ENUMERATION OF IMMUNOMAGNETICALLY CAPTURED ESCHERICHIA COLI IN WATER SAMPLES USING QUANTUM DOT-LABELED ANTIBODIES

Immunomagnetic separation was coupled with quantum dot (QD) labeling for the rapid, selective and sensitive detection of Escherichia coli in water samples. The target bacteria were recovered from the solution by antibody-coated paramagnetic beads, and sandwich complexes were formed by using secondary antibodies labeled with QDs. The fluorescence intensities, as a result of the capturing of different concentrations of bacteria, were measured, and a linear correlation ( R ²  =  0.976) was obtained between log E. coli concentration ( x ) and the intensity ( y ) with a regression model of y =  26.9x  +  41.1 in a working range of 8.9  ×  10¹ and 1.9  ×  10⁶ cfu/mL. The selectivity of the developed sensor was examined with Enterobacter aerogenes and Enterobacter dissolvens, which did not produce any significant response. The ability of the immunoassay to detect E. coli in real water samples was investigated and the results were compared with the experimental results from plate-counting methods. A good agreement was observed between the QD-enhanced detection and plate counting.

PRACTICAL APPLICATIONS

In this study, a rapid, sensitive and convenient fluorometric assay based on the immunomagnetic separation (IMS) and quantum dot (QD) labeling was employed for the detection of Escherichia coli in water samples. The incorporation of QDs into fluorometric immunoassay techniques has various advantages over labeling with organic dyes and enzymes. In addition, the spectroscopic properties of QDs can allow multiplexed immunoassays coupled with IMS for bacteria detection, which will be investigated in further studies. Here we showed that QD labeling is a promising tool for the detection of E. coli in real water samples containing different components with a lower detection limit.     

'BITTY' CREAM: THE OCCURRENCE AND SIGNIFICANCE OF BACILLUS CEREUS SPORES IN RAW MILK SUPPLIES

SUMMARY: Methods for determining the Bacillus cereus content of milks and of rinses of dairy equipment are described and their limitations discussed. Milk samples from various sources were examined throughout the year for 'bittiness' as well as for B. cereus. The organism was not detected in all samples showing bittiness. There was a marked seasonal variation in the B. cereus index of raw milk supplies; maximum numbers were obtained from July to September and minimum numbers in April and May. Rinses of farm dairy equipment yielded few B. cereus spores but milk cans not uncommonly contained large numbers, especially in the summer months, when 10·5% of cans showed more than 5 × 10³/can. Preliminary observations on other sources of B. cereus are described and some of the problems of the control of this organism in raw milk supplies are discussed.     

PCR-BASED FLUORESCENT METHOD FOR RAPID DETECTION OF SALMONELLA TYPHIMURIUM IN POULTRY SAMPLES

A DNA binding fluorescence method based on polymerase chain reaction (PCR) products was evaluated for rapid detection of Salmonella Typhimurium in poultry products. Wash water samples of chicken carcasses and ground turkey were inoculated with S. Typhimurium to obtain final concentrations of 10° - 10⁵ CFU/mL. One mL of each sample was used to get the DNA template and 5 μL of the sample template was added into 25 μL of SYBR Green PCR Master Mix and two specific Salmonella ompC gene primers. The negative control was the same except 5 μL of each wash solution was added instead of 5 μL sample template. The reaction was carried out in a thermocycler. Finally, the fluorescence signal of each PCR product was measured using a fluorometer. The PCR products were also confirmed by ethidium bromide agarose gel, and the DNA concentrations of the PCR products were measured by a filter fluorescence photometer. The results showed that when bacterial cells increased from 0 to 2 CFU/mL, the fluorescence signal increased significantly. The PCR-based fluorescence method could detect the target bacteria in minutes after PCR amplification compared to hours by gel electrophoresis and also could be done at an earlier time during PCR amplification. The detection limit of this method for S. Typhimurium in the poultry samples was 2 CFU/mL without any enrichment.     

METHODS OF ASSESSING THE SPORICIDAL EFFICIENCY OF AN ULTRA-HIGH-TEMPERATURE MILK STERILIZING PLANT. II. EXPERIMENTS WITH SUSPENSIONS OF SPORES IN MILK

SUMMARY: The sporicidal efficiency of an ultra-high-temperature (UHT) milk processing plant has been tested using spores of a strain of Bacillus subtilis in milk. With the inoculum and volume of milk adjusted to obtain a countable number of survivors by a conventional dilution counting method, a temperature of 130·5° with the minimum time setting of the plant was found necessary to give a destruction of 99·99999%. This temperature was lower than that found previously (135°) for spores suspended in water and evidence is produced to support the suggestion that UHT milk may be inhibitory to the germination and/or subsequent growth of heated spores.     

