ERK/DTK Live Bee & Salt Solution Sampling Protocol

Project Plan 

This sampling project is based on the sampling procedures for the National Honey Bee Disease survey.  While the sampling procedure is largely the same it does differ in some important ways. Please download and watch the training video prior to initiating sampling. BE SURE TO READ THESE INSTRUCTIONS CAREFULLY BECAUSE THERE ARE SOME IMPORTANT DIFFERENCES BETWEEN THE NATIONAL HONEY BEE DISEASE SURVEY AND THIS ONE. If you have any questions or need additional information, email, 301-405-3799.

Emergency Response Kits and Diagnostic Testing Kits are designed for sampling 16 colonies total, 8 from healthy colonies and 8 from weak or crashing colonies. You should not expect results to be a definitive diagnosis as to the causes of honey bee mortality in your operation from the processing of these kits. Rather, we should be able to rule out factors that have not contributed to your losses.


  • large Priority Mail flat rate boxes (to mail salt water samples)
  • postage for above boxes (standard postage rate is $16.85 per box)
  • transparent shipping tape
  • writing pen
  • marker or sharpie
  • 1 pair of scissors

PROVIDED ITEMS (See Figure 1):

  • 2 live bee shipping boxes
  • 3 pre-addressed mailing labels for live bee shipping boxes, pollen (if sampling) & concentrated salt water solution samples
  • 2 data sheets
  • 16 small (125 mL) bottles with concentrated salt water solution
  • queen candy in petri dish on bottom of the live bee shipping box (food supply)
  • 2 capped 15 mL tube filled with water and a sponge in live bee box (water supply)
  • zip lock bags (place sample bottles and data sheets in bags prior to shipping)
  • 1 funnel
  • ¼ cup measuring cup
Fig 1

Figure 1: Sampling Kit

Overview: The goal is to collect 2 samples of adult bees, 1 sample from 8 strong colonies in each apiary and 1 sample from 8 weak or crashing colonies. You will need to open 16 colonies and remove 1 frame that contains young, developing brood (if available) and shake the adult bees into the box used to the emergency response kit. Scoop the bees into the measuring cup. You will collect three, ¼ cup scoops of bees from each colony.  Two, ¼ cup scoops of bees will go into salt water bottle and then sealed for an individual salt water sample, and the third, ¼ cup scoop of bees will be placed in the live bee box making a combined live bee sample from 8 colonies. This procedure will be repeated a second time for the 8 unhealthy hives. You will leave the apiary with 2 combined samples of live bees (one box containing the assumed healthy colonies and one box containing the assumed sick colonies), 16 samples of bees in salt water solution in the small salt water bottles. You will want to provide each colony with an identification to label the hives that you sampled. Please also label each salt water solution bottle to match your colony ID and include on that label whether the colony was ‘healthy’ or ‘crashing’.


Please note it is best to ship live bees between Monday and Wednesday so they do not arrive at the Bee Research Lab in Maryland on the weekend when there is no one available to receive them. Please do not sample and ship live bees on Thursday.


  1. Open box and remove wax paper covering queen candy (in petri dish glued to floor of box). See Figure 2.
  2. Remove cap from 15 mL tube containing the wet sponge to allow the bees access to a water source (Figure 3). Place tube back in the live bee box (Figure 4) and secure it with tape to the inside of the box. Close the box making sure the two sides of the box with metal screens match up to ensure proper ventilation

    Fig 2 and 3 and 4

    Figure 2: Preparing Fondant and Food

  3. Be sure to keep the live shipping box in the shade and out of direct sunlight and wind while collecting remaining samples.
  4. Open colony to the brood nest and examine for disease and queen status/condition. Record any disease/queen status, or unusual conditions present on the data information sheet. Remember to record only healthy colony data on the same sheet and crashing colony data on the second sheet.
  5. Find a frame containing uncapped brood. If uncapped brood is not available, find a frame from the center of the cluster.
  6. Shake bees from frame into the original shipping box and scoop two, ¼ cup of adult bees (about 300) (Figure 5) into the funnel inserted into the lid of the live bee shipping box. In order to prevent bees from escaping during samping between hives cover the hole of the funnel. You can set the saltwater cap over the hole in the funnel if necessary.
  7. Scoop two more ¼ cups of bees from the box into the funnel inserted in the small bottle containing salt water, tap the bottle and funnel to force bees into the bottle for the individual salt water sample. This amount of bees should fill the bottle halfway. Close the bottle tightly, shaking it to make sure bees are fully dampened with salt water and place to the side.Fig 6Fig 5
  8. Repeat steps 3 through 6 until 8 strong colonies have been sampled. Then begin at step 1 with second provided live bee box for weak colonies until a total of 16, 8 strong and 8 weak colonies have been sampled.
    1. Note we are sampling at the apiary level for the live bee sample, so samples from first 8 strong colonies are being placed in the same live shipping box and 8 samples for weak colonies will be placed in a second, separate box. You will be collecting 3 scoops of live bees from each hive and placing them in either the live bee shipping box or the individual salt water bottles.
    2. You will fill out 2 data sheets. One for the healthy colonies and a second one for the weak or crashing colonies.
  9. Place the small salt water bottles (containing bees) into large ziploc bags to contain any leaks from the salt water before placing them into the large flat rate shipping box. Please make sure lids are tightly sealed to prevent leakage. Group healthy samples in separate bags than weak or crashing samples, labeling bags with either ‘healthy’ or ‘weak’ and label each sample with your name, date and hive number with a marker or sharpie before placing in return shipping box. Please also include a listing of the hives sampled on a separate sheet of paper with pencil in the event of bottle leakage during shipping. Package should be shipped ground. (Ex. Jane Bee, 10/15/12, #346)
  10. Ensure both data collection sheets are completely filled out and legible and place in return shipping box.
  11. Place the mailing label (Attn: Heather Eversole – University of Maryland Honey Bee Lab, 4291 Fieldhouse Drive, Plant Sciences Building Rm. 4112, College Park, MD 20742) over the now sealed hole on the top of live bee shipping box and secure it with tape. Write FROM and your return address on the upper left corner of the box. Write the date on the front of the box. Please also write healthy colonies or weak/crashing colonies on the outside of the box.
  12. Email: Research Assistant Heather Eversole ( within 24 hours of shipment or call 301-405-3799 to notify personnel that a live bee shipment is expected. Please also email Heather within 24 hours after shipping salt water and filtered samples to notify the Bee Research Lab that a shipment is to be expected.

This sampling protocol is based on the USDA AHPIS National Honey Bee Survey. For additional information on this effort please visit

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