Title: Testing Packaged Ice for Total Coliforms and E. coli is easy and affordable!

Authors: Gil Dichter and Chris Dunn Is testing your packaged ice once a month sufficient to insure the quality of the many lots produced within the month? If the results came back indicating a positive result for pathogenic bacteria, it may require a recall on all the ice produced since the previous test date. Wouldnt you rather have the peace of mind knowing that each lot produced is tested in order to prevent a possible large recall? Why is testing for coliforms important? The US Food and Drug Administration has determined that testing for coliform bacteria is one of the best indicators of the overall microbiological safety of a food product. While coliforms (Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., Serratia spp., and others) by themselves may not be life-threatening, they can do not normally cause serious illness and, when these are present, but serve as a red flag n indicator for the overall microbiological quality of the product and the sanitation quality of the manufacturing process. They also indicate the possibility of the presence of other pathogenic organisms of fecal origin. Thus, any time coliforms are found, you also need to look for E. coli, the pathogenic strains of which are dangerous and life threatening. What methods are used? For more than a hundred years microbiologist use two primary types of coliform tests. First is the Multiple Tube Fermentation (MTF). Samples are put in three different tubes with growth mediums and incubated. Visible growth and the creation of gas in the tubes confirm that coliforms are present. You get estimates, but not actual numbers of colonies growing. The second is Membrane Filtration (MF). A 100ml volume of a water sample is drawn through a 0.45 m pore size membrane filter. The filter is then placed in a petri dish with selective agar media (food for bacteria) and incubated at 44.5 degrees C. If there are any fecal coliforms, they grow and produce an acid that reacts with the agar resulting in a color change. Which color depends on the type of selective media used and the type of indicator present. The color change denotes the presence or absence of the coliforms along with enumeration of colonies.

(Multiple Tube Fermentation MTF)

Membrane Filtration (MF) Both of these methods can take four to five days to complete and both require sophisticated laboratory facilities and trained technicians. Hence, in 1991 the US Environmental Protection Agency, the group that approves testing methods for drinking water, approved the first rapid detection Presence/Absence tests called Colilert. Rapid Detection of Coliforms One of the best known tests, recently demonstrated at the IPIA convention in New Orleans, is called Colilert. It uses an enzymatic procedure based on Defined Substrate Technology. In 24 hours you get simultaneous detection of the Presence/Absence of total coliforms and E. coli.2 The technology used for these tests is based on colorimetric and fluorogenic enzyme substrates, ONPG and MUG. These are the major sources of carbon in coliform bacteria. As coliforms grow in the Colilert reagent, they use enzyme -galactosidase to metabolize ONPG and change the sample from colorless to yellow.

E. coli use enzyme -glucuronidase to metabolize MUG and create a blue fluorescence under a 365 nm UV light. Since most non-coliforms do not have these enzymes, they are unable to grow and interfere.

Potential False Positives that could be triggered by a few non-coliforms that do have these enzymes are selectively suppressed by Colilert's specifically formulated matrix. 2

Doing these tests yourself in your packaged ice plant One of the major advantages of rapid testing methods like Colilert is that they can be done in your plant, with minimal equipment and very basic training. What you need for testing: A small incubator or oven capable of maintaining 35 0.5C (available online for $200$500). A 365nm UV light source (online for less than $150). Test kit (IDEXX.com starting at about $8.00 each with volume discounts available) Lab supplies: sterile scoop to collect the ice; a sterile bag or container for the ice to melt (you need enough ice so that when it melts you have 100ml of liquid); sterile, transparent, nonfluorescing test container with secure cap, disposable latex gloves and a marking pen. Procedure to melt the Ice: Aseptically (use sterile gloves and scoop) withdraw 25-35 ice cubes into a sterile wide mouth vessel or plastic bag. Allow ice to melt and pour 100 mL into the test vessel. This should be done within 2 hours at room temperature. PThe procedure for Determining Presence-Absence Result: Wash hands and put on the latex gloves. Carefully separate one blister pack of test nutrient from the strip of five following the package insert instructions. Add the contents of the pack to the test container with the melted ice sample and then cap the vessel. Shake until the powdernutrient is dissolved. Incubate for 24 to 28 hours at 35 0.5C. At the end of the 24 - 28 hours, read the results according to the interpretation table below.

