Effect of minocycline on subgingival plaque bacteria

The effects of minocycline on subgingival plaque samples from patients with chronic periodontitis were investigated in vitro. Minocycline concentrations as low as 1.0 microgram/ml inhibited 95.7% of the cultivable bacteria in the samples but 256 micrograms/ml was necessary to inhibit all of the cultivable bacteria in the samples. Although up to 99.9% of bacteria in the plaque samples were killed by a 6 h exposure to 8.0 micrograms/ml of minocycline, large numbers of viable bacteria remained. These results imply that adequate reductions in the numbers of viable subgingival plaque bacteria are unlikely to occur after exposure to minocycline at concentrations attainable in gingival crevicular fluid after systemic administration.     

Effect of minocycline on subgingival plaque bacteria

The effects of minocycline on subgingival plaque samples from patients with chronic periodontitis were investigated in vitro. Minocycline concentrations as low as 1.0 μg/ml inhibited 95.7% of the cultivable bacteria in the samples but 256 μg/ml was necessary to inhibit all of the cultivable bacteria in the samples. Although up to 99.9% of bacteria in the plaque samples were killed by a 6 h exposure to 8.0 μg/ml of minocycline, large numbers of viable bacteria remained. These results imply that adequate reductions in the numbers of viable subgingival plaque bacteria are unlikely to occur after exposure to minocycline at concentrations attainable in gin-gival crevicular fluid after systemic administration.     

Microbiology of Anaerobic Sludge Fermentation: I. Enumeration of the Nonmethanogenic Anaerobic Bacteria1

An anaerobic medium containing sludge supernatant fluid and glucose was used for enumeration of bacteria from the sludge fermentation. Comparison of viable counts from several separate samples consistently showed 10 to 100 times more anaerobic than aerobic bacteria. However, viable counts of the various samples differed by as much as 10 times; this variation probably reflects a change in the natural environment or sampling errors, or a combination of the two. Direct microscopic counts yielded values of about 10(10)/ml. The discrepancy between viable (10(8) to 10(9)/ml) and direct counts may be due to large numbers of dead cells. Random isolates of representative colonies from high dilutions exhibited the ability to ferment sugars and are not likely to be methane bacteria.     

Variation in colony counts of total viable anaerobic rumen bacteria as influenced by media and cultural methods.

Volume and type of medium, carbohydrate concentration, carbohydrate ratios, and inoculum level were investigated as possible factors influencing total colony counts of anaerobic rumen bacteria obtained in roll tubes (18 by 150 mm). Colony counts were lower when the rumen fluid was clarified by centrifugation before inclusion in the medium; however, decreasing the volume of 40% rumen fluid glucose-cellobiose-starch-agar medium (RGCSA medium with 0.025% each of glucose and cellobiose and 0.05% starch, 4 ml per tube) was compared to the clarified rumen fluid medium and non-rumen fluid medium (medium 10) of Caldwell and Bryant (1966), 9 ml of each per tube. Total counts of rumen contents from sheep consuming four different types of rations were higher with the 4 ml of RGCSA medium than with the other two media. Dilution of the basal medium as a result of inoculum volume, as much as 1.5 ml per 4 ml of medium, did not appear to affect total counts. Colony counts and the simplicity of medium preparation and inoculation would favor the present method for routine use in estimating numbers of total viable anaerobic rumen bacteria, especially when large numbers of samples are involved.     

Incidence of Prosthecate Bacteria in a Polluted Stream

Water samples were collected aseptically several times throughout the year at nine stations on the Red Cedar River, a stream flowing through farmland and receiving effluent from several municipalities in central Michigan. Total prosthecate bacteria were enumerated by both direct and viable counting techniques. By direct techniques, these bacteria accounted for 0.62 to 1.1% of the total microflora during the study. The predominant type of appendaged bacteria was the caulobacters (Caulobacter, Asticcacaulis, and the fusiform caulobacter), which accounted for 64 to 93% of the total prosthecate forms. The others of importance were prosthecomicrobia (< 1 to 24%), including Prosthecomicrobium and Prosthecochloris; hyphomicrobia (< 1 to 15%), including Hyphomicrobium and Rhodomicrobium; and Ancalomicrobium (< 1 to 6%). The viable counts of heterotrophs also indicated that the caulobacters were the most numerous prosthecate bacteria in the stream. They ranged from fewer than 1 per ml to a maximum of almost 4,000 per ml. During the coldest period, when the total viable counts decreased to about 10(4) per ml compared to their summer high of over 10(7) per ml, the caulobacters actually increased in numbers. In December (temperature 0 to 1 C), they comprised from 0.09 to 1.0% of the viable microbial count, and in March (6.0 to 8.0 C) they accounted for 0.14 to 2.8%. The other heterotrophic prosthecate bacteria were generally found at numbers less than 1 per ml, with the exception of the December study when Hyphomicrobium was present in numbers as high as 2,400 per ml. There was no consistent correlation between the frequency of prosthecate bacteria and total coliforms in the stream during the investigation.     

