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 use of the Catalasemetre in assessing the quality of milk

The Catalasemetre, for assessing the quality of raw and pasteurized milk, has been studied. No correlation was found between catalase activity and bacterial counts for farm bulk tank milks within the range 5.2 ± 10²-5.4 ± 10⁵ cfu/ml. Similarly, no relation was observed between catalase activity and somatic cell counts of milk (range of counts from 0.08 to 3.5). However, the catalase activity and bacterial count of pasteurized milks which had been pre-incubated at 21.C for 25 h in the presence of crystal violet-penicillin-nisin to inhibit Gram-positive bacterial growth were significantly related. Thus, the use of this pre-incubation procedure coupled with the Catalasemetre to estimate bacterial growth, has potential in assessing the keeping quality of pasteurized milk samples within 25.5 h of production. Results on the thermostability of native milk catalase are also presented.     

ATP bioluminescence rapid detection of total viable count in soy sauce

The adenosine triphosphate (ATP) bioluminescence rapid determination method may be useful for enumerating the total viable count (TVC) in soy sauce, as it has been previously used in food and beverages for sanitation with good precision. However, many factors interfere with the correlation between total aerobic plate counts and ATP bioluminescence. This study investigated these interfering factors, including ingredients of soy sauce and bacteria at different physiological stages. Using the ATP bioluminescence method, TVC was obtained within 4 h, compared to 48 h required for the conventional aerobic plate count (APC) method. Our results also indicated a high correlation coefficient (r = 0.90) between total aerobic plate counts and ATP bioluminescence after filtration and resuscitation with special medium. The limit of quantification of the novel detection method is 100 CFU/mL; there is a good linear correlation between the bioluminescence intensity and TVC in soy sauce in the range 1 × 10²–3 × 10⁴ CFU/mL and even wider. The method employed a luminescence recorder (Tristar LB‐941) and 96‐well plates and could analyse 50–100 samples simultaneously at low cost. In this study, we evaluated and eliminated the interfering factors and made the ATP bioluminescence rapid method available for enumerating TVC in soy sauce. Copyright © 2011 John Wiley & Sons, Ltd.     

Observations on the effect of raw milk quality on the keeping quality of pasteurized milk

Raw milk was stored for up to 14 d at 4°C and pasteurized on days 1, 3, 4, 7, 9 and 14. Precautions were taken to eliminate post-pasteurization contamination. The pasteurized milks were stored at 4°C and analysed at weekly intervals for standard plate counts (SPC), psychrotrophic counts (PC) and aerobic spore counts (ASC). The initial raw milk quality was very good and the keeping quality of all the pasteurized milks tested was greater than 22 d. In some cases the milk still had acceptable SPC after 42 d storage, which shows the keeping quality that can be achieved when the process is well controlled. However, the best keeping quality resulted from milk pasteurized on the third and fourth days. Even milk pasteurized on the seventh and ninth had superior keeping quality to that pasteurized on the first day. The lactoperoxidase anti-microbial system in raw milk may be most active around days 3 and 4.     

EVALUATION OF ADENOSINE TRIPHOSPHATE (ATP) BIOLUMINESCENCE FOR ESTIMATING BACTERIA ON SURFACES OF BEEF CARCASSES

A rapid (<15 min), inexpensive and simple method has been developed to estimate the concentration of bacteria on surfaces of beef carcasses using adenosine triphosphate (ATP) bioluminescence. Surfaces (5x5 cm²) of beef carcasses (n= 159) were collected by excision. An ATP assay and aerobic plate count were performed on each sample. A significant (p < 0.001) positive linear relationship (r = 0.83) between plate count and ATP assay was obtained for 159 beef carcass samples. When thresholds levels were set at 1 × 10⁴, 1 × 10⁵ and 1 × 10⁶ CFU/cm², there was moderate to good agreement between the ATP bioluminescence assay and the aerobic plate count as determined by the k-statistic. The application of this ATP bioluminescence test to HACCP systems for beef slaughter processes is discussed.     

