Bacterial flora in the digestive tract of cattle. I. Comparison of nonselective culture medium and changes in fecal bacterial flora with age.

Studies were made on nonselective culture medium and the method of culture for the investigation of the bacterial flora in the digestive tract of cattle. With their results, further studies were done to clarify changes in the fecal bacterial flora in eight calves less than 6 months of age with the lapse of time. Three roll-tube media were used in the gas jet method. They were modified VL agar (VL medium), rumen fluid glucose cellobiose agar (RGCA medium), and Medium 10 (M 10). Moreover, glucose liver blood agar (BL medium) was used in the anaerobic jar method. In this method the steel wool method was applied after the substitution of carbon dioxide. Of the four media used, VL medium was proved to be the most efficient. It was followed by RGCA medium and M 10. BL medium was much less efficient than any other medium. When the fecal bacterial flora was examined in calves for changes with the advance in age, the total bacterial count and the enteric bacterial count decreased in the second half of the experimental period. The lactobacillary group count remained almost at a constant level of 7 approximately 9 (logarithmic value) per gram in breast-fed calves, but decreased to a level of 5 (logarithmic value) per gram in bottle-fed calves at about 2 months of age or later. The streptococcal group count showed no particular tendency to change. When the organic acid contents of the feces were estimated in calves in every stage of growth, the amount of total organic acids and that of propionic acid were larger in bottle-fed than in breast-fed calves.     

Effect of γ-Irradiation on the Microflora of Freshwater Fish: I. Microbial Load,Lag Period,and Rate of Growth on Yellow Perch (Perca flavescens) Fillets

Microbial flora were compared in irradiated and nonirradiated yellow perch fillets. These studies included effects of irradiation on the total microbial population, the lag phase, and rate of growth in this freshwater fishery product. The work was conducted concurrently with sensory and chemical evaluation, and constituted part of an investigation designed to evaluate the effect of substerilization doses (0.3 and 0.6 Mrad) of Co⁶⁰ γ rays on the storage life of yellow perch fillets at 1.0 or 6.0 C. In five storage tests, total plate counts prior to irradiation did not exceed 8.7 × 10⁵ per gram of sample; this count was reduced nearly 100% by irradiation with either 0.3 or 0.6 Mrad. Progressively lower maximal bacterial populations and lengthened lag phases were obtained as more radiation was used. The growth rate of the population did not appear to decrease significantly. Microbial data obtained in these studies confirmed the sensory and chemical studies, by indicating that irradiation can significantly extend the refrigerated shelf life of freshwater fish.     

Effect of γ-Irradiation on the Microflora of Freshwater Fish: I. Microbial Load, Lag Period, and Rate of Growth on Yellow Perch (Perca flavescens) Fillets

Microbial flora were compared in irradiated and nonirradiated yellow perch fillets. These studies included effects of irradiation on the total microbial population, the lag phase, and rate of growth in this freshwater fishery product. The work was conducted concurrently with sensory and chemical evaluation, and constituted part of an investigation designed to evaluate the effect of substerilization doses (0.3 and 0.6 Mrad) of Co⁶⁰ γ rays on the storage life of yellow perch fillets at 1.0 or 6.0 C. In five storage tests, total plate counts prior to irradiation did not exceed 8.7 × 10⁵ per gram of sample; this count was reduced nearly 100% by irradiation with either 0.3 or 0.6 Mrad. Progressively lower maximal bacterial populations and lengthened lag phases were obtained as more radiation was used. The growth rate of the population did not appear to decrease significantly. Microbial data obtained in these studies confirmed the sensory and chemical studies, by indicating that irradiation can significantly extend the refrigerated shelf life of freshwater fish.     

Bacterial Cell Production from Hexadecane at High Temperatures

On mineral medium with hexadecane as the sole carbon source, stable mixed bacterial enrichment cultures were obtained from soil inoculum at 25, 35, 45, 55, and 65 C. Cell yields (grams of dry cells per gram of hexadecane) were determined for each of the enrichment cultures grown at the temperature at which they were enriched, and also for the 55 and 65 C cultures grown at various temperatures. In all cases, cell yields decreased with increasing growth temperature. The highest yield obtained at 65 C was 0.26, and the lowest yield obtained at 25 or 35 C was 1.02. Slower growth was observed at higher temperatures.     

