Comparison of the Precision Obtained in Counting Viable Bacteria by the Spiral Plate Maker, the Droplette and the Miles & Misra Methods

The Spiral Plate Maker yielded counts of viable bacteria in pure suspensions that were at least as precise as those obtained under comparable conditions by the Droplette and Miles & Misra methods. A tendency of the Miles & Misra method to produce erroneous counts of Escherichia coli was demonstrated and the advantages of the mechanical method over established counting methods is discussed.     

Evaluation of the spiral plate and laser colony counting techniques for the enumeration of bacteria in foods

Summary (1) In a parallel study, samples of food and dairy products, bacterial cultures and spore suspensions were examined by two operators using both the spiral plate and surface drop techniques for counting bacteria. (2) Statistical analyses of the results showed no differences between the methods at the 5% level of probability; regression and correlation coefficients were highly significant. A variation between paired counts of less than 0.5 log₁₀ cycles was given by 95% of the samples. (3) The replicate variances of both methods were <0.006, indicating good agreement betweeen duplicate plates. (4) An electronic laser counter used in this study was found to give comparable results (r=0.966) to the grid-method of colony counting in a substantially shorter time. (5) Analysis of operation times and material requirements for each method showed that significant savings in cost, time, space and support labour could be achieved with the spiral plate method over conventional techniques.     

Evaluation of methods for the enumeration of Campylobacter jejuni and Campylobacter coli bacteriophages

The Spiral Plater System used for enumeration of bacteria was evaluated for the titration of Carnpylobacter phages. Twelve phages of C. jejuni were titrated using the conventional surface droplet technique, soft agar overlay-pour plate technique, and the Spiral Plater System. Phage counts obtained by the three methods were similar but the Spiral Plater System showed greater precision than the other methods.     

Evaluation of new culture media for rapid detection and isolation of salmonellae in foods.

Conventional methods for Salmonella detection in foods can require up to 6 and at least 4 days. We have observed that the total analysis time can be reduced to 48 h by using Salmosyst broth as a liquid medium for both preenrichment and selective enrichment and Rambach agar (RA), a new selective plate medium. In samples of artificially contaminated ground beef Salmonella enteritidis was detected at a concentration of 0.4 CFU/g (10 CFU/25 g) by both a conventional method and the new method. Of 519 samples of foods for sale, 38 were Salmonella positive by both methods while 471 were negative. Nine samples which were negative by the conventional method were positive by the Salmosyst-RA method, while one sample positive by the first method was negative by the last. Therefore, the Salmosyst-RA method showed 97.9% sensitivity compared with the 81.2% sensitivity of the conventional method. The new method was also highly specific (98% specificity) in presumptive identification of Salmonella colonies. Furthermore, a 6-h preenrichment in Salmosyst broth has been proved sufficient for the repair of heat-injured Salmonella cells and for subsequent recovery by selective enrichment. In conclusion, the Salmosyst-RA method shows several advantages over both conventional and rapid noncultural methods: (i) only two media are required instead of the five media for conventional methods; (ii) in real time it is comparable to other rapid noncultural methods, which require 30 to 31 h; (iii) it is highly sensitive and specific; and (iv) it allows the isolation of Salmonella strains which can be characterized by appropriate phenotypic and genotypic typing methods for epidemiological investigations.     

Enumeration of airborne bacteria and fungi using solid phase cytometry

Conventional methods for the enumeration of airborne micro-organisms are inaccurate and time-consuming, hence the interest in novel approaches is increasing. In the present study, the use of solid phase cytometry (SPC) was evaluated for the enumeration of airborne micro-organisms. A 4 h SPC procedure based on viability staining was applied to samples from 50 locations and compared with an optimised culture-based method. Plate counts after air sampling were repeatable but strongly dependent on sampling volume. Samples with low or high microbial load were difficult to analyse using the culture-based method, unlike with SPC. Results show that SPC can be considered superior to the culture-based method because of its much higher dynamic range, its speed and its ability to enumerate not only culturable but all viable micro-organisms.     

