Rapid selective enumeration of bacteria in foods using a microcolony epifluorescence microscopy technique

The growth patterns of microcolonies of 59 different pure cultures were studied on eight selective solid media. A method of growing microcolonies on the surface of polycarbonate membrane filters, placed on the selective agar media, followed by staining and examination by epifluorescent microscopy was developed. The patterns of growth of the pure cultures as microcolonies were studied on the eight selective media. Only four media proved to be reliable for this purpose and the relationship between the microcolony count and plate count was studied on these media together with nutrient agar. Microcolony counts using three of these media (enriched lauryl sulphate aniline blue, pseudomonas selective agar (C-F-C) and Baird-Parker medium) were capable of giving reliable estimates of coliforms (r = 0.89), pseudomonads (r = 0.93) and staphylococci (r = 0.92) after incubation at 30 degrees C for 3 or 6 h (staphylococci) at contamination levels of above 10(3) bacteria/g in a variety of foods. The results are available within a working day and should allow the more efficient management of food supplies.     

Microcolony epifluorescence microscopy for selective enumeration of injured bacteria in frozen and heat-treated foods

A rapid (less than 6 h) method for selectively enumerating coliforms, pseudomonads, and staphylococci has been developed which involves counting microcolonies grown on the surface of polycarbonate membranes under selective conditions. The method was not directly applicable to foods containing injured bacteria due to the poor formation of or an inability to form microcolonies under selective conditions. However, the introduction of a 3- to 5-h resuscitation step in tryptone soya broth allowed the method to give reliable estimates of these organisms in a variety of frozen and heat-processed foods. Under nonselective conditions, i.e., for total counts, the microcolony method enabled a rapid count to be made of viable bacteria in heat-treated foods, but these results were also made more consistent by the introduction of a resuscitation step. This method makes results from these foods available far faster than conventional enumeration methods.     

Microcolony epifluorescence microscopy for selective enumeration of injured bacteria in frozen and heat-treated foods.

A rapid (less than 6 h) method for selectively enumerating coliforms, pseudomonads, and staphylococci has been developed which involves counting microcolonies grown on the surface of polycarbonate membranes under selective conditions. The method was not directly applicable to foods containing injured bacteria due to the poor formation of or an inability to form microcolonies under selective conditions. However, the introduction of a 3- to 5-h resuscitation step in tryptone soya broth allowed the method to give reliable estimates of these organisms in a variety of frozen and heat-processed foods. Under nonselective conditions, i.e., for total counts, the microcolony method enabled a rapid count to be made of viable bacteria in heat-treated foods, but these results were also made more consistent by the introduction of a resuscitation step. This method makes results from these foods available far faster than conventional enumeration methods.     

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.     

Detection of viable, but non-culturable Pseudomonas fluorescens DF57 in soil using a microcolony epifluorescence technique

Abstract Kanamycin-resistant Pseudomonas fluorescens DF57-3 cells (Tn5 modified) inoculated in soil microcosms rapidly lost their culturability, as defined by visible colony formation on Kings B agar supplemented with kanamycin. Thus, after 40 days only 0.02–0.35% of the initial inoculum was culturable. A microcolony epifluorescence technique was developed to determine the viable, but non-culturable subpopulation. A suspension of bacteria from the soil was prepared in salt solution after a sonication procedure and a sample was filtered onto a 0.2 μm Nuclepore filter. The filter was then placed for 3–4 days on the surface of Kings B agar before staining with acridine orange for epifluorescence microscopy. By staining and washing the filters carefully, disruption of microcolonies could be avoided. A majority of the microcolonies resulted from 2–3 cell divisions during the first 2 days of the incubation period, after which the cell divisions stopped. These microcolonies were taken to represent a population of viable, but non-culturable cells and comprised about 20% of the initial inoculum. A similar recovery was obtained when the filters were incubated on the surface of citrate minimal medium or soil extract medium. A few microcolonies showed continued growth on the filters, however, and their number corresponded well with that of visible macrocolonies. Observation by microscopy of a few (2–3) cell divisions (microcolony epifluorescence technique) is proposed for determination of subpopulations of viable, but non-culturable bacteria in soil.     

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.     

