Automated dead-end ultrafiltration of large volume water samples to enable detection of low-level targets and reduce sample variability

Aims

A Portable Multi‐use Automated Concentration System (PMACS) concentrates micro‐organisms from large volumes of water through automated dead‐end ultrafiltration and backflushing. The ability to detect microbial targets from ground, surface and cooling tower waters collected using standard methods was compared with samples from the PMACS in this study.

Methods and Results

PMACS (100 l) and standard grab samples (100–500 ml) were collected from sites in Florida and South Carolina, USA. Samples were analysed for the presence of faecal indicator bacteria (FIB; ground and surface water) or Legionella pneumophila (Lp; cooling tower water). FIB were enumerated by growth on selective media following membrane filtration or in IDEXX defined substrate media. Lp cells were detected by direct fluorescence immunoassay using FITC‐labelled monoclonal antibodies targeting serogroups 1, 2, 4 and 6. FIB were found in PMACS samples from ground and surface waters when their concentrations were below detection limits in grab samples. The concentrations of Lp in cooling tower samples collected over 5 months were more consistent in PMACS samples than grab samples.

Conclusions

These data demonstrate that PMACS concentration is advantageous for water monitoring. FIB were detected in PMACS samples when their concentrations were below the detection limits of the standard methods used. PMACS processing provided more representative samples of cooling tower waters reducing sample variability during long‐term monitoring.

Significance and Impact of the Study

This study highlights the utility of PMACS processing for enhanced monitoring of water for low‐level microbial targets and for reducing sample variability in long‐term monitoring programmes.     

Microbiological and chemical-sensory characteristics of three coffee varieties processed by wet fermentation

This work evaluated the bacterial diversity during coffee wet fermentation of the three coffee varieties—Mundo Novo (MN), Ouro Amarelo (OA), and Catuaí Vermelho (CV). Isolates were identified by polyphasic techniques: biochemical tests, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and DNA sequencing. Chemical compositions were determined by high (HPLC) and gas chromatography-mass spectrometry (GC-MS) and the roasted beans were sensorial evaluated using the cupping test. Thirty-six mesophilic bacteria and six lactic acid bacteria were identified. Lactobacillus plantarum and Leuconostoc mesenteroides were often found in all varieties. Citric acid was the acid detected in higher concentrations. The volatile profile of the green coffee beans changed during the fermentation in the tank, but more significantly, during the roasting process. These volatiles belonged to the classes of acids, alcohols, aldehydes, and hydrocarbons. Temporal dominance of sensations analysis showed sensorial sensations of acidity (OA and CV), bitterness, chocolate, nuts (MN), and sweetness (CV). The characteristics of each coffee variety were distinct, mainly in relation to total bacteria population, volatile compounds, and sensorial profile. In conclusion polyphasic methodology was efficiently done for bacteria identification; the dominant bacteria might be used for starter cultures and the chemical and sensory analyses helped to understand the changes in coffee fermentation. Our findings are relevant to future select starter bacteria for coffee processing to improve quality and standardization of quality.     

蛭弧菌的分离及其生长条件和裂解能力的研究

噬菌蛭弧菌具有裂解病原菌、净化水体的功效,从海洋环境中分离到4株Bh04系列蛭弧菌,对其生长条件进行了测定,同时研究了它们对61株菌株的裂解能力。结果表明,在1%～3%的盐度范围内,蛭弧菌均可生长,最适盐度为3%;在15～30℃温度条件下蛭弧菌也可生长,但最适培养温度为20-25℃;只有在使用活的宿主菌的培养条件下,蛭弧菌才能生长。4株蛭弧菌分别可裂解21、24、40、43株菌,各占总试验菌数(61)的34.4%、39.3%、65.6%和70.5%。4株蛭弧菌一起,则可裂解55菌株,占总试验菌株的90.2%。研究结果揭示了蛭弧菌在消除海洋环境中有害细菌方面的潜在应用价值。     

