Inoculated Pack Process Bio-Assay:

The inoculated pack is a bio-assay technique in which a batch of product or containers of product are inoculated with a precise concentration of test organisms, usually heat-resistant spores, and are then subjected to processing conditions of varying intensities. The processed product or containers of the product are then evaluated for either the presence or absence of the test organisms either by incubation and observation, or by direct enumeration of surviving organisms. These techniques are used to either design or evaluate processes that cannot be reliably simulated or measured by the process authority’s more traditional means of heat penetration testing and calculation. They can also be used to validate processes determined by other means. The techniques usually employ microbial spores that have been grown and been calibrated for their resistance to heat or other sterilants by IEH for this specialized use as measuring tools.

We use two types of inoculated experimental packs as process design or verification tools: Count Reduction and Inoculated Pack.

The count reduction technique involves the inoculation of containers of product with relatively high numbers of spores having a moderate to high heat resistance (D-value). The intent is to process the product using the processing conditions under evaluation and to have a readily detectable and accurately enumerable quantity of spores survive the process. By comparing the initial count to the count of survivors and knowing the thermal resistance of the spores when heated in the product under study, it is possible to measure the lethality of the entire process and to relate that measurement to calculated values obtained by other methods.

The inoculated pack method is similar to the count reduction method in that containers of product are inoculated with a known count of spores of a test organism and then processed, but the intent of the inoculated pack is slightly different from the Count Reduction method, and therefore, varies from it in the following manner. The organism chosen for the assay is usually one that is a significant potential natural spoilage organism of the product and one that is a surrogate for significant organisms of public health significance.

For example, low-acid foods packed in hermetically sealed containers are processed primarily to destroy the spores of the toxigenic, heat-resistant, spore-former Clostridium botulinum ( the public health target organism), but beyond that they are processed to attain the condition of commercial sterility, i.e. the absence of microorganisms capable of outgrowth in the product under “normal” conditions of storage and distribution. It is not advisable to introduce spores of Clostridium botulinum into the plant environment to do an inoculated pack, so we use a surrogate, the non-toxigenic spore-former Clostridium sporogenes, and a close relative.

The pack is conducted by inoculating approximately 100 containers for each of at least five processing variables. The containers are processed and then placed in an incubator to allow the outgrowth of any surviving spores. The processing variables used are designed so that some result in complete failure, others in partial failure, and still others in which the containers remain completely sound. An inoculated pack is intended to provide information on both the lethality of a particular set of processing conditions, as well as the effects of the product environment on the death rate kinetics of the test organism. It is well known that many food product properties affect the heat resistance and outgrowth characteristics of many microorganisms and some of that important information can be derived from the results of a well-designed and executed inoculated pack.

If your product or process will require an inoculated pack or a count reduction study to verify the appropriateness of its design, we are able to advise and assist you in making the right choices. Our staff is uniquely trained and positioned with the expertise to help you succeed using these valuable assay tools.