Isolation and characterization of heterotrophic,aerobic bacteria from oil storage caverns in northern Germany

Aerobic heterotrophic bacteria were enriched and isolated from three oil storage caverns of the German national oil reserve at different distances from the oil/brine interface. Microscopically no bacteria were found in the original samples, but colony counts showed more than 100 colony-forming units (cfu)/ml in two samples, whereas 0 to 4 cfu/ml were found in the other samples. Enrichments using defined mineral salts medium or complex medium revealed culturable organisms in all samples. All colony types were isolated and further separation of organisms during isolation was completed microscopically. Enrichments in media containing complex organic compounds led to higher numbers of isolates in samples near the oil/brine interface than enrichments with oil as the sole source of carbon. Micro-organisms that could utilize oil as the sole source of carbon were isolated from all enrichment cultures. Identification of the isolates revealedBacillus strains in all samples and coryneform bacteria in the samples from cavern 123.     

Microbiological characterization of a fuel-oil contaminated site including numerical identification of heterotrophic water and soil bacteria

Seven soil samples and seven groundwater samples from a site contaminated with fuel-oil were investigated using several chemical and microbiological techniques. In soil samples, 500 to 7,500 mg/kg of total hydrocarbons were found. These samples contained no n-alkanes but iso- and branched chain alkanes. No polychlorinated biphenyls could be detected. Microbiological investigations included estimations of total cell counts, viable cell counts on different media, and numbers of methylotrophic, denitrifying, sulphate reducing, anaerobic (with the exception of methanogenic organisms), and hydrocarbon degrading bacteria. Viable and hydrocarbon degrading bacteria were found in all samples. A total of 1,366 pure cultures was characterized morphologically and physiologically and identified by numerical identification using a data base of more than 4,000 reference strains. Groundwater samples were dominated by gram-negative bacteria of the generaPseudomonas, Comamonas, Alcaligenes, andAcinetobacter, which were also found in soil samples. In addition, more grampositive bacteria belonging to the generaArthrobacter, Nocardia, andBacillus could be isolated from soil samples.     

Resuscitation and quantification of stressed Escherichia coli K12 NCTC8797 in water samples

The aim of this study was to investigate the impact on numbers of using different media for the enumeration of Escherichia coli subjected to stress, and to evaluate the use of different resuscitation methods on bacterial numbers. E. coli was subjected to heat stress by exposure to 55 degrees C for 1h or to light-induced oxidative stress by exposure to artificial light for up to 8h in the presence of methylene blue. In both cases, the bacterial counts on selective media were below the limits of detection whereas on non-selective media colonies were still produced. After resuscitation in non-selective media, using a multi-well MPN resuscitation method or resuscitation on membrane filters, the bacterial counts on selective media matched those on non-selective media. Heat and light stress can affect the ability of E. coli to grow on selective media essential for the enumeration as indicator bacteria. A resuscitation method is essential for the recovery of these stressed bacteria in order to avoid underestimation of indicator bacteria numbers in water. There was no difference in resuscitation efficiency using the membrane filter and multi-well MPN methods. This study emphasises the need to use a resuscitation method if the numbers of indicator bacteria in water samples are not to be underestimated. False-negative results in the analysis of drinking water or natural bathing waters could have profound health effects.     

Bacterial population in Russian space station "Mir"

We had the opportunity to investigate the bacterial population in air samples, condensation water, and inner wall swabs from the Russian space station Mir. From the first and second air samples during the mission, 29 and 7 bacterial colonies were collected, respectively. The values were equivalent to 16.8 and 4.0 cfu/100 liter air, respectively. Condensation water was collected from three different sites. The total viable bacterial counts were 2.1 x 10(6), 5.2 x 10(2), and 3.0 x 10(1) cfu/ml. The phylogenetic position of each isolate was determined by total 16S rDNA sequencing. Bacteria from air samples were mainly Gram-positive (35/36 colonies), and staphylococci occupied dominant specifically (23/36 colonies). On the other hand, Gram-negative bacteria were mainly isolated from condensation water samples. Most strains were thought to be opportunistic pathogens or environmental bacteria (such as those that inhabit soil, water, or air) found on earth. However, 6 of 23 isolates were suspected to be new species according to phylogenetic analysis and quantitative DNA-DNA hybridization data. The isolation of the other levels 3 and 2 bacteria, using specific selective media, was unsuccessful because all samples were heavily contaminated with fungi. To overcome this situation, PCR methods were applied to survey most levels 3 and 2 pathogenic bacteria in the condensation water samples. Up to 380 different primers for bacterial pathogens were used in this study. Only Mycobacterium avium 16S DNA sequences, however, could be amplified from the three water samples. The average bacteria count was estimated to be about 10(4) organisms/ml water.     

