Microbiological evaluation of the intraruminal in sacculus digestion technique

The influence of nature of the feed sample, feeding frequency and pore size on the influx of bacteria and protozoa into synthetic fiber bags suspended in the rumens of sheep fed different diets was studied. Counts of total culturable bacteria in bags with a pore size of 10 microns were less than 30% of the ruminal counts for animals that were fed the lucerne hay and high-roughage diets. The maximum count (62 and 82% of the ruminal count) for these specific diets was obtained by using bags with a pore size of 53 microns. Protozoal counts in bags with pore sizes of 30 and 53 microns were equal to or higher than the ruminal counts for the lucerne hay and high-roughage diets but less than half of the ruminal count for the low-roughage diet. An interaction between incubation time, feeding frequency of the host animals, and the microbial populations developing inside the bags was also demonstrated. The results clearly show that the microbial population inside the bag differed from that of the surrounding ruminal ingesta and that caution must be taken in interpreting results on feed evaluation and especially on rates of degradation when using the in sacculus technique. Factors influencing the influx of bacteria and protozoa into bags with different pore sizes and containing a variety of substrates are discussed together with suggestions for the use of this technique.     

Elasticity of the sacculus of Escherichia coli.

Preparations of purified peptidoglycan of Escherichia coli (i.e., sacculi) were studied by low-angle laser light scattering. Control experiments and theoretical calculations based on the Rayleigh-Gans theory showed that the mean sacculus surface area could be accurately inferred from measurements with our apparatus by using computer routines developed previously. Large changes in the mean saccular surface area resulted from alterations in the stress caused by varying the net charge on the sacculi. The net charge was affected by altering the suspending medium pH, causing carboxyl and amino groups in the peptidoglycan to gain or lose protons, or by acetylation or succinylation of the amino groups. A preponderance of either plus or minus charges caused an expansion of the mean sacculus surface area. The largest increase in area probably represents the elastic limit of the peptidoglycan and was 300% above the area of isoionic sacculi. This degree of expansion is consistent with possible conformations of the intact peptidoglycan structure without necessitating rupture of the wall fabric. Our findings concerning saccular elasticity provide support for the surface stress theory. It provides a mechanism so that bacteria can grow and divide while maintaining turgor pressure, without the necessity of having and using proteins to do the mechanical work.     

Arrangement of glycan chains in the sacculus of Escherichia coli.

A novel of Escherichia coli endopeptidase was used for a selective partial hydrolysis of the peptide bridges which interlink the glycan chains in E. coli sacculi. The loosening of the murein network revealed, in the electron microscope, a preferential orientation of the glycan chains, more or less perpendicular to the length axis of the cell. Control incubations with E. coli transglycosylase or egg-white lysozyme did not leave ordered structures behind.     

ATPase activity of myosin in hair bundles of the bullfrog''s sacculus.

Mechanoelectrical transduction by a hair cell displays adaptation, which is thought to occur as myosin-based molecular motors within the mechanically sensitive hair bundle adjust the tension transmitted to transduction channels. To assess the enzymatic capabilities of the myosin isozymes in hair bundles, we examined the actin-dependent ATPase activity of bundles isolated from the bullfrog's sacculus. Separation of 32P-labeled inorganic phosphate from unreacted [gamma-32P]ATP by thin-layer chromatography enabled us to measure the liberation of as little as 0.1 fmol phosphate. To distinguish the Mg(2+)-ATPase activity of myosin isozymes from that of other hair-bundle enzymes, we inhibited the interaction of hair-bundle myosin with actin and determined the reduction in ATPase activity. N-ethylmaleimide (NEM) decreased neither physiologically measured adaptation nor the nucleotide-hydrolytic activity of a 120-kDa protein thought to be myosin 1 beta. The NEM-insensitive, actin-activated ATPase activity of myosin increased from 1.0 fmol x s-1 in 1 mM EGTA to 2.3 fmol x s-1 in 10 microM Ca2+. This activity was largely inhibited by calmidazolium, but was unaffected by the addition of exogenous calmodulin. These results, which indicate that hair bundles contain enzymatically active, Ca(2+)-sensitive myosin molecules, are consistent with the role of Ca2+ in adaptation and with the hypothesis that myosin forms the hair cell's adaptation motor.     

Microbiological evaluation of processed rice consumed in Japan

Samples of processed rice from four different brands showed counts of mesophilic aerobic bacteria from 1 × 10² to 5 × 10³ for milled rice and from 1 × 10⁶ to 6.8 × 10⁶ cells g^–1 for brown rice. Rice seeds contaminated by milling had extensive counts including faecal coliforms. In cooked rice, no microbial growth was noted during 4 days at room temperature (spring season, 15–20 °C) and at least 2 weeks at 4 °C. No contamination was detected in cooked rice packs. A rapid and highly reliable procedure for detection of microorganisms in cooked rice is proposed.     

