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National Honey Bee Pests and Diseases Survey

Sampling Protocol

Overview

Detailed information about the National Honey Bee Pests and Diseases Survey, including the project 

plan, training videos, and all protocols, is located at the Animal and Plant Health Inspection Service 

(APHIS) Survey of Honey Bee Pests and Diseases webpage.

This document outlines the sampling protocol to collect adult bees and conduct Tropilaelaps spp. 

“bump” tests in eight randomly selected colonies in each participating apiary.

Please read this protocol carefully and watch the training video accessible using the link below prior 

to initiating sampling.

https://www.aphis.usda.gov/plant_health/plant_pest_info/honey_bees/downloads/National%20Bee%2

0Survey%20Video_small.wmv

For additional information, email Rachel Fahey (University of Maryland) at nhbs@umd.edu or Josie Ryan 

(United States Department of Agriculture Animal and Plant Health Inspection Service) at 

josie.k.ryan@usda.gov.
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List of Equipment Needed for Sampling/Shipping

Figure 1: Reusable equipment needed for sampling apiaries (provided by the participating state or 

territory). Also needed are basic beekeeping protective equipment (e.g. coveralls, veil, etc.) and 

beekeeping tools (e.g. smoker, hive tool, etc.), which are not provided.

For all apiaries (to be provided by your state 

unless otherwise noted; Fig 1)

 1 plastic wash tub

 1 metal pan

 1⁄4 cup measuring cup

 2 radiator funnels

 1 gallon jug (to be filled with water)

 1 squeeze bottle (to be filled with 

water)

 1 gallon of chlorine bleach (to be used 

for sterilizing equipment) (not pictured)

 1 gallon sized bucket

 mesh strainer

 binder clips

 scissors

 writing pen

 permanent marker

 1-inch wide transparent shipping tape

Per apiary (contained in your live bee sampling 

kit provided by the UMD; Fig 2)

 1 live bee shipping box

 1 pre-addressed mailing label

 1 data sheet

 Sample ID labels

 1 large (500 mL) bottle with alcohol

 1 small (125 mL) bottle with alcohol

 1 cloth filter 

 queen candy in petri dish on bottom of 

the live bee shipping box

 1 capped 15 mL tube filled with water 

and a sponge

 brochure for the beekeeper
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Procedure Summary

Before beginning, ensure all equipment in the “List of Equipment” is on hand as well as beekeeping 

protective gear and beekeeping tools. Remember to fill a one-gallon bottle and squeeze bottle with 

water. 

Identify an apiary with at least ten colonies of bees (eight of which will be sampled, with two extra in 

case an inspector encounters dead outs or queen-less colonies during inspection). Dead outs and queenless colonies should not be included in the survey sampling. Prioritize sampling from queen producers, 

package/nuc producers, honey producers, and apiaries used for crop pollination. Select apiaries in areas 

at high risk for exotics invasion (near deep water shipping ports, international airports, and high traffic 

areas for migratory beekeeping). Apiaries should be chosen to give as close to an equal representation 

of the entire state as possible. Ideally, a state will be sectioned into four quadrants with apiaries 

randomly chosen from each quadrant.

When sampling an apiary, it is critical to select colonies at random rather than haphazardly or regularly 

spaced. Colonies should not be preferentially selected because they seem “healthy” or “sickly”. One way 

to select colonies at random is to “assign” them numbers in ascending order. Use a random number 

generator app on your phone to select each colony you will sample. 