The enumeration of proteolytic bacteria in foods

Summary For the enumeration of proteolytic bacteria in foods, regularly or sometimes obtained by microbial fermentation, a surface plating technique, using Frazier's gelatin/agar, incubated for two days at 31±1°C. and thereupon developed with sublimate solution, appeared useful.     

Rapid enumeration of microorganisms in foods by the direct epifluorescent filter technique.

Filtration of "stomachered" food suspensions through nylon filters (pore size, 5 microns) removed most of the food debris without affecting the recovery of microorganisms. Two to ten milliliters of these prefiltered suspensions could be filtered in the direct epifluorescent filter technique (DEFT). The technique takes less than 30 min to complete and has a lower sensitivity of less than 60,000 microorganisms per g for all products examined. Vegetative bacterial cells, spores, fungal hyphae, and yeasts could be distinguished with the technique. For fresh meat and fish, the DEFT count of prefiltered suspensions agreed well with the plate count of unfiltered suspensions over the range of 10(4) to 10(10)/g (correlation coefficient of 0.91). For frozen meat and fish and frozen vegetables, the two counting methods had correlation coefficients of 0.87 and 0.66, respectively. The poor correlation for frozen vegetables was due to the inclusion in the DEFT count of nonviable bacteria killed by the blanching process used to inactivate enzymes. Good agreement was obtained between the prefiltered DEFT count and unfiltered plate count for cooked meats, cream doughnut, and whole peppers. Possible reasons for the poor agreement between the DEFT count and plate count for certain products are discussed.     

海洋病毒荧光显微计数法的优化与应用

【目的】建立一种快速、稳定、可靠的海洋病毒计数方法。【方法】海水水样经福尔马林固定后,滤过孔径为0.02μm的Anodisc Al2O3膜。滤膜经SYBR Green I染色后,在相应波长的激发光下进行观察。借助荧光显微镜目镜网格尺,计数视野中的病毒颗粒,换算后获得样品中病毒的浓度。【结果】对具体实验方法进行了优化,可快速、稳定地对海水中的病毒计数。【结论】建立了一种适用于国内实验条件的、可靠的海洋病毒计数方法。     

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.     

Semi-automated counting of bacteria and somatic cells in milk using epifluorescence microscopy and television image analysis

An epifluorescent microscope fitted with a 'Chalnicon' closed circuit television camera linked to an Optomax System III image analyser was used to count bacteria and somatic cells on membrane filters prepared from milk by the direct epifluorescent filter technique (DEFT). For both bacteria and somatic cells, the semi-automated DEFT count of low count milk exceeded the visual DEFT count but this difference became proportionately less as the count increased. There was close agreement between the semi-automated and visual DEFT counts over the range 10⁵-5 times 10⁶/ml for bacteria and 3 times 10⁵-5 times 10⁶/ml for somatic cells. The semi-automated DEFT count of bacteria and somatic cells correlated well with the plate count and Coulter count respectively, with correlation coefficients of 0.83 and 0.81.     

Simultaneous enumeration of different bacteria using reverse sample genome probing technique

Quantitative reverse sample genome probing (RSGP) with lambdaDNA as an internal standard was used to enumerate the total numbers of Rhizobium sp. CCRC 13560, Rhizobium meliloti CCRC 13516 and Bradyrhizobium sp. CCRC 13585. K(lambda)/Kx ratios varied between the three species but also in response to the amounts of lambdaDNA or genomic DNA used in the labeling mixture or fixed upon the membrane. Comparative enumerations of pure cultures revealed higher counts using genomic probing as compared with growth-based colony forming units (CFU; 3.4+/-1.7-fold higher for R. meliloti, 6.4+/-7.8-fold higher for Rhizobium sp. and 0.34+/-0.17-fold higher for Bradyrhizobium sp.). In mixed cultures, the estimated cell numbers using genomic probing were 126+/-172-, 85+/-83- and 4.0+/-3.4-fold higher (same respective order) than the growth-based assay. By replacing the klambda/kx ratio with k'lambda/k'x (slope from signal intensity of differently diluted lambdaDNA/slope from signal intensity of differently diluted target DNAxf(x)/flambda), significant improvement in the accuracy of the estimation was achieved. The calculated cell numbers via the genomic probe technique were 0.99+/-0.13-, 1.25+/-0.23- and 0.18+/-0.11-fold higher than the respective CFUs in pure cultures of R. meliloti, Rhizobium sp. and Bradyrhizobium sp. In samples containing mixed populations, the estimated numbers from genomic probing were 1.25+/-0.51-, 45.9+/-14.8- and 0.27+/-0.07-fold higher than the CFU-derived cell count (same respective order).     