Interactions of Escherichia coli membrane lipoproteins with the murein sacculus.

Bifunctional cross-linking reagents were used to identify cell envelope proteins that interacted with the murein sacculus. This revealed that a number of [3H]leucine-labeled proteins and [3H]palmitate-labeled lipoproteins were reproducibly cross-linked to the sacculus in plasmolyzed cells. The results suggested that most of the cell envelope lipoproteins, and not only the murein lipoprotein, mediate interactions between the murein sacculus and the inner and/or outer membrane of the cell.     

Receptor current in ciliate cells of the frog sacculus

The present study aims to provide further contribution towards identifying the ions which actually sustain the receptor current in labyrinthine sensory cells. The experiments were carried out on isolated sacculi of the frog. The macular epithelium of the sacculus was positioned in a two compartment chamber which allows the fluid bathing the inside and the outside of the sacculus to be replaced separately with solutions having different ionic composition. The potential across the epithelium was clamped to zero and both the receptor current and the postsynaptic potentials in response to mechanical stimuli were first recorded when the endolymphatic fluid had a normal ionic composition and then, at different time intervals, after replacing the endolymph with solutions deprived of K+ (replaced with Na+, Rb+, Cs+ and Ca++) and Na+ (replaced with choline and saccharose). The results have shown that both the receptor current and the postsynaptic potentials are abolished after replacing the endolymphatic K+ with Na+, Cs+ or Ca++, whereas are partially preserved when K+ is replaced with Rb+. These findings strongly suggest that the receptor current in labyrinthine sensory cells is carried almost exclusively by K+ and that this current flows across specific K-channels.     

Microbiological evaluation of wet and dry floor sanitization systems in hospital patient rooms.

A new system for sanitizing floors in hospital patient rooms has been developed. The method consists of dry dusting with a cotton-blend, chemically treated (10% by dry mop weight) dust mop. This method was compared with a conventional cleaning protocol consisting of an initial predusting with the same nongermicidal chemical (3% by dry mop weight) followed by wet mopping with a fresh solution of a quaternary ammonium disinfectant-detergent. Each of six rooms was sampled by using RODAC plates (Becton Dickinson Labware, Oxnard, Calif.) on 10 consecutive days for each of the two methods. The study was initially performed during the summer and then repeated during the winter. Results imply that there is no significant difference between the new dry method (81.9% CFU reduction) and the conventional method (83.1% CFU reduction). Furthermore, the initial dry dusting step in the conventional method accounted for virtually all of the reduction by that method. Thus, wet mopping with a germicidal chemical produced no additional significant reduction of natural microbial populations on environmental surfaces beyond that achieved by dry dusting with dust-suppressant chemicals.     

DNase I digestion as a tool for the quantitative evaluation of C-heterochromatic-DNA in situ.

The amount of DNA resisting the C-banding pre-treatments (C-heterochromatic-DNA) was found to account for the interspecific differences of genome size in different Primate groups. The evaluation of this parameter is therefore of great interest in cytotaxonomy. In this work, DNase I digestion was used instead of the pre-treatments C-banding, in an attempt to set up a suitable method for the quantitative evaluation of C-heterochromatic-DNA in both metaphase chromosomes and interphase chromatin. In fact DNase I is known to preferentially digest "active or potentially active" chromatin, and the highly repetitive and inactive DNA in C-heterochromatin should characteristically resist DNase I cleavage. As a model system, differently fixed mouse splenocytes were treated with DNase I for various times, and the digestion was monitored by flow cytometry after propidium iodide staining. In addition, mouse metaphase preparations from lymphocyte cultures were also digested with DNase I, and the amount of residual DNA was evaluated by static microfluorometry. Under controlled conditions of fixation, enzyme concentration, time and temperature, the same limit-digest can be obtained in both interphase nuclei and metaphases, which corresponds to the amount of residual DNA after C-banding and has a C-banding-like pattern in chromosomes.     

Microbiological aspects of anaerobic digestion of swine slurry in upflow fixed-bed digesters with different packing materials

Samples taken from liquid influents, liquid effluents, bottom sediments and biofilms attached to the supports of three laboratory anaerobic, fixed-bed, upflow digesters, filled with wood chips, PVC or expanded-clay support media and fed with swine slurry, were tested microbiologically. The numbers of anaerobic heterotrophic, anaerobic cellulolytic, acidogenic-peptone-glucose fermenting and methanogenic bacteria were determined. For each digester the biogas production was monitored.The highest biogas production, referred to the volatile solids concentration in the feed, was obtained in the digester with wood chips, while production was almost nil in the digester with expanded clay.     