Identify the nearest US Post Office (USPS) or other shipping facility (and confirm their operating hours)

to this apiary so that you can mail the live sample of bees immediately. If you plan to mail several boxes, 

ensure that all boxes are ready to be mailed before the Post Office or shipping facility closes. 

a. It is best to ship live bees between Monday and Wednesday so they do not arrive at the

University of Maryland (UMD) Honey Bee Lab on the weekend when there is no one 

available to receive them. Please do not sample and ship live bees on Thursday or 

Friday.

b. It is legal to mail live bees by ground transportation through USPS. Please tape the box 

well and ensure the air vents are not covered. Western states may experience long 

ground transportation transit times (5 days or more). You may need to use another 

shipping company, such as United Parcel Service (UPS), to mail live bees. We will contact 

you if your bees are dead on arrival (unsuitable for molecular testing). If difficulty arises, 

there is an alternative solution available which requires additional training. Please 

contact Rachel Fahey for more information on alternative sampling methods 

(nhbs@umd.edu).

Important: Split up your sampling trips so you are not sampling 

all of your beekeepers within the same season. Take samples in 

the spring, summer, and fall. This provides a more 

comprehensive pest and disease baseline for your state.
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c. Go to the USPS webpage for Mailable Live Animals for more information on the postal 

service regulations.

Once you have identified the apiaries that you will sample, explain the sampling process to the 

beekeeper. Show the beekeeper the brochure and answer any questions they may have. 

Set-up

Figure 2(A and B): A live bee shipping box which contains all the materials provided by UMD to collect 

and ship samples in one apiary.

1. Open one live bee mailing box (Fig 2), it should contain:

a. Data sheet.

b. Sample ID labels.

c. Large bottle and small bottle containing ethanol.

d. Petri dish glued to box floor containing queen candy.

e. Capped 15 mL tube filled with water and a wicking sponge.

f. Cloth filter.

g. Mailing label.

2. Place sample identifying labels onto (Fig 3):

a. Data sheet.

b. Large collection bottle containing ethanol.

c. Small collection bottle containing ethanol.

d. Live bee shipping box.
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Figure 3: Place identification label onto data sheet, large bottle, small bottle, and mailing box.

3. Fill out the data sheet. An electronic version of the datasheet is available at the APHIS Honey 

Bee webpage. Please scan and email the completed data sheet to nhbs@umd.edu. 

4. Set up sampling equipment:

a. Remove and discard the plastic top of petri dish and the wax paper covering the queen 

candy (Fig 4).

b. Remove and discard the cap from the 15 mL tube containing the wet sponge to allow 

bees access to the water source. Place tube in live bee box (Fig 5).

c. Close the shipping box, making sure the two sides with metal screens match up to 

ensure proper ventilation for live bees, and place a funnel into the hole on the top of 

the box (Fig 6A). 

d. BE SURE TO KEEP THE LIVE SHIPPING BOX IN THE SHADE AND OUT OF DIRECT 

SUNLIGHT AND WIND, otherwise the sampled bees may overheat and die.

e. Open the large alcohol bottle and place a second funnel into its mouth so it can receive 

bees (Fig 6B). It may be helpful to place the alcohol bottle in the center of a roll of duct 

tape to stabilize the bottle.
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Figure 4: To ensure queen candy does not dry out before use, it has been sealed with wax paper in the 

petri dish. Remove and discard the petri dish cover and the wax paper before placing bees into the live 

shipping box. 

Figure 5(A and B): Bees require water during transport, so the test tube with the sponge and water 

should be UNCAPPED before placing bees into the live shipping box.

Figure 6A: Live bee shipping box with funnel inserted to receive bees. Figure 6B: Large bottle with funnel 

ready to receive bees.
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Live Bee Sample

5. Open the colony to the brood nest and examine for disease and queen status/condition. Record 

any disease/queen conditions on the data sheet.

6. Find a frame containing at least some uncapped brood (Fig 7). Ensure that the queen is not 

present on this frame. If no brood is present, and another colony is available, do not sample the 

brood-less colony. If no other colony is available, take an adult bee sample from a frame in the 

center of the brood nest.

Figure 7: A frame containing sufficient adult bees, uncapped and capped brood for sampling.

