Farming BSFL |Monitoring for Coliform Pathogensby Terry Green on 04/18/15
Black Soldier Fly larvae (BSFL) grown on food scrap, or on other biodegradable waste products, destined for incorporation into animal feedstock, or as food for human consumption, and BSFL byproducts destined for use as agricultural amendments, should be screened for contaminating pathogens before they are marketed (see, Farming BSFL - Foodborne Pathogens & Safety | What You Need to Know). This blog describes a practical and inexpensive way of screening BSFL and their byproducts for coliform contamination.
Start by building a microbial culture incubator using three-quarter inch thick plywood assembled with a groove cut on the front panels for insertion of a sliding door to make it easy to open and close the incubator while keeping the temperature in check. For heat inside the incubator box, mount a light socket on the upper top panel and insert a small 25 watt incandescent heating bulb into the socket (see Fig. 1, - Upper Left and Upper Right panels). Avoid using a higher wattage bulb. Too much wattage will result in too much heat generated inside the incubator.
The dimensions of the incubator should be approximately 16 inches (H) x 12 inches (W) x 9 inches (D). Drill a small hole (~ one quarter inch in diameter) through the top panel of the incubator to vent off excess heat. If necessary, the sliding door can also be opened slightly to vent off more heat in achieving the desired temperature in culturing plates or films placed inside the incubator.
The temperature inside the incubator can be easily monitored using a remote wireless thermometer placed inside the incubator. With this design you should be able to operate the incubator at a constant temperature anywhere within the range of 30° to 35° C ± 2° C without difficulty.
Testing is straightforward. After applying 1 ml of test sample to the coliform test film (3M Petrifilm, kit #6410), the sterile cover sheet gets folded gently back over the top of the plate. Air bubbles trapped under the film are dispersed, and the film spreader is then gently dragged over the top of the film to disperse sample uniformly across the surface of the gel film. Place the film inside the incubator and leave it for 24 hours at 30° C (or at 35° C, if preferred).
After a 24 hour incubation period the film can be read for coliform positive CFUs (colony forming units) following interpretation guidelines provided with the test kits. Other commercial coliform screening kits can be used with the incubator, similarly. Follow the instructions accompanying the screening kit used in your screening protocol.
The lower limits of detection for the rapid 3M Petrifilm, test kit #6410, is 100 CFU per 100 ml of sample. Lower coliform limits at 20 CFU’s per 100 ml sample can be achieved using alternate test films purchased from 3M. The latter more sensitive plates are bigger and accept up to 5 ml of a test sample which accounts for the increased sensitivity of the film.
A positive coliform sample should be run in parallel with you sample(s) screened for coliforms to ensure good quality control. This can be easily done by drawing water from a source known to be contaminated with fecal matter. A water slurry taken from a barnyard where cattle, chickens or other animals are housed and contaminated with fecal matter, for example, works fine as a positive control, or simply draw a water sample from the bowl of a toilet (Fig. 2).
The positive control helps confirm that the coliform test system is working. Test culture plates, tubes or films that have outdated should not be used since they may not be reliable in yielding interpretable results.
Fig. 1. Setup for screening coliform contaminants using 3M Petrifilm CC plates (kit # 6410). Upper Left and Upper Right, layout of incubator made from plywood and a small 25 watt incandescent heating bulb. Lower Left, 3M Petrifilm, freshly drawn BSFL leachate fraction from BSF bin ready to be tested and spreader plate (far left) which comes with Petrifilm coliform kit; Lower Right, application of 1 ml of test leachate to Petrifilm plate. (copyright(c) 2015, Terry Green, all rights reserved.)
Fig. 2. Coliform positive 3M Petrifilm. A 1 ml water sample drawn from a toilet bowl was applied to the film and subsequently incubated at 30° C for 24 hours. Arrows point to examples of positive coliform CFU’s evidenced by formation of carbon dioxide bubbles and acid formed by coliforms fermenting lactose incorporated in the gel layered on the film. A tetrazolium dye indicator further highlights CFU’s evidenced by formation of dark purple-red spots where colonies are located on the plate. CFU’s are TNCC (too numerous to count). (copyright(c) 2015, Terry Green, all rights reserved.)
On running a coliform screen in parallel with the positive control on leachate drawn from our BSFL food scrap growing bins, we obtained negative test results (Fig. 3). The absence of coliforms in the leachate sample indicates that the leachate is free of fecal contaminants, and hence free of enteric pathogens. Since the leachate is also an excretion product of BSFL feeding on the food scrap waste, the negative test results further indicates that the larvae are likewise free of coliforms.
Fig. 3. Coliform negative test results obtained on screening BSFL leachate drawn off from BSFL food scrap bins used for harvesting BSFL. The 3M Petrifilm screen (#6410) was set up in parallel with the coliform positive control (see Fig. 2) except 1 ml of BSFL leachate was used as the source sample applied to the film. No coliform CFU’s are evident on the film following 24 hours incubation at 30° C. (copyright(c) 2015, Terry Green, all rights reserved.)
Keep a record of your coliform test results. Over time this will provide a valuable quality control record and history concerning the safe management of your operation in farming BSFL.
Check back for more to follow on the management and strategies in scaling up BSFL production. Comments on this blog, or any of our other blogs, are always welcome. Follow us through our RSS feed. For additional information or follow-up questions, visit our Forums page, or Contact Us (http://www.dipterra.com/).