Response of the Extract-Release Volume and Water-Holding Capacity Phenomena to Microbiologically Spoiled Beef and Aged Beef

The aging of ground beef was effected by storing in gas-impermeable, sterile plastic bags with incubation at 7 and 15 C. Control meat from the same preparations was wrapped in aluminum foil and stored at the same temperature. In three experiments where control meat was tested, aged meat did not attain a log bacterial number of ca. 8.4 per gram until an average of 6 days after this level was reached in control meats. This degree of difference was shown in values for both extract-release volume (ERV) and water-holding capacity. The previously reported ERV value of around 25, which was found to correspond to an average log bacterial number of ca. 8.5 per gram for ground beef allowed to spoil in aluminum foil and freezer paper, was approximated for aged meats, which required an average of 9.7 days to attain this number compared with 4.1 days for unaged meats. Plate count methods indicated the predominant flora of aged beef to be gram-negative, facultatively psychrophilic rods.     

Effect of Controlled Atmosphere on Growth of Mold on Synthetic Media and Fruit

Growth of seven spoilage molds on agar plates at several temperatures in both controlled atmosphere (CA) and in air was studied. Each mold responded somewhat differently to CA at each temperature; however, there were some general tendencies. The lag phase was generally increased by CA and, in some cases, was substantially extended when incubation was just above the minimum growth temperature. The mycelial structure of molds seems to be different when grown in CA than when grown in air. With only two exceptions of 24 holding conditions, the maximum amount of mycelia was always less in CA than in air. Spore development varied with each mold at each temperature; generally, it was considerably less in CA than in air. CA storage of cherries above 34 F (1 C) did not retard mold infection to any extent; at 34 F, mold growth was inhibited and storage life was extended several days as compared to air storage. CA storage of strawberries at 34 F resulted in a mold-free product after 7 days of incubation, whereas the air-stored berries were slightly infected. However, when mishandled berries showing some mold growth were stored at 34 F, CA did not stop further mold growth.     

Potential Role of Pyrophosphate:Fructose 6-Phosphate Phosphotransferase in Carbohydrate Metabolism of Cold Stored Tubers of Solanum tuberosum cv Bintje

To gain a better understanding of the mechanism of cold induced sweetening, sugar accumulation in potato, Solanum tuberosum cv Bintje, was compared to the maximum activity of inorganic pyrophosphate (PPi):fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) and the concentration of two regulatory metabolites. Mature tubers accumulated reducing sugars and sucrose at an almost linear rate of 13.4 and 5.2 micromole per day per gram dry weight at 2°C and 4.5 and 1.3 micromole per day per gram dry weight, respectively, at 4°C. During storage at 8°C sugar accumulation was nil. Sugar accumulation was preceded by a lag phase of about 4 days. The accumulation of reducing sugars persisted for at least 4 weeks, whereas sucrose accumulation declined after 2 weeks of storage. The ratio of glucose:fructose changed concomitantly with sugar increase from 65:35 to equimolarity. The maximum activity of PPi:fructose 6-phosphate 1-phosphotransferase was 2.51 and 2.25 units per gram dry weight during storage at 2 and 8°C, respectively. The temperature coefficient of this enzyme from potatoes kept at 2 or 8°C was 2.12 and 2.48, respectively. The endogenous concentration of fructose 2,6-biphosphate increased from 0.15 to 1 nanomole per gram dry weight during storage at 2 and 4°C but remained the same throughout storage at 8°C. After exposure to 2°C an initial increase in the concentration of PPi was observed from 4.0 to 5.6 nanomoles per gram dry weight. Pyrophosphate concentration did not change during storage at 4°C but decreased slightly at 8°C. All observed changes became annulled after transfer of cold stored tubers to 18°C. These data strongly indicate that PPi:fructose 6-phosphate 1-phosphotransferase can be fully operational in cold stored potato tubers and the lack of increase in PPi concentration supports the functioning of this enzyme during sugar accumulation.     

