Automated food microbiology: potential for the hydrophobic grid-membrane filter.

Bacterial counts obtained on hydrophobic grid-membrane filters were comparable to conventional plate counts for Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus in homogenates from a range of foods. The wide numerical operating range of the hydrophobic grid-membrane filters allowed sequential diluting to be reduced or even eliminated, making them attractive as components in automated systems of analysis. Food debris could be rinsed completely from the unincubated hydrophobic grid-membrane filter surface without affecting the subsequent count, thus eliminating the possibility of counting food particles, a common source of error in electronic counting systems.     

USE of an ELECTRONIC HGMF INTERPRETER SYSTEM FOR ENUMERATION of NALIDIXIC ACID RESISTANT SALMONELLAE IN CHICKEN CAECA

An electronic counting system using hydrophobic grid-membrane filters (HGMF) and the HGMF Interpreter was evaluated for its usefulness in enumerating nalidixic acid resistant Salmonella in frozen chicken caeca. Salmonella recovery was equivalent on both Hektoen Enteric and EF-18 agars. However, the color of the Salmonella growth on EF-18 agar was more easily differentiated by the HGMF Interpreter electronic counting system. the study showed that a 4 h resuscitation on a nonselective medium was required in order to maximize the subsequent recovery on Hektoen Enteric agar, though not on EF-18 agar. Using the EF-18 agar as the Salmonella selective medium, a method was established that recorded counts of nalidixic acid resistant Salmonella rapidly and easily in electronic data files, for subsequent retrieval and manipulation.     

ICMSF methods study. XVII. An international comparative study of the direct plate and hydrophobic grid-membrane filter methods for enumeration of Escherichia coli in foods. International Commission on Microbiological Specifications for Foods

Eight laboratories compared counts of Escherichia coli from naturally or artificially contaminated ground beef, other meats and poultry, vegetables, fish and shellfish, cheese, and diverse sources such as swabs, by the Anderson-Baird-Parker direct plate (DP) and a hydrophobic grid-membrane filter (HGMF) method. For five of the eight laboratories overall counts by HGMF were significantly low (51-83%) compared with those by DP. Counts by HGMF tended to be lower for naturally contaminated samples; several possible causes were investigated. In a subsidiary study, analyst variation in counting HGMF ranged from 0.8-7.3%, with little evidence of effects from counting positive versus negative grid cells or from the fullness of growth or staining intensity.     

Improved detection of coliforms and Escherichia coli in foods by a membrane filter method.

Analytical procedures based on filtration of homogenates through membrane filters, and particularly hydrophobic grid-membrane filters (HGMF), offer definite improvements in the enumeration of Escherichia coli and coliforms in foods. Whereas the counted specimen in pour plates may not usually be greater than 0.1 g, up to 1.0 g of ground beef, green beans, potato, cod, strawberries, or grapes could be filtered and counted on HGMF. Greatly improved limit of detection, reduced interference by noncoliforms, and complete removal of growth inhibitors such as polyphenols were demonstrated for HGMF, using violet red bile and mFC agars. In addition, counting on HGMF eliminated a false-positive reaction caused by sucrose in ice cream.     

Improved detection of coliforms and Escherichia coli in foods by a membrane filter method.

Analytical procedures based on filtration of homogenates through membrane filters, and particularly hydrophobic grid-membrane filters (HGMF), offer definite improvements in the enumeration of Escherichia coli and coliforms in foods. Whereas the counted specimen in pour plates may not usually be greater than 0.1 g, up to 1.0 g of ground beef, green beans, potato, cod, strawberries, or grapes could be filtered and counted on HGMF. Greatly improved limit of detection, reduced interference by noncoliforms, and complete removal of growth inhibitors such as polyphenols were demonstrated for HGMF, using violet red bile and mFC agars. In addition, counting on HGMF eliminated a false-positive reaction caused by sucrose in ice cream.     

Machine for printing hydrophobic grids on membrane filters

An apparatus for printing wax grids on membrane filters is described. The machine produced more and better hydrophobic grid-membrane filters much more rapidly than by hand.     

Automation of microbial enumeration: development of a disposable hydrophobic grid-membrane filter unit.

A disposable filter unit containing a hydrophobic grid-membrane filter (HGMF) was developed. The unit is liquid tight to serve as a specimen transport container and, by removal of the funnel extender (175- or 300-ml capacity), the unit becomes less than the height of two stacked petri plates to save space during in situ incubation. The polyethylene mesh which supports the HGMF facilitates rinse removal of any substance(s) that would interfere with microbial growth. The correlations between a pour plate, a conventional square HGMF, and a disposable filter unit on microbial enumeration were examined. Characteristics (e.g., clumping, spreading, etc.) of some microorganisms limit the linear counting range to less than 1,000 CFU per filter.     