RAPID DETECTION OF SALMONELLA TYPHIMURIUM IN FOOD SAMPLES USING A BIENZYME ELECTROCHEMICAL BIOSENSOR WITH FLOW INJECTION

A bienzyme (tyrosinase and horseradish peroxidase) electrochemical biosensor was developed for detection of Salmonella typhimurium, and evaluated for application in a flow injection system coupled with immunomagnetic separation for food samples. Parameters for immunomagnetic separation, enzymatic reaction, flow injection and electrochemical detection were determined using pure culture samples. The selectivity was tested in the presence of Listeria monocytogenes, Campylobacter jejuni and E. coli 0157:H7. The results showed a linear relationship for logarithmic values between peak current ratio and the cell number of S. typhimurium in the range of 10³ 10⁵ cfu/mL, with R²= 0.99. The detection limit of this method was 1.09 × 10³ cfu/mL for S. typhimurium and the detection time was 2.5 h. Samples of chicken carcass wash water and ground beef were used to evaluate the biosensor. The results demonstrated that this biosensor has a potential for rapid detection of different pathogens in various food samples.     

METHODS OF ASSESSING THE SPORICIDAL EFFICIENCY OF AN ULTRA-HIGH-TEMPERATURE MILK STERILIZING PLANT. III. LABORATORY DETERMINATIONS OF THE HEAT RESISTANCE OF SPORES OF BACILLUS SUBTILIS IN WATER AND IN MILK

SUMMARY: Thermal death curves for spores of Bacillus subtilis 786 have been determined in water and in milk. Generally a non-logarithmic order of death was observed. Numbers of survivors were lower in milk than in water, suggesting that there may be inhibitory factors in UHT sterilized milk which affect the germination and/or subsequent growth of heated spores.
The thermal death curves for spores suspended in milk yielded Q₁₀ values of about 30 in the range 110–120°. This is higher than the figures previously reported in the literature for R. subtilis spores. Spores of a number of strains of B. subtilis were compared with strain 786 and all gave high Q₁₀ values.
The results obtained in this work have been used to predict the destruction of spores at higher temperatures in a UHT plant (Burton et al. 1958). The calculated values agree well with the results obtained in the plant by Franklin et al. (1958).     

METHODS OF ASSESSING THE SPORICIDAL EFFICIENCY OF AN ULTRA-HIGH-TEMPERATURE MILK STERILIZING PLANT I. EXPERIMENTS WITH SUSPENSIONS OF SPORES IN WATER

SUMMARY: A method of assessing the sporicidal efficiency of a UHT milk sterilizing plant operating on water is described. Water heavily contaminated with spores of a strain of Bacillus subtilis was filtered, after treatment in the plant, through membrane filters and the surviving spores estimated by incubation of the membranes in nutrient agar. With this plant a temperature of c . 135° caused a 99·99999% kill of B. subtilis spores. Confirmation of the lethal effects of temperatures above 135° was obtained by passing treated water into 10 gal churns containing sterile concentrated nutrient broth and incubating the churns.     

AN IMPROVED METHOD OF CELL ENUMERATION FOR FILAMENTOUS ALGAE AND BACTERIA1

A new simple method for estimating the number of individual cells per milliliter in suspensions of filamentous microorganisms is described. This 2-part procedure utilizes a standard microscopic counting chamber and is independent of filament length or individual cell size. A statistical analysis of the method is also presented.     

APPLICABILITY OF RAPID PAPER KITS FOR TOTAL AND COLIFORMS/E. COLI COUNTS OF RAW MILK

The paper test kits developed for total bacterial count in 18–24 h (PKTs 3 formula) and for total coliforms/E. coli counts in 12–18 h (PKCs 10 formula) were evaluated against pure cultures often strains of bacteria: Escherichia coli, Enterobacter aerogenes, Citrobacter freundii, C. diversus, C. amlonaticus, Klebsiella pneumoniae, Salmonella Typhimurium, S. Typhi, S. Paratyphi A, Shigella flexneri, Bacillus cereus and Staphylococcus aureus. The cell suspensions in 0.85% sterile saline water were loaded onto paper kits and incubated at 30C for total counts and 37C for total coliform/E. coli counts. Simple regression analysis of the data showed a strong correlation between the paper kits and standard methods (P < 0.05) with r² values of 0.98 for PKTs and 0.97 for PKCs. Other noncoliforms such as S. Typhimurium, S. Typhi, S. Derby, S. Paratyphi A, S. flexneri, Gram positive B. cereus, and S. aureus, could not grow on PKCs. Both PKTs and PKCs were very simple and easy to use by either trained or untrained personnel in laboratories and in fieldwork. The cost per fieldwork sample is very low, therefore it shows great promise as an alternative method for the enumeration of total bacterial and coliforms/E. coli counts of raw milk.     