Association of germfree mice with intestinal microfloras obtained from "normal" mice

A cultured microflora obtained from the caecum of a "normal" mouse was given to 4 groups of germfree mice and was supplied 1x, 2x, 3x and 4x respectively at 5-day intervals. Another group received a 10(-7) dilution of the caecal flora while a group associated with an 'SPF' flora served as control. The difference (measured by 8 parameters) between mice supplied with the cultured flora or with a 10(-7) dilution, both given once only, was small. Supplying the flora 3x resulted in more 'normal' mice compared with mice which received the flora once or twice. The caeca of specified-pathogen-free mice contained more bacteria per gram (microscopic bacterial count), less aerobic and anaerobic bacteria per gram (viable counts), while the yield as percentage of the microscopic bacterial count was lower as compared with the group to which a cultured flora was supplied 4 times.     

Levels of Indole-3-Acetic Acid in Lemna gibba G-3 and in a Large Lemna Mutant Regenerated from Tissue Culture

Large changes in indole-3-acetic acid (IAA) levels occur during growth of Lemna gibba G-3 in sterile culture. The levels of IAA were measured in plants during a 45 day growth cycle using HPLC and isotope dilution analysis followed by selected ion current monitoring GC-MS analysis with ¹³C₆-IAA as the internal standard. Even though the rate of plant growth remained constant over the entire growth period, IAA levels ranged from a high of 222 to a low of 6 nanograms per gram fresh weight. A Lemna mutant (jsR₁) which has a giant phenotype was obtained by regeneration from primary callus cultures. Microspectrofluorometry of diamidino-2-phenylindole stained cells showed that jsR₁ has the same amount of DNA per nucleus as the parent line (PL). All jsR₁ cell types measured are about 1.5 times larger than in PL. The endogenous levels of IAA per gram fresh weight were higher in jsR₁ at several stages of the plant culture cycle as compared to PL. This difference ranged from 1.2 to over 100 times as much. While PL showed only one high peak at day 9, jsR₁ had IAA levels of 480 and 680 nanograms per gram fresh weight at days 9 and 45, respectively. Throughout the midculture stage of the growth cycle (20-28 days) both jsR₁ and PL had IAA levels in the range of 9 to 14 nanograms per gram fresh weight. In contrast to PL, at day 45, jsR₁ had no detectable ester or amide conjugates of IAA. These changes in IAA levels were determined in sterile plant cultures and thus cannot be attributed to bacterial or fungal activity.     

Isolation and identification of adherent epimural bacteria during succession in young lambs.

Successive changes in aerobic and anaerobic bacterial counts and changes in the generic composition of the epimural community in lambs from 1 to 10 weeks were determined. Bacterial culture counts revealed a predominantly anaerobic community, with the mean anaerobic count being 1.4 X 10(7) CFU/cm2 of tissue surface. The aerobic count was highest at 1 week of age and declined significantly thereafter to a mean of 1.8 X 10(4) CFU/cm2, thus representing only 0.13% of the mean anaerobic count after week 1. Of the 345 strains isolated anaerobically at 1, 2, 4, 6, 8, and 10 weeks of age, 47, 32, 12, 32, 2, and 5% were capable of growth in a partially reduced medium, indicating a reduction in the number of facultative anaerobes with time. The majority of isolated strains were identified as belonging to genera commonly isolated from rumen contents. In some instances, however, strains did not correspond to previously described species, and some genera were present in proportions different from those expected in rumen fluid. At three of the sampling times, one genus was dominant, constituting 45 to 55% of the isolates. These dominant isolates were Streptococcus bovis, Bacteroides sp., and an anaerobic Streptococcus sp. for weeks 1, 2, and 10, respectively. During the transition period (weeks 4 to 8), two or more groups were codominant.     

Isolation and identification of adherent epimural bacteria during succession in young lambs

Successive changes in aerobic and anaerobic bacterial counts and changes in the generic composition of the epimural community in lambs from 1 to 10 weeks were determined. Bacterial culture counts revealed a predominantly anaerobic community, with the mean anaerobic count being 1.4 X 10(7) CFU/cm2 of tissue surface. The aerobic count was highest at 1 week of age and declined significantly thereafter to a mean of 1.8 X 10(4) CFU/cm2, thus representing only 0.13% of the mean anaerobic count after week 1. Of the 345 strains isolated anaerobically at 1, 2, 4, 6, 8, and 10 weeks of age, 47, 32, 12, 32, 2, and 5% were capable of growth in a partially reduced medium, indicating a reduction in the number of facultative anaerobes with time. The majority of isolated strains were identified as belonging to genera commonly isolated from rumen contents. In some instances, however, strains did not correspond to previously described species, and some genera were present in proportions different from those expected in rumen fluid. At three of the sampling times, one genus was dominant, constituting 45 to 55% of the isolates. These dominant isolates were Streptococcus bovis, Bacteroides sp., and an anaerobic Streptococcus sp. for weeks 1, 2, and 10, respectively. During the transition period (weeks 4 to 8), two or more groups were codominant.     

