Skim milk powder contains the proteins lactalbumen and caseinogen. Lactalbumen makes the skin that is such an annoyance when making hot chocolate. Caseinogen is very abundant and is seen as fine suspended blobs which we see as the opacity and white colour of milk. Sporulating P. larvae produce proteolytic enzymes that reduce the opacity caused by suspended protein blobs. The concentration of skim milk powder solution doesn't seem to be too critical. There are stories of people adding a scant tablespoon of milk powder to a glass then filling to so as to make a 'cloudy but not opaque' solution. You can try to get as close to 1% solution by first making a stock solution by adding warm (37 C) sterile water to a very clean jar loaded with 1 g of milk powder until the total volume (powder plus water) is 100 ml. Agitate to suspend the milk into the 'solution'. Don't add hot water to the milk powder. High temperatures will denature the milk proteins. Use any multiples of these numbers you want. Dispense your finished solution into smaller but uniform containers for the testing. Add infected material to your test solution in your uniform containers. Don't use your fingers. Brand new toothpicks from a freshly opened package will make good tools to inoculate your test medium. Do multiple tests. Try not to get bits of material not associated directly with the 'diseased' material into the testing solution. (Ideally you would be working with isolated, pure cultures all the while using proper aseptic technique.) Incubate at 37 C for 20 minutes. Try a water bath with a thermometer to monitor the temperature. Don't let water bath water get into your testing containers. Temperature is important. Not too warm, not cool: 37 C is about the temperature in your armpit. (And, yes, I have used my armpit as an incubator. This aseptic technique deserves a paper.) A positive test is a clearing of the milk suspension (cloudiness vanishes). I recommend a negative control (one to which the infected material is not added) for comparison. A clot is not a positive. Lot's of different kinds of bacteria will clot milk. So will acids. The 'positiveness' is likely variable and open to subjective interpretation without a negative control. I wouldn't consider a 'positive' as PROOF but enough evidence for a presumptive P. larvae, especially if prior gross in-field examination showed signs of AFB. I have concerns that any kitchen-counter testing will yield false, silly and invalid results. The Holst Milk Test, like any diagnostic test, assumes you are working with an isolated, pure culture of a suspected pathogen. You need autoclaved glass ware, specialized growth medium, bacteriological loops, microbiologists...etc. I've never used the Holst Milk Test in my lab, but then again, my veterinarian clientele have never asked me to confirm a suspected case of AFB. I've never experienced AFB personally or know anyone locally who will admit to a recent encounter. Strange that. I'd like to try to isolate this wee nasty. May be this year I'll get 'lucky'. Richard Goetze :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: -- Visit www.honeybeeworld.com/BEE-L for rules, FAQ and other info --- ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::