Automation of microbial enumeration: development of a disposable hydrophobic grid-membrane filter unit

A disposable filter unit containing a hydrophobic grid-membrane filter (HGMF) was developed. The unit is liquid tight to serve as a specimen transport container and, by removal of the funnel extender (175- or 300-ml capacity), the unit becomes less than the height of two stacked petri plates to save space during in situ incubation. The polyethylene mesh which supports the HGMF facilitates rinse removal of any substance(s) that would interfere with microbial growth. The correlations between a pour plate, a conventional square HGMF, and a disposable filter unit on microbial enumeration were examined. Characteristics (e.g., clumping, spreading, etc.) of some microorganisms limit the linear counting range to less than 1,000 CFU per filter.     

Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe.

A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera.     

Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe

A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera.     

Improved Aerobic Colony Count Technique for Hydrophobic Grid Membrane Filters

The AOAC International official action procedure for performing aerobic colony counts on hydrophobic grid membrane filters (HGMFs) uses Trypticase soy-fast green FCF agar (FGA) incubated for 48 h. Microbial growths are various shades of green on a pale green background, which can cause problems for automated as well as manual counting. HGMFs which had been incubated 24 or 48 h at 35°C on Trypticase soy agar were flooded underneath with 1 to 2 ml of 0.1% triphenyltetrazolium chloride (TTC) solution by simply lifting one corner of the filter while it was still on the agar and adding the reagent. Microbial growths on HGMFs were counted after color had been allowed to develop for 15 min at room temperature. With representative foods, virtually all colonies stained pink to red. Automated electronic counts made by using the MI-100 HGMF Interpreter were easier and more reliable than control HGMF counts made by the AOAC International official action procedure. Manual counting was easier as well because of increased visibility of the microbial growths. Except in the case of dairy products, 24-h TTC counts did not differ significantly from 48-h FGA counts, whereas the FGA counts at 24 h were always significantly lower, indicating that for many food products the HGMF TTC flooding method permits aerobic colony counts to be made after 24 h.     

Membrane filtration of food suspensions.

Factors affecting the membrane filtration of food suspensions were studied for 58 foods and 13 membrane filters. Lot number within a brand, pore size (0.45 or 0.8 micrometer), and time elapsed before filtration had little effect on filterability. Brand of membrane filter, flow direction, pressure differential, age (microbiological quality) of the food, duration of the blending process, temperature, and concentration of food in the suspension had significant and often predictable effects. Preparation of suspensions by Stomacher (relative to rotary blender) addition of surfactant (particularly at elevated temperature) and prior incubation with proteases sometimes had dramatic effects of filterability. In contrast to popular opinion, foods can be membrane filtered in quantities pertinent to the maximums used in conventional plating procedures. Removal of growth inhibitors and food debris is possible by using membrane filters. Lowering of the limits of detection of microorganisms by concentration on membrane filters can be considered feasible for many foods. The data are particularly relevant to the use of hydrophobic grid-membrane filters (which are capable of enumerating up to 9 X 10(4) organisms per filter) in instrumented methods of food microbiological analysis.     

Enumeration of High Numbers of Bacteria Using Hydrophobic Grid-Membrane Filters

Printing a wax grid on a conventional membrane filter yields a device functioning as a most probable number apparatus (MPN), used at a single dilution but with a very large number of growth compartments (e.g., 3,650). By restraining the lateral spread and confluence of colonies, the hydrophobic grid-membrane filter (HGMF) allows growth- or colony-forming units (GU) to be resolved at levels far above those which produce an uncountable lawn on a conventional membrane filter. It also eliminates the size variation of normal bacterial colonies. As a result, the HGMF can give more accurate estimates of the concentration of GU. The method by which grid-cell count observations can be used to obtain MPN estimates of the number of GUs is described, and estimates obtained using the MPN method on the HGMF are compared with those resulting from conventional colony count procedures on membrane filters. A linear relation was observed between MPNGU and the number of GUs, at levels up to 30,000 GUs, for pure cultures of bacteria and for samples of natural waters. The HGMF has great potential for reducing the labor required in quantitative microbiology, since it allows, with one filter, enumeration of microorganisms over a very large concentration range and therefore reduces the need to make dilutions.     

Use of nuclepore filters for counting bacteria by fluorescence microscopy.

Polycarbonate Nuclepore filters are better than cellulose filters for the direct counting of bacteria because they have uniform pore size and a flat surface that retains all of the bacteria on top of the filter. Although cellulose filters also retain all of the bacteria, many are trapped inside the filter where they cannot be counted. Before use, the Nuclepore filters must be dyed with irgalan black to eliminate autofluorescence. Direct counts of bacteria in lake and ocean waters are twice as high with Nuclepore filters as with cellulose filters.     

Use of nuclepore filters for counting bacteria by fluorescence microscopy.

Polycarbonate Nuclepore filters are better than cellulose filters for the direct counting of bacteria because they have uniform pore size and a flat surface that retains all of the bacteria on top of the filter. Although cellulose filters also retain all of the bacteria, many are trapped inside the filter where they cannot be counted. Before use, the Nuclepore filters must be dyed with irgalan black to eliminate autofluorescence. Direct counts of bacteria in lake and ocean waters are twice as high with Nuclepore filters as with cellulose filters.     

Improved immunological membrane filter method for detection of food-borne Salmonella strains.