Bacterial Distribution and Phylogenetic Diversity in the Changjiang Estuary before the Construction of the Three Gorges Dam

The bacterial community structure in the Changjiang estuary was studied for comparison with future changes, related to the construction of the Three Gorges Dam. Population densities of bacteria in the surface water at station C1 estimated by CFU on marine agar plates and by DAPI direct count, were 2.8 x 10(4) ml(-1) and 4.2 x 10(5) ml(-1), respectively. Physicochemical properties of water, such as temperature and salinity, suggested that station C1 was affected by freshwater from the Changjiang River. Cluster analysis of the PCR-RFLP patterns obtained from 9 samples showed that the bacterial community structure at station C1 was different from the structure at the other stations. Bacterial diversity in the surface water at station C1 was studied based on the genotypes of the 250 clones of 16S rRNA, and on the phenotypes generated on Biolog GN plates for 70 isolates. Sequences of bacteria from two common marine groups, alpha- and gamma-Proteobacteria, were frequently observed. Some other divisions, including the beta-Proteobacteria, C/F/B group, low G+C gram positive, high G+C gram positive, chloroplasts, and relatives of Verrucomicrobia were also observed. The putative dominant species based on both genotype and phenotype analyses were close relatives of Alteromonas macleodii or Roseobacter spp. These results reflected the nutrient-rich environment at station C1.     

Storage Stability of Clostridium botulinum Toxin and Spores in Processed Cheese

Growth initiated from detoxified spores of Clostridium botulinum 62A resulted in toxin production of 50 to 10,000 mouse lethal doses (MLD) per gram of processed soft surface-ripened cheese. Regular assays during subsequent storage of toxic samples at 2 to 4 C revealed a characteristic two- to fivefold increase in toxin titer during the initial 1 week to 12 months of storage. Thereafter, the toxin titer remained constant for 2 to 4 years, after which the toxicity declined rapidly. At the end of 6 years of storage at 2 to 4 C, the samples still contained 20 to 5,000 MLD of toxin per gram, with the usual toxin level at 200 to 500 MLD. Toxic culture filtrates of C. botulinum incorporated into cheese and stored at 30 C for 60 days showed no decline in toxin in processed type I cheese, but toxin decreased slightly in processed type II and type III cheese. The surface flora of these cheeses did not attack but, on the contrary, protected C. botulinum toxin during storage at 30 C. Initial difficulties in recovering C. botulinum organisms from type I cheese were traced to growth inhibitory activity which could be removed by washing with distilled water in a centrifuge. Viable spores or vegetative cells could be recovered from all samples after 4 to 5 years of storage at 2 to 4 C. After 6 years, organisms were recovered from all except three samples of type I cheese. Two other samples showed a large decrease in viable organisms. In type III cheese, spores remained remarkably stable for 6 years at the level of the initial inoculum, i.e., approximately 10(5) spores per gram.     

Resistance of Bacillus subtilis Spores to Inactivation by Gamma Irradiation and Heating in the Presence of a Bactericide: I. Suitability of Viable Count Procedures

