An improved method for the automated enumeration of fluorescently labelled bacteria in human faeces

The study aimed to improve microscopy-based automated recognition of faecal bacterial cells labelled with 16S rRNA-targeted oligonucleotides and 4',6-diamidino-2-phenylindole (DAPI). Based on the software KS400 (Carl Zeiss Vision, Hallbergmoos, Germany), designed for automising microscopy-based image capture and image analysis, a routine was developed that affords the recognition of doubly stained bacteria and the rejection of artefacts. The accuracy of the automated enumeration was investigated by comparing the resulting data with those obtained by manual counting. The newly developed method was subsequently used to compare the total bacterial counts in human faecal samples using the domain specific probe Eub338 alone and a mixture of 5 domain-specific probes, respectively. Faecal samples from 90 healthy volunteers were analysed. The cell counts obtained with Eub338 were 10% lower than those obtained with the probe mixture. Since the cells detected with the probe mixture covered a wide range of signal intensities, a dynamic analysis routine was developed to effectively detect the whole range of bright to weak signals within the same image, while at the same time reliably rejecting artefacts.     

A modified tube method for the cultivation and enumeration of anaerobic bacteria.

A new type of tube (the Lee tube) has been developed for use in the cultivation and enumeration of obligate anaerobes. The Lee tube is a double-walled, screw-capped tube which allows the formation of a thin cylinder of agar medium between the two walls. Anaerobiosis is achieved through deoxygenation of the deep cylinder of agar during sterilization, a minimum of head space, and use of a reducing agent to absorb oxygen introduced during the inoculation procedure. For several species of Clostridium, Bacteroides fragilis, Fusobacterium necrophorum, Veillonella alcalescens, and Pectinatus cerevisiiphilus, colony counts of cultures in the Lee tubes were comparable with those obtained in pour plates incubated in a BBL GasPak system and in anaerobic roll tubes.     

Development and validation of an automated high-throughput system for zebrafish in vivo screenings

The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism.     

Development of a miniaturized four-culture method for the rapid enumeration of four bacterial groups in ground beef

AIMS: A simplified and rapid method was developed to identify and enumerate total mesophilic microbial load, gram-negative bacteria, coliforms, and Escherichia coli in ground beef, using a single module. METHODS AND RESULTS: A 96-well microtiter plate was divided into four sections of two lanes (24 wells), with each section dedicated to each of the four critical bacterial groups. Double strength of four different selective broths (100 microl) was added to each well and an aliquot of sample (100 microl) from the first well was transferred to the next to achieve a twofold serial dilution in each series (24 wells). Initial bacterial load was calculated using the final consecutive positive reaction exhibiting color change or fluorescence. To validate the method, ground beef (n = 32), inoculated with a three-strain mixture of E. coli, together with 30 samples of commercial ground beef, were assayed by the four-culture method and conventional plating. For all media tested, the coefficients of determinations (r2) between the results derived from the four-culture method and those of conventional plating method ranged from 0.83 to 0.92. CONCLUSIONS: The high degree of association between the two methods indicates that the four-culture method could be easily applied to the enumeration of four critical bacterial groups in ground beef. SIGNIFICANCE AND IMPACT OF THE STUDY: This method will facilitate sanitation monitoring at meat processing plants by shortening time required for results, reducing consumable material costs, simplifying procedures, reducing the space required, and cutting the cost of start up equipment.     

Rapid and automated enumeration of viable bacteria in compost using a micro-colony auto counting system

The micro-colony method was used to enumerate viable bacteria in composts. Cells were vacuum-filtered onto polycarbonate filters and incubated for 18 h on LB medium at 37 degrees C. Bacteria on the filters were stained with SYBR Green II, and enumerated using a newly developed micro-colony auto counting system which can automatically count micro-colonies on half the area of the filter within 90 s. A large number of bacteria in samples retained physiological activity and formed micro-colonies within 18 h, whereas most could not form large colonies on conventional media within 1 week. The results showed that this convenient technique can enumerate viable bacteria in compost rapidly for its efficient quality control.     