7. Shake bees from the frame into the wash tub (Fig 8).

8. Knock the wash tub to gather bees into one corner of the tub (Fig 9).

Important: The goal is to sample at the apiary level. You will be 

collecting two scoops of live bees from each of the eight 

colonies. You will place one scoop in the live bee box and the 

other scoop in the large bottle of alcohol. You will also “knock” a 

single brood frame from each colony over the large metal tray 

and then wash the resulting debris into the smaller bottle of 

alcohol. It is critical to execute the “knocking” procedure 

effectively because this debris sample is used to screen for the 

invasive mite Tropilaelaps. Samples from all colonies are being 

placed in the same live shipping box and large alcohol collection 

box, and debris from 8 frames from 8 different colonies are 

being knocked into the same metal tray.
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Figure 8: Shake bees from frame into wash tub. Figure 9: Knock bees into corner of tub to facilitate easy 

removal of 1⁄4 cup samples of live adult bees.

Figure 10: A brimming 1⁄4 cup of bees.

9. Scoop 1⁄4 cup of adult bees (Fig 10) into the funnel inserted into the lid of the live bee shipping 

box (Fig 11). Gently tap the box to force the bees from the funnel into the box (Fig 12). Do not 

collect more than 1⁄4 cup of bees because overcrowding will result in mortality during shipping.

a. BE SURE TO KEEP THE LIVE SHIPPING BOX IN THE SHADE AND OUT OF DIRECT 

SUNLIGHT AND WIND. 
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Figure 11: Place bees from cup into funnel inserted in the live bee shipping box. Figure 12: Gently tap live 

bee shipping box and funnel so bees are forced into the collection box.

Alcohol Bee Sample

10. Scoop another 1⁄4 cup of bees from the tub into the funnel inserted in the large bottle containing 

alcohol; tap the bottle and funnel to force bees into the bottle (Fig 13).

Figure 13: Place a second 1⁄4 cup of bees into large bottle of alcohol.

Pro Tip: It may be helpful to put the lid from the 500 mL alcohol 

bottle over the opening of the funnel to reduce the risk of live 

bees escaping. This may also help you keep track of the lid.
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Tropilaelaps Sample

11. Examine the frame of brood from which the bees were removed in Step 8 and look for any mites 

that could be scurrying across the surface. Tropilaelaps mites move quickly in comparison to 

Varroa and are 1/3rd of their size. If you observe this, please take photos, make note of it on the 

Notes section at the bottom of the data sheet and contact UMD immediately.

12. Hold the frame of brood over the metal collection pan with one surface of the frame facing 

down (Fig 14). Firmly knock the outer edge of the frame in the pan to dislodge mites, beetles 

and other hive debris from the frame into the collection pan (Fig 15). Furthermore, please 

inspect the debris in the pan as you bump each frame. If there is anything out of the ordinary 

(mites that should not be there) and if you view what is a suspected Tropilaelaps mite, please 

photograph it, collect the suspect mite, and contact us immediately.

Figure 14: Firmly knock the frame over the metal collection pan by allowing one end bar to hit the metal 

collection pan. Figure 15: Debris dislodged from frames after being knocked in collection pan.

13. Knock the frame a second time.

14. Flip the frame so that the frame surface facing down in Step 13 is now facing up.

15. Knock the frame twice in the same manner outlined in Steps 13 and 14.

16. Switch the frame so you are now holding the opposite end of the frame and repeat Steps 13-16. 

Each frame should be flipped 2 times, so that the frame is knocked a total of eight times (two

knocks, flip over, two knocks, switch the frame end to end, two knocks, flip over, two knocks).

17. Place the brood frame back into the colony and move any bees remaining in the collection wash 

tub back to the colony.

18. Close the colony.

19. Repeat Steps 5 through 19 until eight colonies have been sampled.
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Sample Preparation

Figure 16: Close the large sampling bottle containing 2 cups of bees (1/4 cup from eight different 

colonies), tightly seal the lid of the container, and tip the bottle several times to ensure all bees are damp 

with alcohol.

20. Close the large bottle containing alcohol and tip the bottle several times, dampening the bees

inside (Fig 16).

