American Foul Brood (AFB)
AFB Test Device
Disease Diagnosis
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Holst Milk Test for AFB in Honey Bees

American foul brood disease

The disease is caused by a bacteria... Paenibacillus (formerly Bacillus) larvae subspecies larvae, often simply referred to as just P. larvae, is the bacterium that causes American foulbrood disease(AFB).

Paenibacillus larvae Paenibacillus larvae is a gram-positive bacterium shaped like a rod with rounded or slightly bulbous ends (as right) that shows a tendency to grow in chains. The rod varies in length, from about 2.5 µm to 5 µm, it is approximately 0.5 µm in width.

Paenibacillus larvae spores The bacteria is spore forming, with oval spores roughly twice as long as they are wide, (approx. 0.6 µm x 1.3 µm).The spores may aggregate together to form clusters or rows. The illustration is not to the same scale.

The spores can be stained with carbol fuchsin, the spores will have reddish-purple walls and clear central regions. As many as 2.5 billion spores may be produced in each infected larva.

Holst milk test

The Holst milk test (Holst 1946) is a simple procedure that can easily be carried out in the field, it is based on the fact that high levels of proteolytic enzymes are produced by sporulating Paenibacillus larvae.

The test is conducted by suspending a suspect scale or a smear of a diseased larva in a test tube or glass vial containing 3 or 4 ml of 1-percent powdered skim milk in water. The tube is then incubated at 37° C. If AFB is present, the suspension should clear in 10 to 20 minutes. It should be noted that this test is not totally reliable, but any doubt should be expelled by using another additional testing method.

Items you will need

Spatula, tweezers or cocktail sticks,
Tubes or vials to conduct the test in,
Calibrated eye dropper,
Disposable surgical gloves,
Cleaning and sterylising materials.



Place the scale or macerated larval remains in the tube or vial using spatula tweezers or cocktail stick, add 4 ml of milk solution using the eye dropper, shake to mix and incubate the stoppered tube in suitable pocket in your bee suit to await results.



Randy Oliver states that fresh milk, even skimmed works equally well. There is a video of Randy here. This test is probably more appropriate to countries that don't have such a good bee inspection service as we have in England and Wales. I would not advise inexperienced beekeepers to use this test in case they get a negative result, which is possible if the larva or scale is not in the right condition. R.P.

 Written... 16 April 2007,
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