革兰阴性菌引起的血培养阳性标本直接细菌鉴定和药敏的应用

目的 评估血培养阳性标本直接细菌鉴定和药敏可行性.方法 将血培养瓶放入Bact/Alert 3D 60血培养系统进行培养筛选.选取78份含革兰阴性杆菌的阳性血培养瓶进行试验.抽取培养液,用BD真空分离管离心血细胞.在收集到足量菌液后,用VITEK-32革兰阴性菌鉴定药敏卡做直接鉴定药敏试验.用标准方法及亚培养后的鉴定药敏试验对直接鉴定药敏试验进行评估.结果 VITEK-32直接鉴定试验,78株中的74株(94.9％)准确鉴定,直接药敏试验标准符合率95.6％.KB法血标本直接药敏试验标准符合率96.2％,但微小错误率高于VITEK-32直接药敏法.结论 Bact/Alert血培养阳性标本直接VITEK-32细菌鉴定和药敏对革兰阴性菌是切实可行的,可大幅度缩短时间,为临床及时修正用药提供依据,具有较高的应用价值.     

Acido-thermophilic Bacteria from Thermal Waters

Three strains of acido-thermophilic bacteria were isolated from hot-spring waters of Tohoku district in Japan. They were aerobic spore-forming bacilli and identified to belong to genus Bacillus. Their characteristics were as follows. They were acidophilic, and grew well in the pH range of between 2.3 and 5.0. Optimal growth conditions were 65°C for temperature and 3.5~4.0 for pH of media. Strains T-4 and T-17 required biotin as growth factor, but T-7 did not require any factors for its growth. These bacteria were different from Bacillus stearothermophilus or B. coagulans in their taxonomic properties.     

A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase

Aims: 1‐Aminocyclopropane‐1‐carboxylate (ACC) deaminase activity is an efficient marker for bacteria to promote plant growth by lowering ethylene levels in plants. We aim to develop a method for rapidly screening bacteria containing ACC deaminase, based on a colorimetric ninhydrin assay of ACC. Methods and Results: A reliable colorimetric ninhydrin assay was developed to quantify ACC using heat‐resistant polypropylene chimney‐top 96‐well PCR plates, having the wells evenly heated in boiling water, preventing accidental contamination from boiling water and limiting evaporation. With this method to measure bacterial consumption of ACC, 44 ACC‐utilizing bacterial isolates were rapidly screened out from 311 bacterial isolates that were able to grow on minimal media containing ACC as the sole nitrogen source. The 44 ACC‐utilizing bacterial isolates showed ACC deaminase activities and belonged to the genus Burkholderia, Pseudomonas or Herbaspirillum. Conclusions: Determination of bacterial ACC consumption by the PCR‐plate ninhydrin–ACC assay is a rapid and efficient method for screening bacteria containing ACC deaminase from a large number of bacterial isolates. Significance and Impact of the Study: The PCR‐plate ninhydrin–ACC assay extends the utility of the ninhydrin reaction and enables a rapid screening of bacteria containing ACC deaminase from large numbers of bacterial isolates.     

A note on the isolation and enumeration of Gram positive cocci from marine and estuarine waters

Ninety-two percent of Gram positive cocci detected in estuarine and marine surface waters were identified as Staphylococcus spp. Micrococcus and Streptococcus spp. were rarely isolated from seawater, except at dump site stations located in the Puerto Rico Trench area of the Atlantic Ocean, where they were present in unusually large numbers. Staphylococci were present in greatest abundance near land, but were also found in open ocean surface waters in low, but relatively constant, numbers. Staphylococcus epidermidis , or strains related to Staph. hominis , were present in all samples. Staphylococci from surface water samples collected at the Puerto Rico Trench dump site degraded a wide range of compounds. It is considered that these cocci were derived from the wastes dumped, as very few were found in unpolluted marine waters and in those cases in which stimulatory factors, such as disposal of wastes, do not play a role, Gram positive cocci will not be recovered unless large volumes of water are filtered. The Gram positive cocci are present in very low numbers in natural seawater of the open ocean and, therefore, are commonly overlooked. The significance of these bacteria in the nutrient cycles of the sea remains to be elucidated. Documentation of a widespread occurrence of Gram positive cocci in North Eastern Atlantic seawater is provided.     

Legionella pneumophila in commercial bottled mineral water

Sixty-eight commercial bottled mineral waters (64 brands, 68 different 'best-before dates') were tested for the presence of bacteria and fungi. Six samples were Legionella antigen positive and six were Legionella pneumophila PCR positive. Two samples were both Legionella antigen and L. pneumophila PCR positive. Legionella cultures were negative. Although the PCR might have detected only dead Legionella cells, the PCR has been described to detect specifically viable but not culturable (VBNC) L. pneumophila cells as well. Whether VBNC bacteria may be present in bottled mineral waters and the risk for infection this may pose for severely immunocompromised patients should be investigated.     