Appearance Clear or Less yellow than the IDEXX comparator Yellow equal to or greater than the IDEXX comparator Yellow and fluorescence equal to or greater than the IDEXX comparator

Result Negative for total coliform and E. coli Positive for total coliform Positive for E. coli

Colilert results are definitive at 2428 hours. In addition, positives for both total coliforms and E. coli observed before 24 hours and negatives observed after 28 hours are also valid. 3

What to do if Coliform or E.coli is present The FDA has published a useful guide to the bottled water industry on what actions should be taken when coliforms and/or E.coli are found in a lot of source water or finished product. (www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/SmallBu sinessesSmallEntityComplianceGuides/ucm206215.htm) If allowable limits are exceeded, labeling with the statement Contains Excessive Bacteria on the package is required. Best practice and the bottled water trade association model code requires zero coliform for any finished product. Any product exceeding this is recalled recalled and warehouse stocks destroyed. The role of PIQCS in Coliform/E.coli testing The International Packaged Ice Association, through the PIQCS manual, encourages its members to adopt a high standard of quality by regularly monitoring the safety of ice throughout the manufacturing and distribution process. To insure that ice is free from harmful microorganisms, PIQCS requires that packaged ice product be sent to a certified laboratory at least each month to test for Total Coliform and Total Plate Count (TPC) bacteria, including E. coli. The requirement is that ice be free of any E. coli bacteria and TPC count does not exceed 500 colonies per ml. With the easy availability and low cost of rapid detection methods like Colilert, coliform testing of each lot is also recommended and can be done in-house with minimum effort and simple training. The results are available within 24 hours. Since ICE is Food, The International Package Ice Association (IPIA) is committed in ensuring that packaged ice is safe to consume. A key part of IPIAs mission has been the development of the Packaged Ice Quality Control Standards, better known as PIQCS, to help members demonstrate to retailers and consumers that there is a difference in quality among packaged ice producers. Members of the association agree to abide by the standard and are subject to annual plant inspections by NSF International, an independent, not-for-profit, third party with expertise in auditing compliance with food safety standards. Conclusions Principle 7 of HACCP requires a packaged ice plant to Establish procedures for verifying the HACCP system is working as intended. In-house micro testing is a fast, easy and cost effective way to help insure you are verifying that your Control Points and Good Manufacturing Practices are working. The ease with which rapid testing for coliform can now be done, requiring very little equipment and capable of being done by non-technicians, offers companies a cost effective program to increase the surveillance and testing of finished product. The cost for in-house testing each production lot is less than ten dollars. 4

Field Code Changed

Colilert is the market leader in Rapid Testing for coliform. In addition to being approved by the US EPA, it is also listed in Standard Methods for Water testing, the AOAC Official Methods of Analysis and referenced by the FDA. However, the EPA has now approved other companies enzyme based methodology products for drinking water coliform testing. There are also variations of rapid test that allow for quantifying the number of colonies in addition to simply determining a presence or absence. There are now similar technologies available for rapid testing of TPC (Total Plate Count), sometimes referred to as HPC for Heterotrophic Plate Count. Cryptosporidium, Guardia Giardia and Pseudomonas aeruginosa can also be done in-house very inexpensively to help plants monitor and control the microbiological purity of their product. This will be reviewed in a future article.

References; 1. International Packaged Ice Association: www.packagedice.com 2. IDEXX Laboratories: www.idexx.com/water 3. NSF International Microbiology Laboratories: www.nsf.org Contact Information Gil Dichter is the Worldwide Technical Support Manager for Water at IDEXX Laboratories (gildichter@idexx.com) Chris Dunn is the General Manager, Beverage Quality for NSF International (cdunn@nsf.org)