An improved membrane filter method that involves the use of an enzyme-labeled antibody stain has been developed for the rapid detection of Salmonella species in foods. The procedure is carried out directly on a hydrophobic grid-membrane filter without requiring transfer by blotting to nitrocellulose. Pure cultures of 54 Salmonella species and 10 foods artificially contaminated with Salmonella colindale gave a positive reaction in which Salmonella colonies were visible as purple dots. Of 11 nonsalmonella organisms, only Citrobacter freundii reacted with Spicer-Edwards antiserum. Of 22 naturally contaminated food samples, 10 were positive for both the hydrophobic grid-membrane filter procedure of the Association of Official Analytical Chemists and the improved enzyme-labeled antibody stain method, and there was perfect agreement between the methods. Of these 10 positive samples, one was negative by the Health Protection Branch method; of the negative samples, two were positive by this latter method. The improved enzyme-labeled antibody stain method allows detection of Salmonella spp. in foods within 48 h, requires little equipment, and is inexpensive, easy to perform, and suitable for automated detection.     

Comparison of fluorescence microscopy and solid-phase cytometry methods for counting bacteria in water

Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 10(6) cells filter(-1). In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 10(5), the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.     

Improved immunological membrane filter method for detection of food-borne Salmonella strains

An improved membrane filter method that involves the use of an enzyme-labeled antibody stain has been developed for the rapid detection of Salmonella species in foods. The procedure is carried out directly on a hydrophobic grid-membrane filter without requiring transfer by blotting to nitrocellulose. Pure cultures of 54 Salmonella species and 10 foods artificially contaminated with Salmonella colindale gave a positive reaction in which Salmonella colonies were visible as purple dots. Of 11 nonsalmonella organisms, only Citrobacter freundii reacted with Spicer-Edwards antiserum. Of 22 naturally contaminated food samples, 10 were positive for both the hydrophobic grid-membrane filter procedure of the Association of Official Analytical Chemists and the improved enzyme-labeled antibody stain method, and there was perfect agreement between the methods. Of these 10 positive samples, one was negative by the Health Protection Branch method; of the negative samples, two were positive by this latter method. The improved enzyme-labeled antibody stain method allows detection of Salmonella spp. in foods within 48 h, requires little equipment, and is inexpensive, easy to perform, and suitable for automated detection.     

Estimating red deer Cervus elaphus populations: an analysis of variation and cost-effectiveness of counting methods

• 1 Different counting methods are currently used to estimate red deer populations in the open range in Scotland, but there are few data available to compare variation in estimates, or relative cost‐effectiveness.
• 2 While it is impossible to determine the accuracy of counts (as real numbers are unknown), variation within and between different methods can be measured by repeat counts of the same area within as short a period as possible.
• 3 This study aimed to quantify the variation observed from repeat counts using each of four methods (ground, helicopter, infrared helicopter and dung‐counting methods) at one of three study sites in late winters 2003, 2004 and 2005. Additional data from digital camera images of groups from counts in other areas of Scotland were also used to assess the accuracy of visual counts.
• 4 Coefficients of variation (CVs) within any method of between 5% and 16% were recorded, consistent with previous comparisons for red deer open range counts in Scotland. CVs were lowest for ground and helicopter counts. The infrequency of optimal conditions was likely to limit the applicability of infrared counts in Scotland.
• 5 In terms of cost‐effectiveness, helicopter counting was the least labour‐intensive, with costs of other techniques depending on the availability of existing manpower as an overhead cost.
• 6 It is concluded that helicopter counts are most likely to minimize errors while maximizing cost‐efficiency. Accuracy can be improved by the use of digital photography for counting larger deer groups. Estimates are likely to be improved further by increasing the frequency of counts and using the same methods, counters and routes for repeat counts.

     

Enumerating bacterial cells on bioadhesive coated slides

Quantifying bacterial abundance and biomass is fundamental to many microbiological studies. Directly counting via epifluorescence microscopy has become the method of choice, especially for environmental samples, and conventional techniques require filtration of cells onto black polycarbonate membrane filters. We investigated the utility of instead capturing stained bacterial suspensions on bioadhesive slides, performing tests using pure cultures of bacteria, mixtures of cultured bacteria, and environmental samples from five habitat types. When compared to the standard filtration and flow cytometric approaches, bioadhesive slides were found to be an accurate and precise platform for rapid enumeration of bacteria. Total bacterial counts made using the three methods were positively correlated for acridine orange and Live/Dead® (L/D) staining (0.81 ≤ r ≤ 0.95, all p ≤ 0.002). All platforms had similar precision, though counts obtained using bioadhesive slides were significantly higher than those made with polycarbonate filters and flow cytometry. The specific bioadhesive slides we used resulted in substantial cell mortality for certain pure cultures and river water samples, limiting their use for L/D determination. Cell enumeration using bioadhesive slides is particularly effective because it is highly precise at a wide range of cell concentrations, allows observation of cells that are not readily discernible on filters, reduces the number of steps and processing materials associated with sample analysis, and increases throughput.