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food for the low level inoculation and 0 cfu/25 g for the uninoculated control samples.
1
The objective was to achieve fractional positive data within the range of 5-15 positives
2
for the low inoculation level. Culture information is provided in Tables 3-7 in the
3
Appendix.
4
A 3-tube Most Probable Number (MPN) analysis of each food was performed. On the
5
day of initiation of analysis, 3 samples of 100g, 20 x 25g, 3 x 5g from each low level
6
were prepared for each food, enriched according to the appropriate reference method and
7
confirmed according to the specified reference method procedures. A different culture of
8
Listeria
was used for each inoculated food. Cultures were tested to confirm biochemical
9
reactions prior to use in the study. In this study all cultures were biochemically typical of
10
the species.
11
Preparation inoculated food
12
For ham, pepperoni, hot dogs, chicken nuggets, cooked shrimp and smoked salmon the
13
culture was heat treated prior to inoculation in order to simulate heat stress during
14
processing. An 18-24h broth culture (10 ml) was placed in a 50°C water bath for 10
15
minutes and the cooled prior to preparing dilutions and plating in triplicate on a
16
nonselective agar (TSA) and a selective agar (MOX). The degree of injury caused by
17
heat-stressing was demonstrated by plating the inoculum in triplicate on selective and
18
non-selective agars. The degree of injury was calculated as:
19
100 )
1(
x
n
n
nonselect
select
−
20
where
n
select
= mean number of colonies on selective agar and
n
nonselect
= mean number of
21
colonies on nonselective agar.
22
23
For fresh foods (vanilla ice cream, soft cheese, bagged mixed salad, regular peanut butter,
24
deli ham, ground beef, deli turkey, liver paté) the broth used for inoculation was not heat
25
treated and for low moisture foods (black pepper) the culture was lyophilized.
26
27
The dilutions for the fresh foods were kept refrigerated overnight and the appropriate
28
inoculum was added drop wise to bulk food, followed by thorough mixing. For
29
cantaloupe, individual melons were placed into sterile bags and inoculated with 100 µL of
30
inoculum onto the blossom and allowed to dry. After drying, the cantaloupes were turned
31
over so that the blossom was on the bottom of the bag. After inoculation,
Listeria
was
32
allowed to equilibrate with the food environment for 48-72 h at 2-8°C in refrigerated
33
foods. Since the two methods, LPT and reference, utilized different enrichment broths,
34
two sets twenty 25g samples or melons were pulled for analysis by each method.
35
Additionally, samples were also pulled for MPN analysis.
36
37
38
47