Comparison of media for enumeration of coliform bacteria and Escherichia coli in non-disinfected water

In this work alternative media for detection and enumeration of E. coli and coliform bacteria were compared to the reference method ISO 9308-1 (LTTC) using non-disinfected water samples with background flora. The alternative media included LES Endo agar medium (LES Endo), Colilert-18 with 51-well Quanti-tray (Colilert), Chromocult Coliform agar (CC), Harlequin E. coli/Coliform medium (HECM) and Chromogenic Escherichia coli/Coliform medium (CECM). A total of 110 samples of groundwater, bathing water and spiked water was used. Our results revealed that confirmation of coliform bacteria counts is necessary, not only on lactose-based LTTC and LES Endo media, but also on the chromogenic agar media tested, due to the growth of oxidase positive colonies. LTTC and CC media also allowed the growth of some morphologically typical coliform colonies containing gram-positive bacteria. The recovery of coliform bacteria was lower on LES Endo than on LTTC. In most cases Colilert, CC, HECM and CECM gave higher coliform counts than LTTC. The use of the LTTC medium led to higher E. coli counts than obtained with any of the alternative mediums. There are three explanations for this: (1) high sensitivity of LTTC, (2) false positives on LTTC or (3) false negatives especially with Colilert, but also with chromogenic agar media. Although LTTC was found to be a very sensitive medium, the high degree of background growth of non-disinfected waters disturbed substantially the use of it. In conclusion, our results suggest that Colilert, CC and CECM are potential alternative media for detection of coliform bacteria and E. coli from non-disinfected water.     

Use of Monodispersed, Fluorescently Labeled Bacteria to Estimate In Situ Protozoan Bacterivory

We have developed a procedure for preparing monodispersed, fluorescently labeled bacteria (FLB), which may be used to measure virtually instantaneous rates of protozoan bacterivory in natural waters. FLB can be prepared both from natural bacterioplankton assemblages and from clonal isolates and can be stored in frozen suspension or freeze-dried without apparent loss of fluorescence intensity. They are not toxic to protozoa and can be metabolized to support bacterivorous protozoan growth rates equal to those on the same strain of unstained, viable bacteria. In experiments comparing uptake of FLB with uptake of fluorescent latex microspheres by protozoan assemblages in a salt marsh tidal creek, we found that both pelagic oligotrichous ciliates and phagotrophic flagellates ingested FLB with a frequency 4- to 10-fold greater than they ingested the microspheres. Consequently, it appears that the use of latex microspheres leads to underestimation of protozoan bacterivory and that the FLB technique is superior for estimating instantaneous rates of in situ protozoan grazing on bacterioplankton.     

Factors associated with detrimental effects of rhizobacteria on plant growth

Some of the factors interfering with the specific response of young common bean plants to two rhizosphere fluorescent pseudomonads were studied. These two bacterial strains produced symptoms in foliar plant parts and reduced yield in beans and several other plant species when inoculated on roots. Sensitivity in the plants subjected to bacterial application was highest at early growth stages (up to ten days old plants) giving typical symptoms for each strain in first expanding leaves and typically stunted plant growth. Symptoms to some extent also appeared on plants treated at the age of 2–3 weeks and the fresh shoot weight was affected also in such plants. Deleterious effects of the tested bacteria were found to be related to the level of inoculum (cfu/ml) used and presence or absence of certain nutrients in the bacterial suspensions used for inoculation. The two tested strains differed in nutritional requirements for affecting plant growth. One strain needed sucrose only. The other tested strain required peptone or yeast extract in addition to sucrose to induce typical symptoms and significant yield reductions. Supply of peptone as the only nutrient source to the bacterial suspensions eliminated the deleterious effects of both bacterial strains as tested on beans under nonsterile conditions. The two strains are assumed to affect test plants by different modes of action.     