A flow cytometry method for rapid detection and enumeration of total bacteria in milk

Application of flow cytometry (FCM) to microbial analysis of milk is hampered by the presence of milk proteins and lipid particles. Here we report on the development of a rapid (/= 0.98) between the FCM assay and the more conventional methods of plating and direct microscopic counting was achieved. Raw milk data showed a significant correlation (P < 0.01) and a good agreement (r = 0.91) between FCM and standard plate count methods. The detection limit of the FCM assay was 相似文献   

Timed-release capsule method for coliform enumeration.

Wax-coated capsules containing selective ingredients (brilliant green and oxgall) were added at the time of inoculation of most-probable-number media (modified lactose broths). The inhibitory ingredients gradually diffused from the capsules into the nonselective media, imparting selectivity to the media. Concentrations of brilliant green did not reach inhibitory levels until 2 or more h had elapsed, which permitted repair of some injured cells. Resuscitation of heat-injured Escherichia coli B cells occurred in the capsule-containing media, but not in conventional brilliant green bile 2% broth or violet red bile agar. No statistically significant differences were noted between coliform counts obtained on two groups of water samples by using the capsule, most-probable-number, membrane filtration, and pour plate methods. The capsule method could be used, however, as a combined presumptive and confirmed test for the examination of water. Improvements are needed to adapt the capsule method to the analysis of some categories of food.     

A rapid method for the enumeration of cultivable bacteria in dental plaque samples

A rapid method for estimating the number of cultivable bacteria in dental plaque samples was developed in which five fluorogenic substrates (4-methylumbelliferyl (4-MU)-α-glucoside, 4-MU-β-glucoside, glycyl-prolyl-7-amido-4-methyl-coumarin (-AMC), tyrosyl-AMC and prolyl-AMC) in 150 μl were incubated with 50 μl of dental plaque suspension. The increase in fluorescence measured at emission and excitation wavelengths of 380 and 460 nm correlated with the colony count per plaque sample. The rapid method allows the number of cultivable bacteria in plaque samples to be estimated and, with the choice of appropriate substrates, it should be useful for estimating the numbers of bacteria in other mixed populations.     

Detection, enumeration, and sizing of planktonic bacteria by image-analyzed epifluorescence microscopy.

Epifluorescence microscopy is now being widely used to characterize planktonic procaryote populations. The tedium and subjectivity of visual enumeration and sizing have been largely alleviated by our use of an image analysis system consisting of a modified Artek 810 image analyzer and an Olympus BHT-F epifluorescence microscope. This system digitizes the video image of autofluorescing or fluorochrome-stained cells in a microscope field. The digitized image can then be stored, edited, and analyzed for total count or individual cell size and shape parameters. Results can be printed as raw data, statistical summaries, or histograms. By using a stain concentration of 5 micrograms of 4'6-diamidino-2-phenylindole per ml of sample and the optimal sensitivity level and mode, counts by image analysis of natural bacterial populations from a variety of habitats were found to be statistically equal to standard visual counts. Although the time required to prepare slides, focus, and change fields is the same for visual and image analysis methods, the time and effort required for counting is eliminated since image analysis is instantaneous. The system has been satisfactorily tested at sea. Histograms of cell silhouette areas indicate that rapid and accurate estimates of bacterial biovolume and biomass will be possible with this system.     

Development and validation of an HPLC-UV method for iodixanol quantification in human plasma

Iodixanol is a widely used iso-osmolar contrast medium agent. Similar to iohexol, it can also be a good exogenous marker for the measurement of glomerular filtration rate (GFR). This article describes the development and validation of an HPLC-UV method for quantification of iodixanol in human plasma. Internal standard, iohexol (20 microl, 1 mg/ml), and perchloric acid (30 microl, 20%, v/v) were added to plasma samples (300 microl), followed by neutralization with 10 microl potassium carbonate (5M). Samples were centrifuged and 10 microl of the supernatant was injected onto a C(18) EPS analytical column (3 microm particle size, 150 mm x 4.6 mm). The extraction method yielded >95% recovery for both iodixanol and iohexol. The mobile phase consisted of 0.1% (w/v) sodium formate buffer and acetonitrile. Iohexol and iodixanol peaks were eluted at approximately 5 and 9 min, respectively using a fast gradient method. The assay lower limit of detection was 2.0 microg/ml and lower limit of quantification was 10 microg/ml. The calibration curves, assessed in six replicates, were linear over an iodixanol concentration range of 10-750 microg/ml. Intra- and inter-day accuracy was >95% and precision expressed as % coefficient of variation was <10%. This method is simple, accurate, precise and robust and can potentially be used for iodixanol quantification in large-scale clinical studies.     

