Usefulness of the stomacher in a microbiological regulatory laboratory.

The relative efficiency of the Waring blender, the Stomacher 400, and the Stomacher 3500 for preparing food samples for microbiological analysis was studied. Comparative aerobic plate count (APC) values were determined on 671 samples, representing 30 categories of foods. Of the 26 categories of nonfatty foods, the blender gave significantly higher geometric mean APC values than those given by the Stomacher 400 and the Stomacher 3500 in 65 and 69 percent of the categories, respectively. In a comparison of the two Stomacher models, the Stomacher 400 gave significantly higher geometric mean APC values than these given by the Stomacher 3500 in 73 percent of the food categories. Addition of Tween 80 to four categories of fatty foods at concentrations of 0.5, 1.0, and 2.0 percent did not raise the APC values given by either model of stomacher to the levels given by the Waring blender. Overall, the efficiency of both models of Stomacher, relative to the blender and to each other, was specific and depended upon the particular food being analyzed.     

Usefulness of the stomacher in a microbiological regulatory laboratory

The relative efficiency of the Waring blender, the Stomacher 400, and the Stomacher 3500 for preparing food samples for microbiological analysis was studied. Comparative aerobic plate count (APC) values were determined on 671 samples, representing 30 categories of foods. Of the 26 categories of nonfatty foods, the blender gave significantly higher geometric mean APC values than those given by the Stomacher 400 and the Stomacher 3500 in 65 and 69 percent of the categories, respectively. In a comparison of the two Stomacher models, the Stomacher 400 gave significantly higher geometric mean APC values than these given by the Stomacher 3500 in 73 percent of the food categories. Addition of Tween 80 to four categories of fatty foods at concentrations of 0.5, 1.0, and 2.0 percent did not raise the APC values given by either model of stomacher to the levels given by the Waring blender. Overall, the efficiency of both models of Stomacher, relative to the blender and to each other, was specific and depended upon the particular food being analyzed.     

ROLL TUBES AND AGAR STRIP TUBES FOR THE BACTERIAL COLONY COUNT OF MILK

SUMMARY: Roll-tube colony counts, using the Astell equipment, were lower than the corresponding Petri dish counts with 27 out of 31 raw milks (87%). The difference between the counts by the two methods was greater than 25% of the plate count for 12 (39%) of the samples.
When the same dilution of milk was used for both strip-tube and plate colony counts, about equal numbers of samples gave counts from the strip tubes above and below about the colony count from plates. When, in order to obtain a more reasonable strip-tube count, the plates and strip tubes were prepared from different dilutions of the milk, the counts from the latter were, with only 3 exceptions out of 35 milks, below those from the former. The difference between the counts was greater than 25% of the plate count for 15 (43%) of the milks, a figure similar to that obtained in comparing roll-tube and plate colony counts.     

A NOTE ON THE THERMODURIC BACTERIAL CONTENT OF FARM WATER SUPPLIES

SUMMARY: Significantly higher thermoduric colony counts were obtained when 5 ml. of water were laboratory pasteurized in 5 ml. of sterile milk or 2 ml. of water were pasteurized in 8 ml. of sterile milk, than when 10 ml. of water were directly pasteurized; 86% of the water samples had higher thermoduric counts after pasteurization in milk. Large differences were not common, only 19% of the ratios being over 5 and 5% over 10. Aerobic sporing rods were dominant in the thermoduric microflora irrespective of the method of pasteurization.     

Two stomacher accessories.

Two useful and easily constructed accessories for the Colworth Stomacher blender are described. The first automatically opens and holds Stomacher bags above a balance of weighing in the sample. The second stores bags on the bench between manipulations and opens and holds them as required for pipetting, etc.     

Two stomacher accessories.

Two useful and easily constructed accessories for the Colworth Stomacher blender are described. The first automatically opens and holds Stomacher bags above a balance of weighing in the sample. The second stores bags on the bench between manipulations and opens and holds them as required for pipetting, etc.     

Thermoduric and Psychrotrophic Organisms on Poorly Cleansed Milking Plants and Farm Bulk Milk Tanks

S ummary . Poorly cleansed pipeline milking plants gave much higher total and thermoduric colony counts than the internal surfaces of refrigerated farm bulk milk tanks, higher counts being recorded for milking plants after circulation cleaning than after cleansing by the acidified boiling water (ABW) method. Psychrotrophic counts were highest for farm milk tanks and lowest for milking plants cleansed by the ABW method. There was much variation in the proportion of thermoduric and psychrotrophic organisms in the total bacterial content of milking plants determined at 30°, thermodurics being much more prevalent in 21% of the rinses and psychrotrophs in 16%. Both groups formed about equal proportions in 9% of the pipeline rinses. The bacterial content of the farm bulk milk tanks was characterized by many psychrotrophs and few thermodurics.     

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk ( r = 0·94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0·1 M, pH 3·0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better ( r = 0·90) than that using standard 2 ml samples ( r = 0·88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.     

The Microflora of Hand Washed Milk Bottles

S ummary . The examination of a series of 713 milk bottles cleansed by hand washing at producer-retailer farm dairies showed that though nearly 60% attained satisfactory bacteriological standards, about 30% gave high colony counts (>600/bottle), while coli-aerogenes organisms were found in 17% and milk spoilage organisms in 25% of them. The microflora of efficiently cleansed bottles, with colony counts of <200/bottle, was dominated by micrococci and aerobic sporeforming rods. Only 3.7% of the 259 cultures from these bottles gave acid reactions in litmus milk in 72 h at 22°. Inefficiently cleansed bottles, with colony counts of >600/bottle, had quite a different type of microflora which was usually dominated by Gram negative rods (achromobacteria, nonfluorescent pseudomonads and flavobacteria). A much higher proportion (19%) of the 393 cultures from these bottles gave acid reactions in litmus milk.     

Use of dawn and dusk sight observations to determine colony size and family composition in Eurasian beaverCastor fiber

The methods used to determine family composition and colony size in Eurasian beaverCastor fiber Linnaeus, 1758 are often poorly described in published reports. Here we show how repeated counts of colony size in a random sample of colonies (n = 19) varied between dusk and dawn, between the months of August and September, and following successive counts. Mean counts at dusk and the following dawn did not vary significantly, though mean colony size was significantly greater in August than September. However, because all colony members are rarely seen during a single dusk or dawn count, successive counts often provided new information about the maximum number in each age class. This allowed us to adjust colony size following each count using the largest values thus far obtained, with the result that the mean adjusted colony size increased during six successive counts over seven weeks from 2.4 to 3.8. Family composition based on information from all six counts was 54% adults, 26% yearlings and 19% kits. Evidence suggests that kits in particular are undercounted by this method. Figures for colony size and composition in beaver should be viewed with caution if not obtained by methods tested for both precision and accuracy.     

THE ASSESSMENT OF THE BACTERIOLOGICAL CONDITION OF MILK BOTTLES

SUMMARY: A study of the relative values of a number of bacteriological tests for assessing the condition of milk bottles indicated that the colony count of the bottle rinse solution on yeastrel milk agar incubated for 4 days at 30°, combined with a clot-on-boiling test applied to 1 ml. of rinse in 9 ml. of sterile milk after incubation for 72 hr. at 19–20°, gave the most useful results.
The mean of the ratios of colony counts at 30° to those at 37° was 15·1, while it was as high as 22·9 for rinses with 37° of over 600 for an unsatisfactory bottle should be retained when the test is done at 30°. The thermoduric colony count of rinses of milk bottles, even when laboratory pasteurized in milk, did not provide any additional information to that given by the colony count at 30° made without pasteurization. A high proportion of the organisms in bottle rinses survived laboratory pasteurization in milk, the survival rate being highest in efficiently treated bottles.
The clot-on-boiling test gave results in general agreement with colony counts and served to indicate the potential influence of badly contaminated bottles on the keeping quality of milk placed in them. A substantial proportion of rinses with satisfactory colony counts reduced methylene blue within 48 hr. at 19–20°.
Colony counts at 37° were on the average much lower for bottles treated with steam than for bottles submitted to detergent treatment in various types of bottle washing machines. Treatment of bottles by steam or hypochlorite was more efficiently done on the farms than at the dairies.     

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk (r = 0.94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0.1 M, pH 3.0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better (r = 0.90) than that using standard 2 ml samples (r = 0.88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.     

ADVISORY MICROBIOLOGICAL STANDARDS AND TOLERANCES FOR RAW MILK

SUMMARY: To study the value of recent modifications of microbiological tests used for advisory purposes, samples of tuberculin tested milk, taken at weekly intervals over a 5 year period from the Trawscoed Experimental Husbandry Farm and selected at random during a 12 months period from farms in Wales, were examined by a temperature compensated keeping quality test at 22°, colony count on Yeastrel milk agar in 3 days at 30° and coli-aerogenes colony count on violet red bile agar in 20–24 hr at 30°.
The results show that milk produced and handled under hygienic conditions can be expected to have colony counts of less than 2 × 10⁴/ml and coli-aerogenes colony counts less than 10/ml when examined within 18 hr of milking.     