Microbial biofilm formation and contamination of dental-unit water systems in general dental practice

Dental-unit water systems (DUWS) harbor bacterial biofilms, which may serve as a haven for pathogens. The aim of this study was to investigate the microbial load of water from DUWS in general dental practices and the biofouling of DUWS tubing. Water and tube samples were taken from 55 dental surgeries in southwestern England. Contamination was determined by viable counts on environmentally selective, clinically selective, and pathogen-selective media, and biofouling was determined by using microscopic and image analysis techniques. Microbial loading ranged from 500 to 10(5) CFU. ml(-1); in 95% of DUWS water samples, it exceeded European Union drinking water guidelines and in 83% it exceeded American Dental Association DUWS standards. Among visible bacteria, 68% were viable by BacLight staining, but only 5% of this "viable by BacLight" fraction produced colonies on agar plates. Legionella pneumophila, Mycobacterium spp., Candida spp., and Pseudomonas spp. were detected in one, five, two, and nine different surgeries, respectively. Presumptive oral streptococci and Fusobacterium spp. were detected in four and one surgeries, respectively, suggesting back siphonage and failure of antiretraction devices. Hepatitis B virus was never detected. Decontamination strategies (5 of 55 surgeries) significantly reduced biofilm coverage but significantly increased microbial numbers in the water phase (in both cases, P < 0.05). Microbial loads were not significantly different in DUWS fed with soft, hard, deionized, or distilled water or in different DUWS (main, tank, or bottle fed). Microbiologically, no DUWS can be considered "cleaner" than others. DUWS deliver water to patients with microbial levels exceeding those considered safe for drinking water.     

Viability of Indigenous Soil Bacteria Assayed by Respiratory Activity and Growth

The bacterial population in barley field soil was estimated by determining the numbers of (i) cells reducing the artificial electron acceptor 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to CTC-formazan (respiratory activity), (ii) cells dividing a limited number of times (microcolony formation) on nutrient-poor media, (iii) cells dividing many times (colony formation) on nutrient-poor agar media, and (iv) cells stained with acridine orange (total counts). The CTC reduction assay was used for the first time for populations of indigenous soil bacteria and was further developed for use in this environment. The number of viable cells was highest when estimated by the number of microcolonies developing during 2 months of incubation on filters placed on the surface of nutrient-poor media. The number of bacteria reducing CTC to formazan was slightly lower than the number of bacteria forming microcolonies. Traditional plate counts of CFU (culturable cells) yielded the lowest estimate of viable cell numbers. The microcolony assay gave an estimate of both (i) cells forming true microcolonies (in which growth ceases after a few cell divisions) representing viable but nonculturable cells and (ii) cells forming larger microcolonies (in which growth continues) representing viable, culturable cells. The microcolony assay, allowing single-cell observations, thus seemed to be best suited for estimation of viable cell numbers in soil. The effect on viable and culturable cell numbers of a temperature increase from 4 to 17°C for 5 days was investigated in combination with drying or wetting of the soil. Drying or wetting prior to the temperature increase, rather than the temperature increase per se, affected both the viable and culturable numbers of bacteria; both numbers were reduced in predried soil, while they increased slightly in the prewetted soil.     

Attached and unattached bacterial communities in a 120-meter corehole in an acidic, crystalline rock aquifer