The estimation of cell numbers of flagellate and coccoid Chlorophyta in natural populations

Methods for the quantitative estimation of the small coccoid and flagellate Chlorophyta in natural algal communities are outlined. Experiments to demonstrate the precision of these methods are discussed and the components of variance are calculated for several experimental procedures, both for phytoplanktonic and benthic associations. The smallest overall variance for phytoplankton estimations is 27% where a large number of samples were collected and bulked to reduce the sampling error. The precision in counting epipelic algae by this method is only 12%. This result suggests that the inclusion of a short period of ultrasonication may reduce the overall variance in estimation by randomising the distribution of the algae prior to plate inoculation. Close agreement exists between direct counts and plate counts.

It is realised that the cultural approach has limitations, but comparison with results obtained from other methods indicates the advantages of cultural techniques, particularly when estimating small or motile algae.     

Evaluation of PetrifilmTM methods for enumeration of aerobic flora and coliforms in a wide range of foods

C. DE W. BLACKBURN, C.L. BAYLIS AND S.B. PETITT. 1996. Petrifilm^TM is a ready-to-use alternative to traditional microbial enumeration methods. The Petrifilm^TM Aerobic Count Plate (ACP) and Coliform Count Plate (CCP) were compared with standard methods for the enumeration of the aerobic mesophilic flora and coliform bacteria in 91 foods covering a wide range of different food commodities. There was good correlation between the Petrifilm^TM ACP and the standard aerobic colony count method ( r = 0.989) and between the Petrifilm^TM CCP and the standard Violet Red Bile Agar plating method ( r = 0.872). In both cases, the Petrifilm^TM methods had a better repeatability than the standard methods. The Petrifilm^TM ACP and CCP were shown to be practical and accurate alternatives to standard enumeration methods in a wide range of foods, with benefits of saving time, labour and incubator space.     

A Refinement in Mechanized Plating

A plating instrument is described for use in the determination of viable counts of micro-organisms in foods and other biological materials. It is based on the linear distribution of a fixed volume of inoculum on the surface of a rotating agar plate in a continuous spiral fashion. The rate of inoculation is controlled so that from the relative position and number of the emerging colonies, viable counts up to 10⁹/ml can be obtained without the necessity to dilute the sample. Counting can be rapid and the time taken for reading the plates can be reduced considerably. The instrument includes controls for the variability in the agar thickness, uneven surface as the result of drying, multi-plate inoculation without re-charging and speedy de-contamination between inoculations.     

A new direct plate method for the enumeration of Escherichia coli in frozen foods

H all , L.P. 1984. A new direct plate method for the enumeration of Escherichia coli in frozen foods. Journal of Applied Bacteriology 56 , 227–235.
A new method has been devised, incorporating a resuscitant stage, which allows direct isolation of Escherichia coli biotype I, Irregular type II and Irregular type VI. Rapid indole tests on the distinctive colonies produced enable determinations of E. coli biotype I to be made within 24 h. This method employs materials of low cost and achieves complete recovery of injured cells. It also detects not only anaerogenic strains but those which are slow in producing acid from lactose or give negative results by other methods. If required, further study of isolates can be made after the indole test. Comparisons were made between conventional methods, the new method and a similar direct plate method. The implications of the higher counts obtained by the two latter methods are discussed in relation to microbiological specifications and standards for frozen foods.     

Spiral plate count method for the examination of raw and pasteurized milk.

The spiral plate count method (SPLPC) was compared with the standard plate count (SPC) method by examining 201 samples of raw and pasteurized milk. Although the means of the two methods differed significantly at alpha = 0.01,the difference was less than 10% and was not considered to be of any practical importance. The pooled replicate variances of both methods were less than 0.003, indicating good agreement between duplicate plates, with the variance of the SPLPC slightly less than that of the SPC. We believe this study indicates that the SPLPC could be substituted for the SPC in the bacteriological examination of milk.     

Monitoring microbial numbers in food by density centrifugation

Some foods contain low numbers of microbes that may be difficult to enumerate by the plate count method due to small food particles that interfere with the counting of colonies. Ludox colloidal silicon was coated with reducing agents to produce a nontoxic density material. Food homogenates were applied to a layered 10 and 80% mixture of modified Ludox and centrifuged at low speed. The top and bottom of the tube contained the food material, and the Ludox-containing portion was evaluated by conventional pour plate techniques. Plate counts of the Ludox mixture agreed with plate counts of the food homogenate alone. The absence of small food particles from pour plates resulted in a plate that was more easily read than pour plates of the homogenate alone. Modified Ludox was evaluated for its effect on bacteria at 4 degrees C during a 24-h incubation period. No inhibition was observed. This method is applicable to food products, such as doughnuts, spices, tomato products, and meat, in which small food particles often interfere with routine plate counts or low dilution may inhibit colony formation. Inhibitory substances can be removed from spices, resulting in higher counts. Ludox is more economical than similar products, such as Percoll. Modified Ludox is easily rendered nontoxic by the addition of common laboratory reagents. In addition, the mixture is compatible with microbiological media.     

