Direct 24-Hour Presumptive Enumeration of Escherichia coli 0157:H7 in Foods Using Hydrophobic Grid Membrane Filter Followed by Serological Confirmation: Collaborative Study

Abstract

Fifteen laboratories took part in a collaborative study to validate a method for enumerating Escherichia coli 0157:H7. The method is based on use of a hydrophobic grid membrane filter and consists of 24 h presumptive enumeration on SD-39 Agar and serological confirmation to yield a confirmed E. coli 0157:H7 count. Six food products were analyzed: pasteurized apple cider, pasteurized 2% milk, cottage cheese, cooked ground pork, raw ground beef, and frozen whole egg. The test method produced significantly higher confirmed count results than did the reference method for milk, pork, and beef. Test method results were numerically higher than but statistically equivalent to reference method results for cheese, cider, and egg. The test method produced lower repeatability and reproducibility values than did the reference method for most food/inoculation level combinations and values very similar to those of the reference method for the remaining combinations. Overall, 94% of presumptive positive isolates from the test method were confirmed serologically as E. coli 0157:H7, and 98% of these were also biochemically typical of E. coli 0157:H7 (completed test). Corresponding rates for the reference method were 69 and 98%, respectively. On the basis of the results of this collaborative study and the precollaborative study that preceded it, it is recommended that this method be adopted official first action for enumeration of E. coli 0157:H7 in meats, poultry, dairy foods, infant formula, liquid eggs, mayonnaise, and apple cider

Methods for the detection and isolation of Shiga toxin-producingEscherichia coli

“…A direct 24 h presumptive enumeration of E. coli 0157:H7 in foods by the Hydrophobic Grid Membrane Filter (HGMF) technique, followed by serological confirmation, has been validated (Entis 1998). The method consists of a 24 h presumptive enumeration on SD-39 agar used with the ISO-GRID HGMF.…”

Differentiation of Salmonella enterica Serovars and Strains in Cultures and Food Using Infrared Spectroscopic and Microspectroscopic Techniques Combined with Soft Independent Modeling of Class Analogy Pattern Recognition Analysis

“…Aliquots (1 ml) of the 10 Ϫ7 dilution of each culture were added to 20 ml of sterile saline solutions. The prepared cell suspensions were placed on a filtration unit (Iso-Grid, Neogen Corporation, Lansing, Mich.), vacuum filtered through an HGMF (0.45 m pore size; Neogen), and then rinsed with 20 ml of saline solution (14). The HGMFs were carefully placed onto Miller-Mallinson (MM) agar medium and incubated at 42 Ϯ 0.2ЊC for 24 Ϯ 2 h. Five to seven colonies were selected from each plate for sample preparation.…”

“…One milliliter of the dilution containing approximately 10 2 CFU/ml was mixed with 10 ml of TSB. The prepared cell suspensions were placed into a disposable filtration unit (ISO-GRID, Neogen) and vacuum filtered through an HGMF (0.45-m pore size) (2,14). The HGMFs were carefully placed onto MM agar and incubated at 42ЊC for 24 h. Colony growth was observed, and HGMFs were peeled from the agar, placed into empty petri dishes, and dried under vacuum for 1 h at 80ЊC.…”

“…The populations of Salmonella were approximately 3.0 ϫ 10 1 CFU/ml of the liquid portion. The liquid portion (1 ml) of each contaminated food was filtered through an HGMF as previously described, and the membrane was then rinsed twice, each time with 20 ml of saline solution (0.85%) (14). Membranes were placed onto MM agar and incubated at 42ЊC for 24 h. Colony growth was observed after incubation, and three isolated colonies per plate were collected to prepare samples as described.…”

“…Hydrophobic grid membrane filters (HGMFs) have been used in methodologies designed to enumerate aerobic microbiota and pathogens in foods (2,14). This filtration system allows for the development of colonies in a distinct and orderly array on agar media by using a membrane filter printed with a grid of hydrophobic material.…”