Southern North Island Beekeeping Group Ltd

American Foul Brood - Holst Milk Test

The disease American foul brood disease (AFB)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 sporesThe 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.


Used cocktail sticks can be disposed of in the smoker, which is an aid to hygiene in a field situation.

Powdered milk is inexpensive, so it is no hardship to prepare some to the correct dilution before leaving for the disease inspection trip, if not used throw it away.

Skim Milk Powder is a product made from the separation of whole milk and the evaporation and spray drying of the skim milk portion.

Typical Analysis of the skim milk suitable for this test... Moisture Max 4.0% Typical 3.0% Fat typical 1.00% to 1.25% Lactose around 51.00%

Liquid skim milk is 9% solids and 91% water, measured by weight.

The skim milk powder tends to dissolve more readily in cool water and can be refrigerated overnight prior to the testing. Reconstitute the skim milk around four hours before intended use if kept at room temperature.

AFB Holst Milk Test Randy Oliver NY Bee Wellness
Randy Oliver of demonstrates how to do a simple test for American Foul
Brood in honey bees. The NY Bee Wellness Workshop, August 7, 2016

Southern North Island Beekeeping Group Ltd