Validation of a Novel Rinse and Filtration Method for Efficient Processing of Fresh Produce Samples for Microbiological Indicator Enumeration

Abstract

Several methods have been described to prepare fresh produce samples for microbiological analysis, each with its own advantages and disadvantages. The aim of this study was to compare the performance of a novel combined rinse and membrane filtration method to two alternative sample preparation methods for the quantification of indicator microorganisms from fresh produce. Decontaminated cantaloupe melons and jalapeño peppers were surface inoculated with a cocktail containing 10(6) CFU/ml Escherichia coli, Salmonella Typhimurium, and Enterococcus faecalis. Samples were processed using a rinse and filtration method, homogenization by stomacher, or a sponge-rubbing method, followed by quantification of bacterial load using culture methods. Recovery efficiencies of the three methods were compared. On inoculated cantaloupes, the rinse and filtration method had higher recovery of coliforms (0.95 log CFU/ml higher recovery, P = 0.0193) than the sponge-rubbing method. Similarly, on inoculated jalapeños, the rinse and filtration method had higher recovery for coliforms (0.84 log CFU/ml higher, P = 0.0130) and E. coli (1.46 log CFU/ml higher, P < 0.0001) than the sponge-rubbing method. For jalapeños, the rinse and filtration method outperformed the homogenization method for all three indicators (0.79 to 1.71 log CFU/ml higher, P values ranging from 0.0075 to 0.0002). The precision of the three methods was also compared. The precision of the rinse and filtration method was similar to that of the other methods for recovery of two of three indicators from cantaloupe (E. coli P = 0.7685, E. faecalis P = 0.1545) and was more precise for recovery of two of three indicators from jalapeño (coliforms P = 0.0026, E. coli P = 0.0243). Overall, the rinse and filtration method performed equivalent to, and sometimes better than, either of the compared methods. The rinse and filtration method may have logistical advantages when processing large numbers of samples, improving sampling efficiency and facilitating microbial detection.

Efficacy of two hygiene methods to reduce soil and microbial contamination on farmworker hands during harvest

“…When filtering volumes less than 1 ml, the funnel (with the vacuum closed) was pre-filled with 20 ml of sterile 0.1% peptone water before the sample was added to allow even sample dispersion across the membrane prior to opening the vacuum (Heredia et al, 2015). Following filtration through duplicate membranes for each serial volume of rinsate, each membrane was placed on a separate Petri dish containing solidified agar for bacterial enumeration.…”

“…Detection and quantification of the universal and human-specific 16S rDNA markers for Bacteroidales were performed using the AllBac and BFD primers and probes as described (Ravaliya et al, 2014). The remaining sample was processed as follows to detect and enumerate coliforms, generic E. coli, and Enterococcus (three common, non-pathogenic bacteria) hereafter called "indicator bacteria" as described (Heredia et al, 2015).…”

Detection of Ralstonia pseudosolanacearum in drain water based on concentration, enrichment and the use of a duplex TaqMan PCR test

“…The analytical sensitivity of the test can be improved by concentration of cells from water and selective multiplication (enrichment) of the target cells. In contrast to plant material, the use of drain water allows rapid concentration of low bacterial densities by centrifugation or filtration (Heredia et al ., 2015; Wang & Salazar, 2016; Lelis & van der Wolf, 2017). Densities of bacteria belonging to the RSSC may be further increased by growth in a (semi‐) selective broth, such as the SMSA broth (Elphinstone et al ., 1996).…”