Morphological and phenotypical characterization of Bacillus sporothermodurans

AIMS: Enumeration of resistant bacteria in ultra-high temperature (UHT) treated milk; morphological characterization and phenotyping of resistant strains by traditional and nontraditional methods and their identification by molecular biology. METHODS AND RESULTS: Modified standard plate count agar (PCA) and modified brain-heart infusion (BHI) agar were used for colony counts. Physiological culture traits were determined as suggested by Bergey's Manual of Systematic Bacteriology or in modified J-broth or in modified BHI agar. Scanning electron microscope (SEM) was used for microscopic examination. Strain identification was carried out by polymerase chain reaction (PCR). A total of 125 (62.81% of 199) samples were positive and the bacterial load was higher than 10(5) CFU ml(-1) in 46 samples (28.80% of 125). The 16S rRNA sequence of bacterial cultures obtained from UHT-treated milk was similar to that of Bacillus sporothermodurans M215 type strain((T)) and different biotypes were found by analysis of colony appearance, cell morphology and physiological traits. CONCLUSIONS: Bacillus sporothermodurans was the predominant sporigenous micro-organisms in UHT milk. SIGNIFICANCE AND IMPACT OF THE STUDY: BHI agar is more suitable than PCA for quality control of milk after UHT treatment. Modified J-broth medium is useful to determine selected physiological traits of B. sporothermodurans. The strains characterized and identified as B. sporothermodurans were significantly different compared with the type strain.     

Real-time PCR detection of Paenibacillus spp. in raw milk to predict shelf life performance of pasteurized fluid milk products

Psychrotolerant sporeformers, specifically Paenibacillus spp., are important spoilage bacteria for pasteurized, refrigerated foods such as fluid milk. While Paenibacillus spp. have been isolated from farm environments, raw milk, processing plant environments, and pasteurized fluid milk, no information on the number of Paenibacillus spp. that need to be present in raw milk to cause pasteurized milk spoilage was available. A real-time PCR assay targeting the 16S rRNA gene was designed to detect Paenibacillus spp. in fluid milk and to discriminate between Paenibacillus and other closely related spore-forming bacteria. Specificity was confirmed using 16 Paenibacillus and 17 Bacillus isolates. All 16 Paenibacillus isolates were detected with a mean cycle threshold (C(T)) of 19.14 ± 0.54. While 14/17 Bacillus isolates showed no signal (C(T) > 40), 3 Bacillus isolates showed very weak positive signals (C(T) = 38.66 ± 0.65). The assay provided a detection limit of approximately 3.25 × 10(1) CFU/ml using total genomic DNA extracted from raw milk samples inoculated with Paenibacillus. Application of the TaqMan PCR to colony lysates obtained from heat-treated and enriched raw milk provided fast and accurate detection of Paenibacillus. Heat-treated milk samples where Paenibacillus (≥1 CFU/ml) was detected by this colony TaqMan PCR showed high bacterial counts (>4.30 log CFU/ml) after refrigerated storage (6°C) for 21 days. We thus developed a tool for rapid detection of Paenibacillus that has the potential to identify raw milk with microbial spoilage potential as a pasteurized product.     

Methods for improved recovery of Listeria monocytogenes from cheese

Method of homogenization (Waring blender versus stomacher), type of diluent (tryptose broth [TB] versus aqueous 2% trisodium citrate), and temperature of diluent (20 versus 40 degrees C) were compared for recovery of Listeria monocytogenes from freshly made and ripened Colby cheese. By using direct plating on McBride listeria agar, significantly higher numbers of L. monocytogenes were recovered when cheese samples were (i) homogenized for 2 min with the blender rather than the stomacher (P less than 0.01), (ii) diluted in trisodium citrate rather than TB (P less than 0.01), and (iii) diluted in diluents at 40 rather than 20 degrees C (P less than 0.05). Based on these results, a new diluent/enrichment medium was developed by adding 2% trisodium citrate to TB (TBC). Despite superior results with the blender, biosafety concerns led to use of the stomacher for homogenization of cheese samples; hence, the stomaching time was increased to 3 min. Results obtained by direct plating indicated that recovery of L. monocytogenes from Colby cheese and from curd samples taken during manufacture of brick cheese increased when samples were diluted 1:10 in TBC at 45 degrees C and stomached for 3 min, as compared with similarly treated samples diluted in TB at 25 degrees C. A similar comparison of both diluents for recovery of L. monocytogenes from cold-pack cheese food yielded bacterial counts which were not significantly different. Recovery of L. monocytogenes from cold-enriched (at 4 degrees C for up to 8 weeks) samples of Colby cheese and cold-pack cheese food was generally similar for samples homogenized in TBC or TB.     

Methods for improved recovery of Listeria monocytogenes from cheese.