保健食品双歧杆菌和乳酸菌计数方法的优化

目的通过稀释液和培养基的选择对保健食品双歧杆菌和乳酸菌计数方法进行优化。方法对2个品种6个批次的益生菌类保健食品,分别采用2种稀释液和3种培养基进行双歧杆菌和乳酸菌计数,并比较结果。结果选择CYS缓冲液为最优稀释液,M-TOS培养基为双歧杆菌计数的最优培养基,BBL培养基为乳酸菌总数计数的最优培养基。结论优化方法所得菌数均明显优于国标方法,可以用于保健食品的双歧杆菌和乳酸菌计数。     

Chlorine injury and the enumeration of waterborne coliform bacteria.

Injury induced in Escherichia coli cells by chlorination was studied from a physiological standpoint. Predictable and reproducible injury was found to occur rapidly in 0.5 mg of chlorine per liter and was reversible under nonselective conditions. There was an extended lag period in the growth of chlorinated cells not seen in control suspensions followed by the resumption of logarithmic growth at a rate equaling that of control cells. The aldolase activity of cells chlorinated in vivo was equivalent to that obtained for control cells. Oxygen uptake experiments showed that chlorinated cells underwent a decrease in respiration that was not immediatedly repaired in the presence of reducing agents. This effect was more pronouned in rich media containing reducing agents. Uptake of metabolities was inhibited by chlorine injury as shown with experiments using 14C-labeled glucose and algal protein hydrolysate.     

Membrane filter enumeration method for Clostridium perfringens.

A membrane filter procedure has been developed for the rapid quantitation of C. perfringens in the aquatic environment. Background growth is inhibited by the use of D-cycloserine, polymyxin B sulfate, and incubation at 45 degrees C. Differential characteristics include the fermentation of sucrose, production of acid phosphatase, and the absence of beta-D-glucosidase activity. The medium is prepared as follows (in grams per 100 ml of distilled water): tryptose, 3.0; yeast extract, 2.0; sucrose, 0.5; L-cysteine, 0.1; MgSO4. 7H2O, 0.01; bromocresol purple, 0.004; and agar, 1.5. The ingredients are dissolved, and the pH is adjusted to 7.6. After autoclaving at 121 degrees C for 15 min, the medium is allowed to cool at 50 degrees C, and the following are added per 100 ml: D-cycloserine, 40 mg; polymyxin B sulfate, 2.5 mg; indoxyl-beta-D-glucoside, 60 mg; 2.0 ml of a filter-sterilized 0.5% phenolpthalein diphosphate solution; and 0.2 ml of a filter-sterilized 4.5% FeCl3.6H2O solution. Enumeration of C. perfringens in a water sample is completed within 18 to 24 h. The verification of typical colonies was 93%. The average recovery from peptone-water spore suspensions of five strains was 79%, and that from filter-sterilized seawater suspensions was 90%. The precision of the method was approximately equal to that expected from random error alone. Confirmed recoveries of C. perfringens from water and sewage samples generally were greater than those by the Bonde pour tube method.     

Effects of betaine on enumeration of airborne bacteria.

The osmoprotectant betaine was incorporated into collection fluid and enumeration medium to determine its effects on the colony-forming abilities of airborne bacteria, which were collected from three separate locations: a wastewater treatment plant, the roof of a laboratory building, and an unobstructed farmland. At all locations, addition of 2 to 5 mM betaine caused a significant increase (from 21.6 to 61.3%) in colonial outgrowth, compared with the growth rate of controls without betaine. The presence of betaine in both the collection fluid and the enumeration medium had an additive effect on the colony-forming ability of airborne bacteria compared with the presence of betaine in either one alone. The effect of various betaine concentrations on the enumeration of aerosolized Pseudomonas syringae was determined. Betaine showed a threshold for maximum effect at a concentration of 2 to 5 mM. At higher concentrations (10 to 20 mM), the effects of betaine were negligible or possibly inhibitory. The significance of these results with respect to the development of protocols for monitoring airborne microorganisms, including genetically engineered microorganisms, is discussed.