THE ENUMERATION OF AQUATIC BACTERIA USING DAPI

SUMMARY

A comparison was made between acridine orange (AO) and 4'6-diamidino-2-phenylindole (DAPI), a highly sensitive DNA fluorescing stain, for enumerating aquatic bacteria. When water samples were processed in a darkened room and a final DAPI concentration of 0,05 μg ml ^?1 was used, there was no significant difference between the population estimates obtained with AO and DAPI. With DAPI, bacteria are more easily distinguished from other particulate matter than when stained with AO. The cyanobacterium, Microcystis aeruginosa, normally fluoresced the blue colour characteristic of the DNA-DAPI complex. Frequently colonies fluoresced pink and were heavily populated by bacteria. DAPI may offer a rapid method to assess the proportion of senescing colonies in a population.     

MINIATURIZED TWO-FOLD DILUTION METHOD FOR ENUMERATING TOTAL MICROBIAL LOAD AND COLIFORMS IN RAW MILK

A rapid and simple method for enumerating total aerobic plate counts (APC) and coliforms in raw milk was developed and compared with conventional plating method. Following two-fold serial dilution of samples in a 96 well microtiter plate, double strength of two different modified media for APC or coliforms was added to each well. The final positive well (purple to yellow color) was determined and converted to dilution factors. The dilution factor of each sample was converted to Log₁₀ DF (Dilution factors) and compared to actual microbial numbers Log₁₀ CFU/mL. The results of 2-fold dilution method (Log₁₀ DF) were strongly correlated to conventional plating method (Log₁₀ CFU/mL) (P < 0.05). The correlation of the scatterplot of spread plating and 2-fold dilution method indicated a high level of agreement between two methods (R²= 0.921 for total counts and R²= 0.916 for forms from raw milk). This 2-fold dilution method is an easy, rapid, and economical method for enumeration of total microbial loads and coliforms in raw milk.     

RAPID DETECTION OF MICROBIAL CONTAMINATION IN TRICLOSAN AND HIGH FLUORIDE DENTIFRICES USING AN ATP BIOLUMINESCENCE ASSAY

Abstract The Celsis ATP Bioluminescence method was optimized and validated to detect the presence of microbial contamination in High Fluoride and Triclosan dentifrice formulations. Several enrichment broths were evaluated by using a 24–27 h incubation period. The ATP concentrations of the enrichment broths were found to a range from 0.012 to 0.040 nM. None of the tested enrichment broths were found to exhibit any sample inhibition/enhancement effects on the ATP Bioluminescence reaction. Dentifrice suspensions were inoculated with bacteria, yeast, and mold. All test microorganisms (ca. 1–15 CFU/g) were detected within a 24–27 h incubation period by using TAT Broth Base enrichment broths containing different concentrations of the following ingredients: Tween 20, Neopeptone, Dextrose, Triton X-100, Thiosulfate, Sodium Dibasic Phosphate, and Glycine. Negative ATP response after 24–27 h of incubation at 35C indicates the absence of contamination from these products.     

THE INCIDENCE AND THERMAL RESISTANCE OF MESOPHILIC SPORES FOUND IN MILK AND RELATED ENVIRONMENTS

SUMMARY: A spore 'spectrum' is described of aerobic mesophiles capable of resisting different heat treatments. It is shown that B. licheniformis is the most common spore former found in bulk milk but since its spores are rapidly destroyed at 100°, the more heat resistant B. subtilis is the dominant surviving spore former in commercial sterilized milk. The thermal resistance of strains of B. subtilis and B. licheniformis isolated from different sources has been investigated and the strains of B. subtilis typed according to the behaviour of their spores when heated at 100°. All strains of B. licheniformis were destroyed more rapidly by boiling for 2 min than strains of B. subtilis but only those strains of the latter which showed some degree of heat activation were more resistant than B. licheniformis . The 'resistant' and heat activated strains of B. subtilis appear to be sparsely distributed in nature and were only isolated from sterilized milk where the heat treatment applied would tend to eliminate other strains. The spore content of bovine faeces was similar to that in bulk milk and the total spore content varied seasonally, the spore content of faeces being on the average a hundred times greater during indoor feeding than during the period when the cattle were fed outside. A faecal infection of the milk in the ratio of 1:10⁴ would infect the milk with spores at about the same concentration as they are found in bulk raw milk, and it is suggested that bovine faeces could be a primary source of spore formers in milk supplies.