Rapid Membrane Filtration-Epifluorescent Microscopy Technique for Direct Enumeration of Bacteria in Raw Milk

Membrane filtration and epifluorescent microscopy were used for the direct enumeration of bacteria in raw milk. Somatic cells were lysed by treatment with trypsin and Triton X-100 so that 2 ml of milk containing up to 5 × 10⁶ somatic cells/ml could be filtered. The majority of the bacteria (ca. 80%) remained intact and were concentrated on the membrane. After being stained with acridine organe, the bacteria fluoresced under ultraviolet light and could easily be counted. The clump count of orange fluorescing cells on the membrane correlated well (r = 0.91) with the corresponding plate count for farm, tanker, and silo milks. Differences between counts obtained by different operators and between the membrane clump count and plate count were not significant. The technique is rapid, taking less than 25 min, inexpensive, costing less than 50 cents per sample, and is suitable for milks containing 5 × 10³ to 5 × 10⁸ bacteria per ml.     

A NOTE ON THE USE OF DEHYDRATED MILK AGAR FOR BACTERIAL COUNTS ON RAW AND DRIED MILKS

SUMMARY: The bacterial colony count of raw milk and of milk powder using milk agar prepared from a proprietary brand of the dehydrated medium has been compared with that obtained using the fresh medium prepared in the laboratory.
No significant difference was found in 17 milks which were examined using the roll-tube technique and 8 replicates/medium but in a second series of 32 milks the plate count (3 replicates/medium) was significantly lower on the reconstituted than on the fresh medium.
Some milk powders gave a much lower plate count on the reconstituted medium, due to the inability of the latter to support adequate growth of Streptococcus thermophilus.     

Use of the Direct Epifluorescent Filter Technique for predicting the keeping quality of pasteurized milk within 24 hours

The keeping quality (KQ) of pasteurized milk stored at 5°C and 11°C was predicted within 24 h by pre-incubating samples and counting bacteria by the Direct Epifluorescent Filter Technique (DEFT). For samples from 5°C storage, 0.03% (w/v) benzalkonium chloride and 0.002% (w/v) crystal violet (final concentration) were added to inhibit the growth of Gram positive bacteria during pre-incubation. The samples from milk stored at 11°C were pre-incubated without the addition of inhibitors. After pre-incubation there was a satisfactory relationship between the DEFT count and the KQ of milks at both 5°C and 11°C. The DEFT count following pre-incubation correctly classified > 80% of pasteurized milks on the basis of KQ.     

Use of the Direct Epifluorescent Filter Technique for predicting the keeping quality of pasteurized milk within 24 hours

The keeping quality (KQ) of pasteurized milk stored at 5 degrees C and 11 degrees C was predicted within 24 h by pre-incubating samples and counting bacteria by the Direct Epifluorescent Filter Technique (DEFT). For samples from 5 degrees C storage, 0.03% (w/v) benzalkonium chloride and 0.002% (w/v) crystal violet (final concentration) were added to inhibit the growth of Gram positive bacteria during pre-incubation. The samples from milk stored at 11 degrees C were pre-incubated without the addition of inhibitors. After pre-incubation there was a satisfactory relationship between the DEFT count and the KQ of milks at both 5 degrees C and 11 degrees C. The DEFT count following pre-incubation correctly classified greater than 80% of pasteurized milks on the basis of KQ.     

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.     

