × 10⁹ organisms added indicated that any terpenes present would participate in the food chain.     

Potential Contribution of Anammox to Nitrogen Loss from Paddy Soils in Southern China

The anaerobic oxidation of ammonium (anammox) process has been observed in diverse terrestrial ecosystems, while the contribution of anammox to N₂ production in paddy soils is not well documented. In this study, the anammox activity and the abundance and diversity of anammox bacteria were investigated to assess the anammox potential of 12 typical paddy soils collected in southern China. Anammox bacteria related to “Candidatus Brocadia” and “Candidatus Kuenenia” and two novel unidentified clusters were detected, with “Candidatus Brocadia” comprising 50% of the anammox population. The prevalence of the anammox was confirmed by the quantitative PCR results based on hydrazine synthase (hzsB) genes, which showed that the abundance ranged from 1.16 × 10⁴ to 9.65 × 10⁴ copies per gram of dry weight. The anammox rates measured by the isotope-pairing technique ranged from 0.27 to 5.25 nmol N per gram of soil per hour in these paddy soils, which contributed 0.6 to 15% to soil N₂ production. It is estimated that a total loss of 2.50 × 10⁶ Mg N per year is linked to anammox in the paddy fields in southern China, which implied that ca. 10% of the applied ammonia fertilizers is lost via the anammox process. Anammox activity was significantly correlated with the abundance of hzsB genes, soil nitrate concentration, and C/N ratio. Additionally, ammonia concentration and pH were found to be significantly correlated with the anammox bacterial structure.     

Plant-Derived Oils Reduce Pathogens and Gaseous Emissions from Stored Cattle Waste

Carvacrol and thymol in combination at 6.7 mM each completely inhibited the production of short-chain volatile fatty acids and lactate from cattle waste in anoxic flasks over 23 days. Fecal coliforms were reduced from 4.6 × 10⁶ to 2.0 × 10³ cells per ml 2 days after treatment and were nondetectable within 4 days. Total anaerobic bacteria were reduced from 8.4 × 10¹⁰ to 1.5 × 10⁷ cells per ml after 2 days and continued to be suppressed to that level after 14 days. If the concentration of carvacrol or thymol were doubled (13.3 mM), either could be used to obtain the same inhibitory fermentation effect. We conclude that carvacrol or thymol may be useful as an antimicrobial chemical to control pathogens and odor in stored livestock waste.     

High-Frequency Phage-Mediated Gene Transfer among Escherichia coli Cells, Determined at the Single-Cell Level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 × 10⁻⁸ to 2 × 10⁻⁶, 1 × 10⁻⁸ to 4 × 10⁻⁸, and <4 × 10⁻⁹ to 4 × 10⁻⁸ per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 × 10⁻⁴ to 1 × 10⁻³, 9 × 10⁻⁴ to 3 × 10⁻³, and 5 × 10⁻⁴ to 4 × 10⁻³ for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

Chlorine Demand and Inactivation of Fungal Propagules

Conidia of filamentous fungi, vegetative yeast cells, and coliform bacteria were tested to determine their chlorine demand and their sensitivity to chlorine inactivation. Levels of chlorine demand for the various conidia, yeast, and coliforms were, respectively, 3.6 × 10⁻⁹ to 3.2 × 10⁻⁸, 1.2 × 10⁻⁹ to 8.0 × 10⁻⁹, and 2.5 × 10⁻¹¹ to 6.3 × 10⁻¹⁰ mg of chlorine per propagule. Preliminary evidence suggests that the chlorine demand per propagule increases as the number of propagules per milliliter decreases. In general, conidia showed greatest resistance to chlorine inactiviation, followed by the yeast and coliforms. Inactivation by chlorine was influenced by pH, with inactivation (chlorine activity) falling in the order pH 5 > 7 > 8.