A statistical evaluation of viable count procedures utilized for obtaining treatment survival curve data for Bacillus subtilis NCTC 8236 spores is described. Within the various recovery conditions tested, incubation on nutrient agar containing 1% dextrose for 48 hr at 37 C was found to promote the highest count of viable spores surviving a variety of bactericidal treatments involving gamma irradiation, heat, and chlorocresol. The count of viable spores on the medium was not significantly altered when the dextrose was added to the nutrient agar either before autoclaving or aseptically at 50 to 55 C from a solution sterilized by filtration. The volume of medium which promoted the highest count of viable spores was 20 ml per 85 mm of diameter in disposable plastic plates. Counts of viable spores were reproducible on successive batches of media. The carry-over of variable concentrations of chlorocresol into the medium from serial dilutions affected the count of viable spores. Spores in the aqueous stock suspension used for all experiments were uniformly distributed after shaking and did not diminish significantly in viability after 16 months of storage at 5 C. Grouping of indexes of dispersion, calculated from quintuplicate plate colony counts, indicated that the suitability of the viable count procedures, employed for the enumeration of spores surviving the various bactericidal treatments, tended to diminish as the level of spore inactivation exceeded 95%.     

Assessment of mouse strain on bacterial translocation from the gastrointestinal tract

The translocation of indigenous bacteria from the gastrointestinal tract to the mesenteric lymphnodes was compared in ten strains of mice. Indigenous Escherichia coli were cultured from the mesenteric lymphnodes of only two of the six mouse strains examined. Thus, spontaneous translocation of indigenous enteric bacteria across the intestinal barrier did not occur to any significant extent in any of the mouse strains examined. Since bacterial overgrowth in the gastrointestinal tract promotes bacterial translocation, bacterial translocation was tested in ten mouse strains including B10 series after antibiotic-decontaminated and subsequent colonization with streptomycin-resistant E. coli C25. E. coli C25 populated the ceca of the mice at levels of 10(8) to 10(9) per gram and translocated to 90-100% of the mesenteric lymphnodes with mean of 10(1.13) to 10(1.86) per mesenteric lymphnode. However, there were no significant differences between mouse strains as to the translocation incidence or the numbers of viable E. coli C25 per mesenteric lymphnode. Thus, genetic differences between mouse strains did not influence bacterial translocation from the gastrointestinal tract to the mesenteric lymphnodes.     

Postharvest behaviour of five Sardinian prune varieties as affected by immersion in heated sodium bicarbonate solution

Storage behaviour of 'Core', 'Core Columbu', 'Fradis' and 'Meloni' white prunes, and a black one ('Sighera') of Sardinian germplasm were evaluated following immersion for 0 (control), 15, 30, 45 or 60 sec in water at 20, 50, 55 or 60 degrees C with or without 2% (w/v) NaHCO3 (SBC). As international varieties, fruit from one white plum ('Shiro') and one black prune ('Stanly') were subjected to the same treatments. Fruit was harvested at commercial maturity, treated and then stored for 1 month at 5 degrees C and 90% RH followed by a simulated marketing period at 20 degrees C and 80% RH for 6 days. Fruit appearance, external damage, firmness and decay percentage were monitored after storage and SMP. Treatments did not induce rind damage (browning or discoloration) to any variety. SBC at 20, 45, 50 or 55 degrees C for 15 or 30 sec was not effective in controlling decay and compared to controls no improvement was observed. Immersion for 45 or 60 sec with SBC at all temperatures improved decay control with respect to controls and best results were obtained at 50 or 55 degrees C. Immersions at 60 degrees C improved decay control, but differences were not significant compared to the control attained with solutions of SBC heated at 55 degrees C. The overall appearance of 'Core', 'Core Columbu', 'Fradis' and 'Shiro' decreased significantly after the SMP period, especially when treated at 55 or 60 degrees C for 60 sec. Fruit shrivel was the main cause of the low rating. SBC did not affect shrivel indicating that heat treatment may be the probable cause. In general, local varieties were less affected by decay than other varieties and they performed well during storage.     

Distribution of aerobic bacteria,protozoa, algae,and fungi in deep subsurface sediments