Production of Enterotoxin A in Milk

Enterotoxin A production in milk was studied by use of variables of milk quality, initial numbers of enterotoxigenic staphylococci, incubation temperature, and time. In both raw and pasteurized milks having a low total viable count, enterotoxin was detected in minimal incubation times of 6 to 9 hr at 35 C, 9 to 12 hr at 30 C, 18 hr at 25 C, and 36 hr at 20 C, after inoculation with 10(6)Staphylococcus aureus cells per ml. When similar milks were inoculated with 10(4)S. aureus cells per ml, enterotoxin was detected in 12 hr at 35 C, 18 hr at 30 C, 24 to 36 hr at 25 C, and 48 to 96 hr at 20 C. In high-count raw milk, enterotoxin was detected only in samples inoculated with 10(6)S. aureus cells per ml and incubated at 35 C. Generally, a concentration of 5 x 10(7)S. aureus cells per ml of milk was reached before enterotoxin A was detected.     

Rapid Enumeration of Bacteria in Heat-treated Milk and Milk Products Using a Membrane Filtration-Epifluorescent Microscopy Technique

A rapid direct epifluorescent filter technique (DEFT), taking ca. 20 min to complete, was used to enumerate bacteria in heat-treated milk and milk-products. The DEFT count could detect as few as 5750 bacteria/ml in pasteurized and skim milk, pasteurized cream, whey and sweet cream butter and was in agreement with the standard plate count. The technique was also used to determine the sterility of cartoned ultra heat-treated milk after incubation at 37°C. The agreement between the DEFT and plate count was poor for evaporated and condensed milks, some reconstituted skim milk powders, pasteurized whey and ripened cream butter. Possible reasons for this are discussed.     

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.     

Bioluminescent assay of total bacterial contamination of raw milk]

A rapid (30-35 min) bioluminescence assay of total bacterial contamination (TBC) of raw milk was optimized. This method includes incubation of milk samples in the presence of Neonol-10 and medical purity grade pancreatin with further removal of nonbacterial ATP by filtration through a membrane filter, cell disruption by treatment with dimethyl sulfoxide, and measurement of ATP concentration in a reaction with the bioluminescent reagent Immolum. The TBC detection threshold is 0.5 x 10(5) colony-forming units (CFU) per ml milk. Coefficients of correlation between the standard plate count method and bioluminescence assay (R) and residual standard deviations (Sxy) in raw milk samples (n = 140) were 0.83 and 0.54, respectively. In sterilized milk samples artificially contaminated with pure cultures of the main representatives of milk microflora (coli-forms, Staphylococcus aureus, Streptococcus thermophilus, and Streptococcus group D), these values were 0.89-0.99 and 0.09-0.29, respectively. The specific content of ATP was found to be (0.8 +/- 0.1) x 10(-18) mol/CFU in coli-forms; (12.0 +/- 8.1) x 10(-18) mol/CFU in S. aureus; (35.2 +/- 16.9) x 10(-18) mol/CFU in S. thermophilus; and (42.5 +/- 1.3) x 10(-18) in Streptococcus group D.     

Prediction of the shelf-life of pasteurized milk at different storage temperatures

Initial psychrotroph counts determined by a Most Probable Number technique were correlated with shelf-lives of pasteurized milk determined at a number of storage temperatures. The initial psychrotroph count was also correlated with a bacterial count carried out on milk agar containing crystal violet penicillin and nisin after previous incubation of the milk at 15°C for 25 h.
Pre-incubation counts carried out at a variety of temperatures and on a variety of media were examined for their relation to shelf-life. Shelf-lives at four pre-set temperatures (2, 6, 10 and 14°C) could best be predicted by pre-incubation of pasteurized milk at 15°C before inoculation on milk agar.
An equation which allows prediction of shelf-life of pasteurized milk at any storage temperature is described.     

Effect of heat treatment on Listeria monocytogenes and Gram-negative bacteria in sheep, cow and goat milks

Sheep milk, compared with cow and goat milk, had a protective effect on Gram-negative bacteria and Listeria spp. heated at 65°C in a test-tube method. This effect was not solely due to fat content as cow milk artificially reconstituted to 10% homologous fat was not as protective. Listeria monocytogenes in whole sheep, cow and goat milks at an inoculum level of 1 times 10⁶ cfu ml^-1 was heated at 68°C for 15 s in the plate pasteurizer and survival was only detected in whole sheep milk after heating. Whole sheep, cow and goat milks containing high levels of L. monocytogenes (1 times 10⁶ cfu ml^-1) could not survive the current HTST plate pasteurization protocol.     