Monitoring Microbial Numbers in Food by Density Centrifugation

Some foods contain low numbers of microbes that may be difficult to enumerate by the plate count method due to small food particles that interfere with the counting of colonies. Ludox colloidal silicon was coated with reducing agents to produce a nontoxic density material. Food homogenates were applied to a layered 10 and 80% mixture of modified Ludox and centrifuged at low speed. The top and bottom of the tube contained the food material, and the Ludox-containing portion was evaluated by conventional pour plate techniques. Plate counts of the Ludox mixture agreed with plate counts of the food homogenate alone. The absence of small food particles from pour plates resulted in a plate that was more easily read than pour plates of the homogenate alone. Modified Ludox was evaluated for its effect on bacteria at 4°C during a 24-h incubation period. No inhibition was observed. This method is applicable to food products, such as doughnuts, spices, tomato products, and meat, in which small food particles often interfere with routine plate counts or low dilution may inhibit colony formation. Inhibitory substances can be removed from spices, resulting in higher counts. Ludox is more economical than similar products, such as Percoll. Modified Ludox is easily rendered nontoxic by the addition of common laboratory reagents. In addition, the mixture is compatible with microbiological media.     

A microbiological evaluation of warm air hand driers with respect to hand hygiene and the washroom environment

A finger rinse technique for counting micro-organisms on hands showed no significant difference in the level of recovered micro-organisms following hand drying using either warm air or paper towels. Contact plate results appeared to reflect the degree of dampness of hands after drying rather than the actual numbers of micro-organisms on the hands. In laboratory tests, a reduction in airborne count of Pseudomonas aeruginosa and Staphylococcus aureus of between 40 and 75% was achieved from 600 readings comparing inlets and outlets of warm air hand driers. In washroom trials, the number of airborne micro-organisms was reduced by between 30 and 75%. Air emitted from the outlet of the driers contained significantly fewer micro-organisms than air entering the driers. Drying of hands with hand driers was no more likely to generate airborne micro-organisms than drying with paper towels. Levels of micro-organisms on external surfaces of hand driers were not significantly different to those on other washroom surfaces. This work shows that warm air hand driers, of the type used in this study, are a hygienic method of drying hands and therefore appropriate for use in both the healthcare and food industry.     

Spiral Plate Method for Bacterial Determination

A method is described for determining the number of bacteria in a solution by the use of a machine which deposits a known volume of sample on a rotating agar plate in an ever decreasing amount in the form of an Archimedes spiral. After the sample is incubated, different colony densities are apparent on the surface of the plate. A modified counting grid is described which relates area of the plate of volume of sample. By counting an appropriate area of the plate, the number of bacteria in the sample is estimated. This method was compared to the pour plate procedure with the use of pure and mixed cultures in water and milk. The results did not demonstrate a significant difference in variance between duplicates at the α = 0.01 level when concentrations of 600 to 12 × 10⁵ bacteria per ml were used, but the spiral plate method gave counts that were higher than counts obtained by the pour plate method. The time and materials required for this method are substantially less than those required for the conventional aerobic pour plate procedure.     

Use of the IUL Countermat Automatic Colony Counter for Spiral Plated Total Viable Counts

An IUL Countermat automatic colony counter was used to enumerate colonies on spiral total viable count plates made with a wide variety of foods. The counter results exhibited a correlation with manual counting results similar to the reproducibility obtained with manually counted spiral plates. Use of this machine results in a large time saving compared with the conventional counting method and is recommended as a generally suitable method for counting spiral total viable count plates.     

