Annex D i ISO 16140 (under revisjon)
General Protocols for Contamination by Mixture and Artificially
Contaminating Food Matrices
This Annex provides examples of how artificial contamination of (food) items can be done. Methods used
by expert laboratories are not limited to methods shown here.
A: Contamination by Mixture
X gram of naturally contaminated sample are mixed up with y gram of non-contaminated sample, in order
to reach the desired level of contamination.
Store the food sample contaminated by mixture at the appropriate storage temperature for that food
matrix. Allow the microbial population to equilibrate in the food matrix for a minimum of 1 day before any
analysis.
B: Artificially Contaminating Food Matrices
B-1: Artificial Contamination of High Moisture Foods with a Liquid (Broth) Culture
Preparation of contaminating microorganism(s) by single sample inoculation
1) culture target strain
Inoculate a tube of non selective enrichment broth with the designated strain. Incubate the
broth at optimal conditions for the strain.
2) adjust level by dilution
After incubation dilute the culture in a suitable diluent till the desired level(s). The level of
dilution required is dependent on the (food) item to be inoculated, level of contamination,
strain used and storage conditions of the food item.
3) inoculate into food by pipetting known volume or spraying known volume.
The diluted culture is inoculated into the (food) item by spraying or pipetting. Inoculation of
individual samples is preferred. The volume of the inoculum should be as low as possible as it
should not influence the aw significantly. As a general rule 0.25 ml per 25 gram of sample is
used.
4) mix to ensure homogeneity
After inoculation the (food) item is mixed thoroughly to ensure homogeneity. In case the
inoculum is added in steps, mixing should be done after each step.
5) apply a stress
Apply an appropriated sample treatment to the samples such as for example:
a) thermal treatment (e.g; 5 min at 50°C) by immersion in given temperature bath
b) freezing treatment (e.g; 72h at -20°C)
c) storage at 4°C (e.g. for1 week)
6) leave sample for stabilisation/stress
Store the (food) item at the appropriate storage temperature (preferably the normal storage
temperature) for that (food) item. Consider also the potential growth or survival of the
organism during the storage period. Examples are: nuts would be stored at room temperature,
orange juice would be stored at 2-8 ºC and ice cream would be stored frozen at less than 0
ºC. Allow the microbial population to equilibrate in the food matrix for a minimum of 2 days
depending on the shelf life of the product involved. For example perishable foods should be
stored for a minimum of 2 days, frozen foods for a minimum of 14 days.
7) check test sample for level of contamination
After the samples have been stored for the appropriate time check the level of contamination
before using the samples in the validation study.
B-2: Artificial Contamination of Low Moisture Foods with a Lyophilized Culture
1) prepare a lyophilised culture
Inoculate a tube of non selective enrichment broth with the designated strain. Incubate the broth at
optimal conditions for the strain. After incubation, collect the bacterial cells by centrifugation. Wash cells
twice with a sterile buffered diluent. Repeat centrifugation and decant the supernatant. Resuspend the
pellet into sterile 10% NFDM (non fat dried milk). Transfer resuspended cells into appropriate containers
for lyophilization.
2) assess level of target organism
Collect the lyophilized cell suspensions in a sterile container. Manually crush the lyophilized culture to
create a homogenous fine powder before assessment of the level of contamination. Use a non selective
method for the determination of the level of contamination and incubate the plates under optimal growing
conditions.
3) inoculate the lyophilised culture into the food to attain the required level
Mix 0.1 g of the lyophilized culture with 10 g of the uninoculated (food) matrix in, for example a sterile
plastic bag. The bag is shaken until the inoculum appeared to be evenly distributed throughout the
food matrix.
Perform serial ten-fold dilutions with the (food) item (e.g. 1 g from first step with 9 g (food) item, etc.) to
dilute the lyophilized culture to the appropriate level. Ensure that proper mixing occurs at each dilution
level.
4) store food
Store the (food) item at the appropriate storage temperature (preferably the normal storage temperature)
for that (food) item. Allow the microbial population to equilibrate in the (food) item. Allow the microbial
population to equilibrate in the (food) item for a minimum of 1 week before any analysis
5) check level
After the samples have been stored for the appropriate time check the level of contamination before
using the samples in the validation study