The bacteria colonizing geologic core sections (attached) were contrasted with those found suspended in the groundwater (unattached) by examining the microbiology of 16 depth-paired core and groundwater samples using a suite of culture-independent and culture-dependent analyses. One hundred twenty-two meters was continuously cored from a buried chalcopyrite ore hosted in a biotite-quartz-monzonite porphyry at the Mineral Park Mine near Kingman, Ariz. Every fourth 1.5-m core was acquired using microbiologically defensible methods, and these core sections were aseptically processed for characterization of the attached bacteria. Groundwater samples containing unattached bacteria were collected from the uncased corehole at depth intervals corresponding to the individual cores using an inflatable straddle packer sampler. The groundwater was acidic (pH 2.8 to 5.0), with low levels of dissolved oxygen and high concentrations of sulfate and metals, including ferrous iron. Total numbers of attached cells were less than 10(5) cells g of core material(-1) while unattached cells numbered about 10(5) cells ml of groundwater(-1). Attached and unattached acidophilic heterotrophs were observed throughout the depth profile. In contrast, acidophilic chemolithotrophs were not found attached to the rock but were commonly observed in the groundwater. Attached communities were composed of low numbers (<40 CFU g(-1)) of neutrophilic heterotrophs that exhibited a high degree of morphologic diversity, while unattached communities contained higher numbers (ca. 10(3) CFU ml(-1)) of neutrophilic heterotrophs of limited diversity. Sulfate-reducing bacteria were restricted to the deepest samples of both core and groundwater. 16S ribosomal DNA sequence analysis of attached, acidophilic isolates indicated that organisms closely related to heterotrophic, acidophilic mesophiles such as Acidiphilium organovorum and, surprisingly, to the moderately thermophilic Alicyclobacillus acidocaldarius were present. The results indicate that viable (but possibly inactive) microorganisms were present in the buried ore and that there was substantial distinction in biomass and physiological capabilities between attached and unattached populations.     

Optimization of isolation and cultivation of bacterial endophytes through addition of plant extract to nutrient media

Many endophytes have beneficial effects on plants and can be exploited in biotechnological applications. Studies hypothesize that only 0.001–1% of all plant‐associated bacteria are cultivable. Moreover, even after successful isolations, many endophytic bacteria often show reduced regrowth capacity. This research aimed to optimize isolation processes and culturing these bacteria afterwards. We compared several minimal and complex media in a screening. Beside the media themselves, two gelling agents and adding plant extract to media were investigated to enhance the number and diversity of endophytes as well as the growth capacity when regrown after isolation. In this work, 869 medium delivered the highest numbers of cultivable bacteria, as well as the highest diversity. When comparing gelling agents, no differences were observed in the numbers of bacteria. Adding plant extract to the media lead to a slight increase in diversity. However, when adding plant extract to improve the regrowth capacity, sharp increases of viable bacteria occurred in both rich and minimal media.     

Some special problems in the determination of viable counts of groundwater microorganisms

Factors affecting viable cell counts in groundwater or sediments were studied with samples from the Segeberg Forest test area in northern Germany. There was very little variation in results with the season (April, August, November) or depth of sampling; generally there were 10³–10⁴ aerobic cells per ml or g sediment. Long incubation times resulted in higher cell counts; groundwater samples required 4–5 weeks, and sediment extracts had to be cultured for 7 weeks. Total cell counts in sediment were 10²–10⁴ cell/g higher than viable cell counts of aerobes. This was explained partly by the additional presence of anaerobes and partly by the observation that some morphotypes may not have grown under our conditions. Viable cell counts were not influenced by cell extraction from the sediment with either Na-pyrophosphate or groundwater extracts. However, iron-precipitating or manganese-oxidizing bacteria were better extracted with sterile groundwater. The microflora of wells was more numerous than that of the free aquifer; consequently it was better to pump off all well water before aquifer water was sampled. The diameter of the well was also important; thinner tubes had higher cell counts than those with wider diameter. For sampling, wells should be at least 1 year old, since young wells contain higher numbers of microorganisms due to underground disturbances from the drilling. Turbid water samples could be clarified by filtration, but this reduced the viable counts by 1–2 orders of magnitude. Two different media inoculated with a sample dilution resulted in the same cell counts, but their microbial diversity was different. Storage of groundwater samples before processing resulted in up to 17-fold increases in cell counts and loss of diversity in the first 24 hours. Cell numbers decreased slowly during longer storage.     

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk (r = 0.94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0.1 M, pH 3.0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better (r = 0.90) than that using standard 2 ml samples (r = 0.88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.     

Diurnal variations in bacterial numbers and fluid parameters in ruminal contents of animals fed low- or high-forage diets.

