method development guidance for reducing incubation in homogenized foods

INTRODUCTION

The InnovaPrep Concentrating Pipette (CP) is an automated, rapid micro-particle concentrator developed for general microbiology use. The system performs ‘mechanical enrichment’ as a front-end to rapid microbial detection in minutes, eliminating the need for centrifugation or enrichment in many sample types - or greatly reducing enrichment times for more difficult matrices such as food homogenates.  

The one-pass method uses tangentially-loaded dead-end filtration to capture micro-organisms in hollow fiber membrane filters within the InnovaPrep CP’s single-use tip. After the sample has been processed and the particles have been trapped, InnovaPrep’s patented Wet Foam Elution™ process is employed to recover the particles from the membrane into a very small final volume.  

Concentrating homogenized food samples is one application of interest for this technology and holds promise for substantially reducing enrichment times, often in half. The concentrated sample pairs directly with classical and modern analytical methods commonly used in food diagnostics today. The following guidance offers a starting point for users exploring new methods to reduce time to detection for same shift results and as a means for reducing costs associated with holding product, especially perishable foods that require refrigeration.

METHOD DEVELOPMENT GUIDELINE

This guidance follows the FDA's Bacteriological Analytical Manual (the BAM) with alteration to the recommended enrichment time for use with the InnovaPrep CP. The BAM is FDA’s preferred laboratory procedures for the detection in food and cosmetic products of pathogens (bacterial, viral, parasitic, plus yeast and mold) and of microbial toxins. The manual is available online:

http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm  ).  It is important to note that even if only a portion of the sample can be concentrated efficiently, a significant concentration factor will be produced that will aid any detection method used.

Please also note, the following protocol is not an AOAC standard. AOAC standards are used globally to facilitate public health and safety.  For AOAC certification, a validation study must be performed for each specific food type/sample matrix. Once a protocol is validated for reduced enrichment times for your desired matrix the AOAC approval process can be initiated.

STUDY DESIGN

Designing a spike study for your matrix to determine limit of detection is the first step in determining how far you can reduce enrichment time using the InnovaPrep CP. For study design guidelines, consult the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces.

MATERIALS REQUIRED:

• InnovaPrep Concentrating Pipette
• CP elution fluid (HC08000 or HC08001)
• CP Pipette filter tip (CC08000, CC08018 or CC08022)
• Food homogenizer such as a blender or stomacher
• Bag Filter (400S by Interscience or similar) pictured at right
• Incubator  
• Supplies and reagents specific to your desired analytical method

STEP 1 - PREPARING THE SAMPLE

1. Collect and prepare food homogenate as directed in the BAM protocol.  
2. Combine the appropriate ratio of sample to enrichment media as directed in the BAM (example: 25g homogenate to 225mL media).
3. Add materials to filter/stomacher bag and process normally
4. Incubate at the recommended temperature for a reduced period.  2/3 or half the normally recommended time is a good starting point.
5. Remove from incubator or water bath.  
6. Pass approximately 50 mL of the incubated sample though the filter section of the Bag Filter.

STEP 2 - CONCENTRATION

7. Once the 50mL sample has been filtered and transferred from the Filter Bag. It is ready for processing on the Concentrating Pipette.
8. Set up Concentrating Pipette as instructed in section 4 of CP user guide.
9. Prime fluid line as instructed in section 5.2.1 of CP user guide.
10. Insert pipette tip and set menu parameters as instructed in section 6.2 of CP user guide for the chosen CP tip type.
11. Lower CP tip into prepared sample.
12. Press – run on menu.
13. When the entire sample has been processed the CP will stop.  
14. Place clean final sample container under tip.  The menu screen will prompt you to press Extract.
15. Press extract. The sample will dispense from the pipette tip into the sample container and is ready for  analysis with your desired method.

HINTS & TROUBLESHOOTING

A technical report entitled “ Reduced Incubation for Detection of Escherichia coli O157:H7 in Experimentally Infected Spinach Samples using the InnovaPrep Concentrating Pipette” is available from the InnovaPrep downloads page at: 

https://www.innovaprep.com/products/concentrating-pipette

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