Separation of micro-organisms from meat and their rapid enumeration using a membrane filtration-epifluorescent microscopy technique

A new method of separating bacteria from beef mince has been developed, in which an alkaline protease, Alcalase 0.6 L, was used to degrade the meat proteins, leaving micro-organisms in suspension. The organisms were then counted, using a membrane filtration-epifluorescent microscopy technique. A correlation coefficient of 0.97 was obtained between this method and the standard plate count, indicating its suitability for use in quality control.     

Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I

Viruses are the most abundant biological entities in aquatic environments, typically exceeding the abundance of bacteria by an order of magnitude. The reliable enumeration of virus-like particles in marine microbiological investigations is a key measurement parameter. Although the size of typical marine viruses (20-200 nm) is too small to permit the resolution of details by light microscopy, such viruses can be visualized by epifluorescence microscopy if stained brightly. This can be achieved using the sensitive DNA dye SYBR Green I (Molecular Probes-Invitrogen). The method relies on simple vacuum filtration to capture viruses on a 0.02-microm aluminum oxide filter, and subsequent staining and mounting to prepare slides. Virus-like particles are brightly stained and easily observed for enumeration, and prokaryotic cells can easily be counted on the same slides. The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously.     

Use of nitrocellulose Synpor filters for counting soil bacteria by epifluorescence microscopy

A modified acridine orange staining method for estimating soil bacterial numbers by epifluorescence microscopy using Synpor filters (VCHZ Synthesia, Czechoslovakia) was elaborated. Comparing with the method of direct count of soil bacteria estimated in a Bürker chamber higher counts of bacteria and a lower variation of results were obtained. To verify the sensitivity of the method, microflora from various soil horizons was tested.     

Comparison of flow cytometry and epifluorescence microscopy for counting bacteria in aquatic ecosystems.

Flow cytometry was used to count bacterial cells from diverse origins: one strain of E. coli, one sample of lake water, and 18 samples of estuary water. To verify the accuracy and the precision of this technique, total bacteria counts made by flow cytometry were compared with counts by direct observation using epifluorescence microscopy. The results of this study showed that flow cytometry was a reliable technique for counting a mixture of bacteria in samples from aquatic ecosystems.     

Rapid enumeration of viable micro-organisms by staining and direct microscopy

A rapid method is described that uses the tetrazolium salt 2-( p -iodophenyl-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT) to stain viable micro-organisms retained on a filter membrane. Good correlation exists between numbers of INT stained cells and aerobic plate counts of single strain cultures of bacteria and yeasts. A pre-treatment step allows the technique to be used for pasteurized milk.     

A medium for enumeration of bacteria in foods containing swarming Bacillus spp.

A new medium containing sodium tripolyphosphate, ethylenediaminetetraacetic acid disodium salt, dihydrate, sodium desoxycholate, supplemented with minerals and nutrient sources was developed, which was very effective in inhibiting swarming of Bacillus spp. This medium polyphosphate-EDTA-desoxycholate agar (PEDA) was compared with tryptone glucose extract agar (Oxoid) for determining the total plate count of various semi-conserved fishery products. While promoting discrete colony formation of Bacillus spp., PEDA provided good bacterial recovery. PEDA is recommended for isolation or enumeration of bacteria in food dominated by swarming Bacillus spp.     

Natural populations of bacteria in Lake Kinneret: Observations with scanning electron and epifluorescence microscopy

The bacterioplankton assemblage in Lake Kinneret, Israel, sampled on 6 occasions representative of different seasonal conditions was studied with scanning electron microscopy (SEM) and epifluorescence microscopy after acridine-orange staining. In near-surface (1–3 m) samples taken in October 1981 and March 1983, several unusual types of budding, appendaged, and filamentous cells were found. During lake stratification, typical large anaerobic forms (including photosynthetic green sulphur bacteria) were observed in samples from the metalimnion and deep (40 m) hypolimnion. Epifluorescence counts indicated that bacteria in the water column ranged from 0.55 to 2.67 × 10⁶ cells ml^–1.