Characterization of bioluminescent derivatives of assimilable organic carbon test bacteria

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.     

Heterotrophic plate counts of surface water samples by using impedance methods.

Membrane filtration, spread plating, and pour plating are conventional methods used to determine the heterotrophic plate counts of water samples. Impedance methods were investigated as an alternative to conventional methods, since sample dilution is not required and the bacterial count can be estimated within 24 h. Comparisons of impedance signals obtained with different water samples revealed that capacitance produced faster detection times than conductance. Moreover, the correlation between heterotrophic plate count and detection time was highest (r = 0.966) when capacitance was used. Linear and quadratic regressions of heterotrophic plate count and impedance detection time were affected by incubation temperatures. Regressions between heterotrophic plate counts based on conventional methods and detection times of water samples incubated at less than or equal to 25 degrees C had R2 values of 0.878 to 0.933. However, regressions using detection times of water samples incubated at greater than or equal to 30 degrees C had lower R2 values, even though water samples produced faster detection times. Comparisons between broth-based versions of R2A medium and plate count agar revealed that the latter correlated highly with heterotrophic plate count, provided that water samples were incubated at 25 degrees C and impedance measurements were conducted with the capacitance signal (r = 0.966). When the linear regression of this relationship was tested with 100 water samples, the correlation between predicted and actual log10 CFU milliliter-1 was 0.869. These results indicate that impedance methods provide a suitable alternative to conventional methods.     

Heterotrophic plate counts of surface water samples by using impedance methods.

Membrane filtration, spread plating, and pour plating are conventional methods used to determine the heterotrophic plate counts of water samples. Impedance methods were investigated as an alternative to conventional methods, since sample dilution is not required and the bacterial count can be estimated within 24 h. Comparisons of impedance signals obtained with different water samples revealed that capacitance produced faster detection times than conductance. Moreover, the correlation between heterotrophic plate count and detection time was highest (r = 0.966) when capacitance was used. Linear and quadratic regressions of heterotrophic plate count and impedance detection time were affected by incubation temperatures. Regressions between heterotrophic plate counts based on conventional methods and detection times of water samples incubated at less than or equal to 25 degrees C had R2 values of 0.878 to 0.933. However, regressions using detection times of water samples incubated at greater than or equal to 30 degrees C had lower R2 values, even though water samples produced faster detection times. Comparisons between broth-based versions of R2A medium and plate count agar revealed that the latter correlated highly with heterotrophic plate count, provided that water samples were incubated at 25 degrees C and impedance measurements were conducted with the capacitance signal (r = 0.966). When the linear regression of this relationship was tested with 100 water samples, the correlation between predicted and actual log10 CFU milliliter-1 was 0.869. These results indicate that impedance methods provide a suitable alternative to conventional methods.     

COMPARISON OF MICROBIOLOGICAL METHODS FOR MONITORING CHICKEN CARCASS QUALITY

Many methods have been developed to determine microbial levels in food products and these include ATP bioluminescence, hydrophobic grid membrane filtration (HGMF), impediometry and turbidimetry. A comparison of these techniques for detecting microbial levels in chicken carcass rinses was conducted.
Only the ATP bioluminescence assay and the HGMF system showed a good correlation with plate counts (r = 0.82 and r = 0.83, respectively). The repeatability of these methods was acceptable. There was also a significant correlation between results obtained with two turbidimetric methods and HGMF as well as between HGMF and ATP bioluminescence data. However, only the ATP bioluminescence assay was able to provide results of microbial levels on a realtime basis (within 10 min). This would be beneficial for HACCP (Hazard Analysis of Critical Control Points) based programs.