Differential carbohydrate media and anaerobic replica plating techniques were used to assess the degrees of diurnal variations in the direct and viable cell counts as well as the carbohydrate-specific subgroups within the mixed rumen bacterial populations in cattle fed maintenance (metabolizable energy) levels of either a high-forage or a high-concentrate diet once daily. The rumen was sampled at 1 h before feeding and 2, 4, 8, 12, and 16 h after feeding, and selected microbiological parameters of the isolated bacterial populations were assessed. Corresponding samples of ruminal fluid were assayed for fermentation acids, carbohydrate, ammonia, and pH changes. The data showed that regardless of diet, total bacterial numbers remained fairly constant throughout the day. The number of viable bacteria declined 40 to 60% after feeding and then increased to a maximum at 16 h postfeeding. Changes occurred in the carbohydrate-specific subgroups within the bacterial populations, and some of the changes were consistent with a predicted scheme of ruminal feedstuff carbohydrate fermentation. Regardless of diet, however, soluble-carbohydrate-utilizing bacteria predominated at all times. Xylan-xylose and pectin subgroups respectively comprised about one-half and one-third of the population when the high-forage diet was given. These subgroups, along with the cellulolytics, constituted lesser proportions of the population when the high-concentrate diet was given. The cellulolytic subgroup was the least numerous of all subgroups regardless of diet but followed a diurnal pattern similar to that predicted for cellulose fermentation. There were few diurnal variations or differences in bacterial cell compositions and ruminal fluid parameters between diets. The observed similarities and dissimilarities of the rumen bacterial populations obtained when the two diets were given are discussed. The data are consistent with the versatility and constancy of the rumen as a stable, mature microbial system under the specific low-level feeding regimens used.     

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk ( r = 0·94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0·1 M, pH 3·0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better ( r = 0·90) than that using standard 2 ml samples ( r = 0·88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.     

THE NORMAL FLORA OF THE BOVINE RUMEN II. QUANTITATIVE BACTERIOLOGICAL STUDIES

SUMMARY: A simple medium and dilution count technique was evolved for determining the viable count of rumen bacteria. Material from three fistulated cows was examined and the viable population found to vary within the range 10⁷-10¹²/g; the most frequently occurring values were 10⁸-10¹⁰/g (94.0% of 350 counts). Observations were made of the homogeneity of rumen contents, of variations in count with time after sampling and between cows, and of variation from day to day, hour to hour, before and after feeding and on different diets. The variations in the viable count, even with gross changes in diet, were never greater than those occurring in a single animal on a fixed diet. Microscopic examinations showed Gram-positive cocci and rods to be the most frequently occurring types of bacteria, Gram-negative organisms of the coli-aerogenes type being present only in low numbers.     

Vertical distribution of bacteria in arctic sea ice

Abstract Heterotrophic bacteria were enumerated in north polar sea ice cores obtained near Point Barrow, Alaska. Highest concentrations of total and viable bacteria were found in the layer containing the sea ice microbial community identified by the maximum chlorophyll a content. Gas vacuolate bacteria were also found in the sea ice, a discovery which is consistent with their recent report from antarctic sea ice microbial communities. The gas vacuolate bacteria comprised 0.2% or less of the viable bacteria isolated from sea ice cores, lower than concentrations reported for most antarctic samples. Most gas vacuolate isolates from the sea ice cores were pigmented pink, orange, or yellow. An ice core from nearby saline Elson Lagoon contained an inverted sea ice microbial community with highest chlorophyll a concentrations and bacterial counts found in the top 0–20 cm of the ice. This surface layer also contained high numbers (up to 186 bacteria/ml) of a nonpigmented, gas vacuolate, elongated rod-shaped bacterium.     

Detection of Legionella species in reclaimed water and air with the EnviroAmp Legionella PCR kit and direct fluorescent antibody staining.

Reclaimed water is an important resource for areas with inadequate water supplies. However, there have been few studies on the variety of microorganisms found in this type of water, since typically reclaimed water is examined only for the presence of coliform bacteria. Many microorganisms, including the legionellae, are known to be more resistant to chlorine than are coliform bacteria. Previously, we detected > 10(3) Legionella cells per ml in primary and secondary sewage effluents and observed no significant reduction in population numbers throughout the treatment process. In this study, we detected Legionella spp. in chlorinated effluent by using an EnviroAmp Legionella PCR kit and direct fluorescent antibody (DFA) staining. However, we were not able to isolate Legionella spp. from either natural or seeded reclaimed water samples. This suggests that the Legionella spp. detected by the PCR and DFA methods may be injured or viable but nonculturable after exposure to the high residual chlorine levels typically found in this type of water source. The numbers of coliform bacteria were low (< 2 cells per 100 ml) in most reclaimed water samples and were not correlated with the presence or absence of Legionella spp. We also collected air samples from above a secondary aeration basin and analyzed them by using the PCR, DFA, and plate culture methods. Legionella spp. were detected in the air obtained from above the secondary basin with all three methods. We concluded that the PCR was superior to the culture and DFA methods for detecting Legionella spp. in environmental water samples.