Method of homogenization (Waring blender versus stomacher), type of diluent (tryptose broth [TB] versus aqueous 2% trisodium citrate), and temperature of diluent (20 versus 40 degrees C) were compared for recovery of Listeria monocytogenes from freshly made and ripened Colby cheese. By using direct plating on McBride listeria agar, significantly higher numbers of L. monocytogenes were recovered when cheese samples were (i) homogenized for 2 min with the blender rather than the stomacher (P less than 0.01), (ii) diluted in trisodium citrate rather than TB (P less than 0.01), and (iii) diluted in diluents at 40 rather than 20 degrees C (P less than 0.05). Based on these results, a new diluent/enrichment medium was developed by adding 2% trisodium citrate to TB (TBC). Despite superior results with the blender, biosafety concerns led to use of the stomacher for homogenization of cheese samples; hence, the stomaching time was increased to 3 min. Results obtained by direct plating indicated that recovery of L. monocytogenes from Colby cheese and from curd samples taken during manufacture of brick cheese increased when samples were diluted 1:10 in TBC at 45 degrees C and stomached for 3 min, as compared with similarly treated samples diluted in TB at 25 degrees C. A similar comparison of both diluents for recovery of L. monocytogenes from cold-pack cheese food yielded bacterial counts which were not significantly different. Recovery of L. monocytogenes from cold-enriched (at 4 degrees C for up to 8 weeks) samples of Colby cheese and cold-pack cheese food was generally similar for samples homogenized in TBC or TB.     

Legionnaires' disease outbreaks and cooling towers with amplifiedLegionella concentrations

This study was designed to investigate the possible association of high colony counts of legionellae from cooling towers and evaporative condensers with Legionnaires' disease outbreaks. We obtained legionellae counts from samples of cooling towers and evaporative condensers that were the likely sources of two different Legionnaires' disease outbreaks and compared these counts with those from cooling towers that were not associated with reports of human disease. Among 675 potential control cooling tower that were samples from 258 facilities, 136 facilities had one or more cooling towers that met our criteria for inclusion into the study. Samples taken from buildings where an outbreak had occurred had much higherLegionella counts than did samples from other buildings. Colony counts from the two outbreak-associated facilities were significantly higher than colony counts from other facilities [Wilcoxon Rank Sum Test (Exact), p<0.01]. The results of the study suggest that, among cooling towers that test positive for the presence of legionellae, higher colony counts are associated with higher risk of Legionnaires' disease.     

The Spleen Colony Technique. Iii. Comparison of the Overlap Effect and of Errors In Cfu-S Determination and the [3H]-Thymidine Suicide Data For Several Strains of Mice

A mathematical model of errors of the spleen colony technique is applied to data obtained from four mouse strains and F₁ hybrids. the variance of the colony counts was close to the Poisson distribution in inbred mice and F¹ hybrids. However, it should be checked regularly. the magnitude of the error in CFU-s determination and of the estimations of the S phase fraction was derived, and is presented relative to the mean colony counts for all mouse strains studied. the optimum spleen colony counts are generally higher than those which are commonly used. However, the utilization of the optimum spleen colony counts requires a correction for the effect of colony overlap.     

Long-term culture of normal human colonic epithelial cells in vitro.

Studies of normal cellular function as well as the understanding of cellular mechanisms of carcinogenesis and other diseases of the large intestine have been limited, particularly due to the lack of long-term culture of normal human large intestinal epithelial cells (NHLIEC). Using the epithelia from surgically resected human colon, we have dissociated a sufficient number of viable NHLIEC and maintained them in in vitro culture for up to 5 months. Normal-appearing human large intestinal mucosal fragments (1 mm2) were treated with 0.01 mg/ml trypsin, 0.2 mg/ml collagenase + 0.1 mM EGTA or 0.1 mg/ml trypsin + 0.1 mM EGTA in a Stomacher laboratory blender to isolate the cells. Compared with other methods, the use of the Stomacher blender combined with low concentrations of proteolytic enzymes yielded greater numbers of cells per gram of tissue, with up to 84% viable cells. Primary and serially passaged NHLIEC were cultured in CMRL-1066, MEM with 5% serum, and serum-free KGM. These media were all supplemented with insulin, hydrocortisone, epithelial growth factor, and bovine pituitary extract. CMRL-1066 was found to be the best medium for NHLIEC. Contaminating fibroblasts were selectively removed by briefly allowing the cells to adhere to the culture vessel and adding 25 U/ml collagenase to the culture media at the first subculture treatment. The epithelial nature and secretory function of the established cells were confirmed by morphological criteria (light microscopy, phase contrast microscopy and electron microscopy), immunoreactivity to cytokeratin, and positive mucin cytochemistry. We propose that using this methodology for the culture and maintenance of NHLIEC for an extended period of time would serve as a valuable model for a variety of investigations.