Microbiological evaluation of the effects of hyperbaric oxygen on periodontal disease

The term periodontitis indicates a variety of clinical manifestations of infectious disorders in which the supporting tissues of the teeth are attacked. The initiation and progression of periodontal disease are attributed to the presence of elevated levels of pathogenic bacteria within the gingival crevice. Approaches to periodontal treatment range from surgical to regenerative therapy and anti-infective chemotherapy. Anti-infective drug therapy should be rationally based on the composition of the pathogenic microbiota. It is also important to recognize that the periodontopathic plaque constitutes a bacterial biofilm infection that may render the resident microorganisms more resistant than the same organisms grown planktonically. Hyperbaric oxygen (HBO) has been successfully used in several medical applications. The therapeutic effect is related to elevated partial oxygen pressure in the tissues. The aim of this study was to evaluate the effects of HBO on a selected number of patients suffering from adult chronic periodontitis in comparison with surgical intervention (scaling and root planning, SRP), as well as the effects of a combination of both therapies on the evolution over time of the microflora of the periodontal pockets. Bacteria were detected either by culture or by a molecular method (PCR). Microbiological data indicate that the combination of HBO and SRP substantially reduced (by up to 99.9%) the gram-negative anaerobe loads of the subgingival microflora. The low values of pathogens persisted for at least two months after the therapy. HBO or SRP alone produced a temporarily more limited effect on periodontal anaerobes. Additional experimental confirmation of these results was provided by molecular detection of the main periodontopathogenic bacteria with a significant reduction in the number of dental sites which harboured them. It is also shown that HBO both alone and in combination with SRP reduced the Gingival Index value to zero and gingival health persisted for 3 months at least. Thus, in parallel with the loss of periodontopathogenic bacteria, a substantial improvement in oral health was observed. In conclusion, this study has shown that HBO may represent a useful aid, especially in combination with SRP, as far as non-surgical periodontal therapy is concerned.     

Turnover and recycling of the murein sacculus in oligopeptide permease-negative strains of Escherichia coli: indirect evidence for an alternative permease system and for a monolayered sacculus.

Turnover of murein in oligopeptide permease-negative Escherichia coli cells appeared to be minimal or nonexistent. In one strain in which it was possible to measure turnover during the first generation of chase, it was found that the rate of turnover was constant throughout a chase of three generations. This result suggests that an "inside-to-outside" mode of growth of the sacculus does not occur in E. coli. Turnover, though minimal, was significantly higher from cells labeled uniformly than from cells labeled only in the lateral wall, suggesting that a significant portion of the observed turnover is related to cell separation. Actually, turnover only appeared to be minimal in opp mutant strains. Tripeptides were being released by turnover at a rate of about 50% per generation and then were efficiently recycled. This suggests that in addition to opp, a low-affinity uptake system for tripeptide derived from the sacculus may exist.     

On the architecture of the gram-negative bacterial murein sacculus

The peptidoglycan network of the murein sacculus must be porous so that nutrients, waste products, and secreted proteins can pass through. Using Escherichia coli and Pseudomonas aeruginosa as a baseline for gram-negative sacculi, the hole size distribution in the peptidoglycan network has been modeled by computer simulation to deduce the network's properties. By requiring that the distribution of glycan chain lengths predicted by the model be in accord with the distribution observed, we conclude that the holes are slits running essentially perpendicular to the local axis of the glycan chains (i. e., the slits run along the long axis of the cell). This result is in accord with previous permeability measurements of Beveridge and Jack and Demchik and Koch. We outline possible advantages that might accrue to the bacterium via this architecture and suggest ways in which such defect structures might be detected. Certainly, large molecules do penetrate the peptidoglycan layer of gram-negative bacteria, and the small slits that we suggest might be made larger by the bacterium.     

Evidence for diffuse growth of the cylindrical portion of the Escherichia coli murein sacculus.

High-resolution autoradiography of thin sections of Escherichia coli cells whose murein was pulse-labeled with [3H]diaminopimelic acid after a period of diaminopimelic acid deprivation indicated that elongation of the murein sacculus occurs by a multisite (diffuse) process. Upon chasing, radioactivity in polar murein was stable, whereas radioactivity in cylindrical murein was reduced, indicating that diffuse intercalation of new murein occurred during cell elongation. Elongation and septation were shown to be overlapping processes.