Dasyclads, cyclocrinitids and receptaculitids: comparative morphology and paleoecology

The cyclocrinitids are an extinct tribe of dasycladacean green algae. They were anatomically very similar to certain Recent dasyclads, even at early growth stages. The morphology and preservation of cyclocrinitids strongly suggest that they had a siphonous cellular organization with extracellular, aragonitic calcification; these features are characteristic of living dasyclads. The light surficial calcification of cyclocrinitids and other dasyclads had important paleoecological effects. It restricted them to low-energy waters, as it provided relatively little structural support. It also confined them to warm, tropical waters; they are good paleoequatorial indicators. The decline of these algae during the late Ordovician and early Silurian may therefore reflect the simultaneous cooling and glaciation. Receptaculitids are entirely unrelated organisms. Their meroms have several distinctive features; they are not homologous to the lateral branches of cyclocrinitids or dasyclads. Receptaculitid calcification was extensive and their thalli were apparently quite sturdy; they often occurred in reefs. Receptaculitids also lived in high-latitude, cold-water environments. Thus, they were ecologically unlike any calcareous green algae, and cannot be used as paleoequatorial indicators. Receptaculitids remain problematical, although the arrangement of meroms suggests plant affinities. □ Calcareous algae, Problematica, Dasycladales, Cyclocriniteae, Receptaculitales, morphology, classification, paleoecology, paleogeography .     

COLD-TOLERANT FERMENTATIVE GRAM-NEGATIVE ORGANISMS FROM MEAT AND OTHER SOURCES

SUMMARY: From various chilled meats, twenty-eight strains of coli-aerogenes bacteria and one Aeromonas were isolated which grew well at +1±5° and some at −1±5°. The optimum growth temperature for most of these strains was nearer 37° than 30°. Nine strains (including the Aeromonas ) fermented lactose rapidly, the remainder slowly or not at all. All the strains which fermented lactose rapidly with the production of gas gave positive presumptive coli-aerogenes tests in MacConkey's broth at 30°, but only five were positive at 37°; none was positive at 44°. Because such organisms can attain populations of millions/cm², they could confuse the interpretation of presumptive coli-aerogenes tests made on chilled meat.     

Occurrence of False-Positive Most Probable Number Tests for Fecal Streptococci in Marine Waters

By the use of the most probable number technique with azide dextrose and ethyl violet azide broths for enterococci, the common occurrence of false-positive tests was noted when marine and estuarine waters were sampled. Organisms isolated included a marine bacterium, gram-positive and gram-negative nonmarine bacteria, and yeasts. All cultures were capable of growth in azide-dextrose, ethyl violet-azide, and KF broths. Representative isolates grew in media containing 0.08% NaN(3). The tentatively accepted most probable number method for fecal streptococci is thus of dubious value in assessment of sewage pollution levels in estuarine waters. All positive tubes must be examined microscopically for the presence of nonstreptococcal forms.     

The Relationship between the Intestinal Microflora and the Effects of Diets containing Raw Navy Beans (Phaseolus vulgaris) on the Growth of Japanese Quail (Coturnix coturnix japonica)

S ummary : Japanese quail chicks died within a few days when fed diet containing raw navy beans, whereas a similar diet containing autoclaved beans supported normal growth. The diet containing raw beans was nonlethal to germfree quail chicks, but did not support such good growth as the heated bean diet. The effects of associating germfree quail on the raw bean diet with various combinations of 149 pure cultures of bacteria isolated from the intestines of ordinary quail have been studied. Strains of Escherichia coli were shown to be responsible for the lethal effects of the raw bean diet.     

Factors Affecting High-Oxygen Survival of Heterotrophic Microorganisms from an Antarctic Lake

We sought to determine factors relating to the survival of heterotrophic microorganisms from the high-dissolved-oxygen (HDO) waters of Lake Hoare, Antarctica. This lake contains perpetual HDO about three times that of normal saturation (40 to 50 mg liter⁻¹). Five isolates, one yeast and four bacteria, were selected from Lake Hoare waters by growth with the membrane filter technique with oxygen added to yield dissolved concentrations 14 times that in situ, 175 mg liter⁻¹. One bacterial isolate was obtained from the microbial mat beneath the HDO waters. This organism was isolated at normal atmospheric oxygen saturation. The bacteria were gram-negative rods, motile, oxidase positive, catalase positive, and superoxide dismutase positive; they contained carotenoids. The planktonic isolates grew in media containing 10 mg of Trypticase soy (BBL Microbiology Systems)-peptone (2:1) liter⁻¹ but not at 10 g liter⁻¹. Under low-nutrient levels simulating Lake Hoare waters (10 mg liter⁻¹), two of the planktonic isolates tested were not inhibited by HDO. Growth inhibition by HDO increased as nutrient concentration was increased. A carotenoid-negative mutant of one isolate demonstrated a decreased growth rate, maximal cell density, and increased cell lysis in the death phase under HDO compared with the parent strain. The specific activity of superoxide dismutase was increased by HDO in four of the five bacterial isolates. The superoxide dismutase was of the manganese type on the basis of inhibition and electrophoretic studies. The bacterial isolates from Lake Hoare possess several adaptations which may aid their survival in the HDO waters, as well as protection due to the oligotrophic nature of the lake.     