The distribution of microorganisms in deep subsurface profiles was determined at three sites at the Savannah River Plant, Aiken, South Carolina. Acridine orange direct counts (AODC) of bacteria were highest in surface soil samples and declined to the 10⁶ to 10⁷ per gram range in the subsurface, but then did not decline further with depth. In the subsurface, AODC values varied from layer to layer, the highest being found in samples from sandy aquifer formations and the lowest in clayey interbed layers. Sandy aquifer sediments also contained the highest numbers of viable bacteria as determined by aerobic spread plate counts (CFU) on a dilute heterotrophic medium. In some of these samples bacterial CFU values approached 100% of the AODC values. Viable protozoa (amoebae and flagellates, but no ciliates) were found in samples with high bacterial CFU values. A variety of green algae, phytoflagellates, diatoms, and a few cyanobacteria were found at low population densities in samples from two of the three boreholes. Low numbers of fungi were evenly distributed throughout the profiles at all three sites. Microbial population density estimates correlated positively with sand content and pore‐water pH, and negatively with clay content and pore‐water metal concentration. A large diversity of prokaryotic and eukaryotic microorganisms was found in samples with high population densities. A survey of bacterial strains isolated from subsurface samples revealed associations of gram‐positive bacteria with high clay sediments and gram‐negative bacteria with sandy sediments. The ability to deposit lipophilic storage material (presumably poly‐ß‐hydroxybutyrate) was found in a high proportion of isolates from sandy sediments, but only rarely in isolates from high clay sediments.     

Moniliformin produced by cultures of Fusarium moniliforme var. subglutinans isolated from swine feed

Feed samples from Iowa suspected of causing vomiting and enlarged vulva as well as mortalities of swine were examined for toxigenic fungi and mycotoxins Fusarium moniliforme Sheldon and F. moniliforme Sheldon var. subglutinans Wollenew. & Reink. accounted for 43% and 18.5%, respectively, of the total count of 4.75×10⁵ propagules filamentous fungi per gram of swine feed, but representatives of various Penicillium spp. and Aspergillus spp. were also found. Eight isolates of F. moniliforme var. subglutinans from the feed produced 51–540 g of moniliformin per g on cracked corn at 25°C for six weeks. Zearalenone was not detected in these corn fermentations. Eight isolates of F. moniliforme from the feed did not produce detectable amounts of either zearalenone or monoliformin on cracked corn. Moniliformin was not detected in the feed samples.     

Lactobacilli in Ensiled High-Moisture Corn

Aerobic mesophilic bacteria, molds, yeasts, and Lactobacillus species were enumerated at various intervals of the ensiling process of high-moisture corn (HMC). A total of 466 isolates of Lactobacillus were identified to determine the species of Lactobacillus associated with ensiled HMC and remoistened corn. The numbers of aerobic bacteria in the ensiled HMC increased to 10(8) per gram within 1 week and remained at this level throughout the storage period. The numbers of aerobic bacteria in the remoistened corn increased to 10(9) per gram and remained at this level during the 60-day storage period, after which they increased rapidly. Yeasts were the predominant microflora of the ensiled HMC. Mold numbers were low in the silos, but were very high in the remoistened corn, especially toward the end of the 60-day storage period. The species of Lactobacillus most frequently isolated from the ensiled HMC were L. plantarum, 52% of the isolates; L. brevis, 19% of the isolates; L. brevis (atypical), 15%; L. fermenti, 11%; and L. buchneri, 3%. The lactic acid bacteria, therefore, are the predominant bacterial flora of ensiled HMC, as they are of other types of plant forages that undergo similar fermentations.     

Fed-batch cultivation of baker's yeast followed by nitrogen or carbon starvation: effects on fermentative capacity and content of trehalose and glycogen

An industrial strain of Saccharomyces cerevisiae (DGI 342) was cultivated in fed-batch cultivations at a specific growth rate of 0.2 h(-1). The yeast was then exposed to carbon or nitrogen starvation for up to 8 h, to study the effect of starvation on fermentative capacity and content of protein, trehalose and glycogen. Nitrogen starvation triggered the accumulation of trehalose and glycogen. After 8 h of starvation, the content of trehalose and glycogen was increased 4-fold and 2-fold, respectively. Carbon starvation resulted in a partial conversion of glycogen into trehalose. The trehalose content increased from 45 to 64 mg (g dry-weight)(-1), whereas the glycogen content in the same period was reduced from 55 to 5 mg (g dry-weight)(-1). Glycogen was consumed faster than trehalose during storage of the starved yeast for 1 month. Nitrogen starvation resulted in a decrease in the protein content of the yeast cells, and the fermentative capacity per gram dry-weight decreased by 40%. The protein content in the carbon-starved yeast increased as a result of starvation due to the fact that the content of glycogen was reduced. The fermentative capacity per gram dry-weight was, however, unaltered.