Examination of Market Foods for Coliform Organisms

Food specimens (490) in nine categories were examined for total aerobic plate count and numbers and types of coliform organisms, including the enteropathogenic Escherichia coli (EEC). The total counts were compared with various suggested standards, and a limit of 100,000/g appeared to be a realistic goal, except for certain food types with a high level of natural flora. Plate counts in VRB were compared to counts obtained by isolation by enrichment in LST Broth, and the latter method produced a higher percentage of isolations. The presence of E. coli was determined by use of EC Medium incubated at 44.5 +/- 0.1 C. Only 40.4% of the positive EC tubes, however, contained E. coli. It appeared that a limit of 10 coliform organisms per g as a suggested standard could be met with several types of foods. Isolation of EEC was obtained only three times, or in 0.6% of the specimens.     

Detection of post-pasteurization contamination of cream by impedimetric methods

Impedimetric methods for evaluating post-pasteurization contamination and shelf-life of cream were assessed. Over 94% of the samples tested were in agreement, using selected cut-offs of 20 h for detection time measured at 21 degrees C with creams containing inhibitors for the growth of Gram positive bacteria on standard plate count agar as growth media, and 3.2 X 10(7) cfu/g for plate counts obtained on cream which had been pre-incubated in the presence of inhibitors for the growth of Gram positive organisms, and on cream stored at 6 degrees C for 7 d. Agreement between the impedimetric method and plate count was not as good if either Brain Heart Infusion or Milk Agar was used in place of Plate Count Agar in the former technique. A poor correlation was obtained between plate count methods for enumerating post-pasteurization contamination and keeping quality with impedimetric measurements on cream alone. It was possible, with a reasonable degree of certainty, to determine if cream had suffered post-pasteurization contamination within 20 h of production.     

Adaptation of an [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay to evaluate the cytotoxicity of the extracellular products of micro-organisms pathogenic to fish

AIMS: Adaptation of a colorimetric assay using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) to evaluate the cytotoxicity of the extracellular products of micro-organisms pathogenic to fish. METHODS AND RESULTS: The optimal conditions for the colorimetric assay were determined and this method was compared with the trypan blue exclusion assay. The protein concentration of extracellular products causing the death of 50% of the cell population (CI50) was determined. CONCLUSIONS: This assay enables quantitative and objective comparison of the cytotoxicity of the extracellular products of micro-organisms pathogenic to fish. It was shown to be more accurate than conventional counting with the trypan blue exclusion assay. SIGNIFICANCE AND IMPACT OF THE STUDY: This method may also be useful for characterizing the cytotoxicity of specific components of extracellular products.     

Detection of post-pasteurization contamination of cream by impedimetric methods

Impedimetric methods for evaluating post-pasteurization contamination and shelf-life of cream were assessed. Over 94% of the samples tested were in agreement, using selected cut-offs of 20 h for detection time measured at 21°C with creams containing inhibitors for the growth of Gram positive bacteria on standard plate count agar as growth media, and 3.2 × 10⁷ cfu/g for plate counts obtained on cream which had been pre-incubated in the presence of inhibitors for the growth of Gram positive organisms, and on cream stored at 6°C for 7 d. Agreement between the impedimetric method and plate count was not as good if either Brain Heart Infusion or Milk Agar was used in place of Plate Count Agar in the former technique. A poor correlation was obtained between plate count methods for enumerating post-pasteurization contamination and keeping quality with impedimetric measurements on cream alone. It was possible, with a reasonable degree of certainty, to determine if cream had suffered post-pasteurization contamination within 20 h of production.     

加工产品中转基因玉米Bt11成分实时荧光PCR定量(性)检测

实验在玉米自身基因和外源基因的边界序列之间设计了具有品种和品系特异性的引物和探针 ,并以实时荧光PCR技术 ,建立了加工产品中转基因玉米Bt1 1成分品系鉴定检测和定量检测的方法。实验对加热条件和时间对检测转基因成分的影响作了探讨 ,并检测了部分市售食品和饲料。检测结果发现 ,加热时间温度越高、时间越长 ,对转基因成分定量检测的影响越大 ;在所检测的样品中可以检测出转基因玉米Bt1 1成分 ,有些样品还同时检出其他转基因成分。本研究实验建立的方法 ,可以用于加工产品中转基因成分的定量检测 ,也可以用于定性检测 ,或作为常规PCR定性检测后的确证实验方法。