3M™ Petrifilm™ Rapid Yeast and Mold Count Plate Method for the Enumeration of Yeast and Mold on Selected Surfaces: AOAC Official Method 2014.05.

BACKGROUND: The 3M™ Petrifilm™ Rapid Yeast and Mold Count (RYM) Plate contains nutrients supplemented with antibiotics, a cold-water-soluble gelling agent, and an indicator system that facilitates yeast and mold enumeration in 48-60 h. OBJECTIVE: The objective of this study is to evaluate the 3M Petrifilm RYM Plate in a matrix extension study for the enumeration of yeast and mold on stainless steel, sealed concrete, and rubber surfaces. METHODS: The 3M Petrifilm RYM Plate was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual, Ch. 18: Yeasts, Molds and Mycotoxins in a paired matrix study for enumeration of yeast and mold on stainless steel, sealed concrete, and rubber environmental surfaces. RESULTS: The 3M Petrifilm RYM Plate demonstrated equivalent performance to the reference method for enumeration of yeast and mold from stainless steel, sealed concrete, and rubber environmental surfaces. There were no significant statistical differences between the 3M Petrifilm RYM Plate and reference method results for the three matrixes evaluated. CONCLUSION: The 3M Petrifilm RYM Plate is an effective plating method for enumerating yeast and mold when analyzing stainless steel, sealed concrete, and rubber surfaces. HIGHLIGHTS: The 3M Petrifilm RYM Plate method allows the user to obtain accurate results within 48-60 h in the matrices evaluated for the presence of yeast and mold when incubated at 25 ± 1°C or 28 ± 1°C. Interpretation and colony counting was straightforward and the 3M Petrifilm RYM Plate method required no additional agar or Petri dishes, creating an easier workflow by cutting down on supplies, sample plating time, and most noticeably, occupying less space in the incubator.

3M™ Petrifilm™ Rapid Aerobic Count (RAC) Plate Method for the Enumeration of Aerobic Bacteria on Selected Surfaces: AOAC Official MethodSM 2015.13.

BACKGROUND: The 3M™ Petrifilm™ Rapid Aerobic Count (RAC) Plate is a sample-ready culture medium system designed to facilitate colony enumeration in 24 h (48 h for dry powders) for selected food and environmental surfaces. OBJECTIVE: The objective of this study is to evaluate the 3M Petrifilm RAC Plate in a matrix extension study for the enumeration of aerobic bacteria on stainless steel, sealed concrete, and rubber surfaces. METHOD: The 3M Petrifilm RAC Plate was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual, Ch. 3 (January 2001): Aerobic Plate Count in a paired matrix study for enumeration of aerobic bacteria on stainless steel, sealed concrete, and rubber environmental surfaces. RESULTS: The 3M Petrifilm RAC Plate showed no statistically significant differences when compared to the reference method for enumeration of aerobic bacteria from stainless steel, sealed concrete, and rubber environmental surfaces. There were no significant statistical differences between the 3M Petrifilm RAC Plate and reference method results for the three matrixes evaluated. CONCLUSIONS: The 3M Petrifilm RAC Plate is an effective plating method for aerobic plate count when analyzing stainless steel, sealed concrete, and rubber surfaces. HIGHLIGHTS: The 3M Petrifilm RAC Plate is robust, quick, and simple to perform, providing enumerable colonies in 22 to 26 h after incubation when compared to the 48 h of the reference method. The small size of the Petrifilm Plate allows for less space to be occupied by plates in the incubators. The visual biochemical and enzymatic indicators make enumeration of colonies a simple task by presenting colonies in either a blue or red color.

Validation of the 3M™ Petrifilm™ Coliform Count Plate for Enumeration of Coliforms in Bottled Water: AOAC Performance Tested MethodSM 082101.

BACKGROUND: The 3M™ Petrifilm™ Coliform Count (CC) Plate is a sample-ready-culture medium system which contains modified Violet Red Bile nutrients, a cold-water-soluble gelling agent, and a tetrazolium indicator that facilitates colony enumeration. OBJECTIVE: To validate the 3M Petrifilm CC Plate method for bottled water through the AOAC®  Performance Tested Method(s)SM program. METHODS: The performance of the 3M Petrifilm CC Plate was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 4, Section III.D for the enumeration of coliforms in bottled water. Matrix data were normalized by log10 transformation and performance indicators included repeatability and difference of means with 90 and 95% confidence intervals. Inclusivity, exclusivity, robustness, and product consistency and stability were evaluated. RESULTS: This study demonstrated that the 3M Petrifilm CC Plate method detects and enumerates coliforms from bottled water. The average log10 counts of the 3M Petrifilm CC Plate method were equivalent to or better than the average log10 counts of the reference method. Results from inclusivity and exclusivity studies demonstrated that the 3M Petrifilm CC Plate method differentiated coliforms from non-coliform strains. In product consistency, stability, and robustness testing, different lots of 3M Petrifilm CC Plates and small deviations in incubation time and temperature did not affect test results. CONCLUSION: The 3M Petrifilm CC Plate method is an accurate and robust method for the enumeration of coliforms in bottled water. HIGHLIGHTS: The 3M Petrifilm CC Plate allows for detection of confirmed coliforms within 24 h. Up to 20 sample-ready plates can be stacked during incubation, providing flexibility and saving space.

Implementation of ATP and Microbial Indicator Testing for Hygiene Monitoring in a Tofu Production Facility Improves Product Quality and Hygienic Conditions of Food Contact Surfaces: A Case Study.

Rapid ATP testing and microbiological enumeration are two common methods to monitor the effectiveness of cleaning and sanitation in the food industry. In this study, ATP testing and microbiological enumeration were implemented at a tofu production facility with the goal of improving cleaning practices and overall plant hygiene. Results from ATP monitoring were used to target areas of the production environment needing additional cleaning; ATP results were verified by microbiological enumeration of aerobic microorganisms, lactic acid bacteria, and yeasts and molds. Products from the production line were enumerated for the same microorganisms to determine if there was an impact on product quality. After the implementation of ATP monitoring and targeted cleaning, there was a statistically lower proportion of swabs that failed to meet established sanitary requirements for ATP, aerobic microorganisms, and lactic acid bacteria (p < 0.05), but not for yeasts and molds. ATP swabs and microbiological enumeration agreed on site hygiene 75.1% (72.3-77.7%, 95% CI) of the time. Product data indicated that unpasteurized finished products contained a statistically lower microbial load of the three groups of organisms following implementation of the practices (p < 0.05).ImportanceCleaning and sanitation are critical to maintaining safe and high-quality food production. Monitoring these activities is important to ensure proper execution of procedure and to assure compliance with regulatory guidelines. The results from monitoring activities can direct targeted cleaning of areas with higher risk of contamination from foodstuffs and microorganisms. The results of this study show that ATP monitoring and microbiological enumeration are useful tools to verify and improve the efficacy of cleaning and sanitation practices, which can have a positive impact on both plant hygiene and product quality. However, testing regimes and critical parameters will vary based on the product and facility.

NlpC/P60 domain-containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan.

A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis (MAP). The MAP genome sequence encodes five NlpC/P60 domain-containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine-155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain-containing proteins than simply hydrolases.

MAP1272c encodes an NlpC/P60 protein, an antigen detected in cattle with Johne's disease.

The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that contains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne's disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were expressed and evaluated with sera from cattle with Johne's disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a distinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis.