Midnite Bee-Beekeeper's: Article Resistence Test (Pettis) Apiary Factsheets Ministry of Agriculture, Food and Fisheries Factsheet #224 APISTAN (FLUVALINATE) RESISTANCE TEST (PETTIS TEST) This test can be used to determine mite resistance when a beehive doesn’t appear to respond to chemical mite control measures. The test uses Apistan, but it can easily be modified to test for resistance to Coumaphos, Bayvarol, Perizin or Apivar. The following materials are required to carry out the test: • 500 ml jar with lid (canning jar) • light metal mesh cover for the jar (8 mesh to the inch hardware cloth) • 75 x 125 mm index card or similar • 9 x 25 mm piece of a new Apistan strip • ¼ cup measure to scoop up bees • 25% alcohol solution or windshield washer fluid • straining cloth and ½ dozen clothespins • plastic or rubber gloves • plastic bucket • sheet of white paper • stapler • sugar cube Step 1 Staple a 9 x 25 mm section of an Apistan strip to the centre of a 75 x 125 mm index card. Make sure to handle the Apistan with gloves. Place the card in the jar with the section of Apistan strip facing inwards. Place a sugar cube in the jar. Step 2 Shake bees from one or two brood combs into an up-turned hive lid or a bucket or box. Scoop up ¼ of a cup (about 150 bees) and put them in the jar, being careful not to damage the bees. Step 3 Replace the solid, round metal section of the canning jar lid with a piece of wire mesh and screw the lid onto the jar to stop the bees from escaping. The holes in the mesh should be large enough to easily let varroa through. Place the jar in an incubator or a warm (30oC) room in the dark for 24 hours. Make sure the lid isn’t covered so air gets into the bees. Step 4 After 24 hours, hold the jar about 10 cm above a piece of white paper and turn it so the mesh lid is facing downwards. Hit the jar with the palm of your hand three times. Count the number of mites that fall on the paper. Factsheet #224 Apistan (Fluvalinate) Resistance Test (Pettis Test) Step 5 Knock the bees to the bottom of the jar. Remove the index card with the attached strip and sugar cube and fill the jar halfway with the alcohol or washer fluid. This should be done outside using gloves. Remove the mesh lid and replace with the original solid lid for the jar. Shake the jar vigorously for 5 minutes or place the jars in a box, put the box in the back of a pickup truck and drive on a rough dirt road for 10 minutes. Step 6 Remove the solid lid and replace it with the mesh lid. Pour the fluid into the straining cloth pinned to the bucket. Refill the jar with fluid, swirl the bees around and pour through the strainer again. Step 7 Count the number of mites recovered on the cloth. If the total number of mites recovered in both samplings (apistan plus fluid) is less than 5, the results should be discarded. Step 8 To calculate the percentage of mites killed by apistan, divide the number of mites that initially fell on the white paper before the bees were killed by the total number of mites recovered (both on the white paper and the straining cloth). % kill by Apistan = ____initial kill______ = X 100 (initial + final kill) If less than 50% of the mites were killed by the Apistan, the mites may be resistant and should be tested with a more sensitive laboratory test. If less than 50% of the mites are killed in the jar by Apistan, the mites may be resistant to Apistan.   Critical Factors For Success of Pettis Resistance Test 1. Prescreen hives using the ether roll technique (250-300 bees) and test only those hives yielding 5 or more mites. This test gives meaningful results only when performed on hives with adequate mite levels. Do not expect levels of resistance to be the same among hives. Select 12 hives per apiary. More is better. This test is not designed to identify individual hives showing resistance. Use apiary averages to assess the results. 2. Perform the test exactly as described. Jar size, size of Apistan pieces and temperature are important. 3. Ensure that bees are mobile in the jars so they contact the strips. Cool temperatures may cause bees to cluster away from strips. If using a darkened incubator it may be helpful to open the incubator periodically to admit light and fresh air to encourage bee movement. 4. It is probably best not to reuse strip pieces or index cards. Wash jars between tests. Do not expose jars with Apistan pieces to sunlight for any length of time. It is best to keep the jars in their storage boxes before and after filling until they are incubated. Sample bees from brood frames. For accuracy and to avoid bee injury, use a measuring scoop. Do not scrape bees directly into jars.