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

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