Quantitative aspects of the Ninhydrin-Schiff reaction on amino cellulose films and tissue sections

Summary In the ninhydrin-Schiff reaction primary amino groups are converted by oxidation with ninhydrin to aldehyde groups which are subsequently stained with pararosanilin. Amino cellulose films, used as a model, and sections of muscle tissue were submitted to this reaction. The amino groups were stained before and after the ninhydrin reaction with dinitrofluorobenzene and the generated aldehyde groups were stained with dinitrophenyl-hydrazine. The molar extinction coefficients used for the calculation of the molar amounts of chromophores from extinction values, and the conditions for maximal staining intensity were determined on the amino cellulose model. With these data the yields of the different steps in the reaction sequence could be calculated in molar amounts from the extinction measurements. The results showed that from the amino groups originally present in the tissue sections less than 40% were converted by ninhydrin. About 90% of the converted amino groups were found as aldehyde groups and from these only 7% reacted with pararosanilin. On amino cellulose similar data were obtained. Attempts were made by modification of the conditions in the ninhydrin oxidation step to increase the overall yield of the reaction. These were only partially successful, but indicate that further quantitative study of other reaction conditions and different aldehyde generating agents could be promising.     

The effect of ozone onLegionella pneumophila and other bacterial populations in cooling towers

Ozone was found to reduce the numbers of bacteria detectable by plate counts and the numbers of presumptiveLegionella pneumophila (DFA-reactive cells), including those that were INT positive, in test cooling towers. The numbers of DFA-reactiveL. pneumophila eventually reached those of the makeup water (Troy, NY, city water). Microbial slime on the interior tower surfaces andPseudomonas populations in the tower waters were also reduced. Water chemistry parameters measured showed no tendency toward a condition that would cause accelerated deterioration of tower material.     

九孔鲍养殖水体及其肠道不同菌群抗药性研究*

为更好地防治鲍鱼病害的发生和流行,对分离自广东汕尾健生鲍鱼养殖场九孔鲍养殖环境及其肠道中不同细菌菌群的耐药性进行了研究。结果表明,四环素、青霉素G、卡那霉素、丁胺卡那霉素和新生霉素均对绝大多数异养菌和弧菌菌株都不敏感或无作用;相反,氟哌酸、红霉素、氯霉素以及环丙沙星等则均对它们比较敏感;复方新诺明、链霉素和多粘霉素B对弧菌菌株均有作用,而且多粘霉素B也对水体中的异养菌群相当有效。     

Applying a most probable number method for enumerating planktonic, dissimilatory, ammonium-producing, nitrate-reducing bacteria in oil field waters

A most probable number (MPN) method was used to enumerate dissimilatory ammonium-producing, nitrate-reducing bacteria (DAP-NRB) in oil field waters and to determine whether they were stimulated by nitrate addition used to control hydrogen sulfide production. An ammonium production medium with 5 carbon and energy sources (acetate, glucose, glycerol, pyruvate, and succinate) and nitrate was used in a 3-tube MPN procedure to enumerate DAP-NRB. These bacteria were detected in 12 of 18 oil field water samples, but they were seldom detected in wellhead samples. Three oil field water samples were amended with nitrate in serum bottles and the numbers of different NRB were determined over a 38-day incubation time. This amendment stimulated increases in the numbers of heterotrophic NRB and autotrophic nitrate-reducing, sulfide-oxidizing bacteria, but DAP-NRB remained a minor portion of these communities. Overall, DAP-NRB were present in many of the oil field waters that were examined but their numbers were low. It appears that DAP-NRB would play a minor role in the consumption of nitrate injected into oil field waters for the control of hydrogen sulfide production.