21. Attach the cloth filter into the strainer using binder clips (Fig 17).

22. Place about two cups of water into the metal collection pan and gently swirl the contents (Fig 

18) before pouring the water and debris through the cloth filter (Fig 19). Be sure to not let any 

water flow over the edge of the cloth filter.

Figure 17: Secure cloth filter to the strainer using binder clips. 

Figure 18: Place water into metal collection pan to dislodge debris.

Figure 19: Pour water and debris from metal collection pan through the cloth filter. Inspect the debris as 

you rinse for any signs of Tropilaelaps mites.
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Figure 20: Using squeeze bottle, ensure last of debris in metal collection pan is filtered through cloth 

filter.

Figure 21: After allowing filter to drip dry for a few minutes, gently fold filter so debris is secure.

Figure 22: Place folded filter into small bottle with alcohol. Secure the bottle lids.

23. Repeat Step 23 until most of the debris in the collection pan has been removed, then using the 

squeeze bottle remove what debris remains and pass this through the cloth filter (Fig 20). Again, 

please inspect the debris as you wash it into the filter to determine if there is anything out of 

the ordinary. If you view what is a suspected Tropilaelaps mite, please photograph it, collect 

the suspect specimen, and contact us immediately.

24. Allow the cloth filter to drip dry for a couple of minutes, and then gently remove it from the 

strainer, folding it so that the filtered debris remains secure in the center of the filter (Fig 21). 

This debris will be examined for mites, specifically Tropilaelaps, upon arrival at the University of 

Maryland Honey Bee Lab.

25. Place the entire gently folded cloth filter into the small bottle containing alcohol and seal the 

bottle tightly to avoid spills during transport (Fig 22). Please make sure all of the filter material is 

in the main part of the bottle and none is on the neck of the bottle. Save the binder clips to be 

reused at subsequent apiaries.
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Shipping Samples

26. Double check the lids of the small and large collections to make sure they are tight. Place both 

the small (containing the cloth filter) and large bottle (containing bees) into large Ziplock bags to 

contain any leaks from the alcohol before placing them into the large Priority Mail flat rate box. 

The samples should be mailed as soon as possible so they can be examined for invasive threats 

to U.S. honey bees. Prior to mailing, place one of the pre-addressed shipping labels (UMD Honey 

Bee Lab, 4291 Fieldhouse Drive, Plant Sciences Building Room 4112, College Park, MD 20742) 

at the appropriate place on the box. Write “FROM” and your return address on the upper left 

corner of the box.

Figure 23: After ensuring the data sheet has been filled out completely, and after making a copy of this 

sheet for your records, place it in its own Ziplock bag and place this Ziplock in the prepaid mailing box 

containing the sample bottles. (Note: Do not put the data sheets in the same Ziplock bag as the sample 

bottles, as the bottles may leak and make the writing illegible.) When ready to ship samples, double 

check all lids are securely fastened, put the sample bottles in Ziplock bags and add packing material (e.g., 

newspaper), if necessary, so bottles are not damaged during shipment. Add sealed Ziplock bag with data 

sheets and seal box with binding tape. 

27. Ensure the data sheet is completely filled out and legible. Please make a scan (there are many 

free phone apps that use a phone camera and can produce a pdf scan) of this sheet for your 

records and email it to nhbs@umd.edu, and then place it into a Ziplock bag (with any other data 

sheets you have already filled out). The Ziplock bag containing the data sheets should be placed 

in the mailing box containing sample bottles (Fig 23). 

Pro tip: Cut a piece of transparent shipping tape and have it 

ready to cover the hole in the live bee box BEFORE you take the 

funnel out. Bees will try to fly out as soon as the hole is opened.
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28. Remove the funnel from the live bee shipping container and cover the hole with the cardboard 

flap (Fig 24).

29. Secure the hole and the lid of the live bee shipping box to the bottom of the box with the 

transparent shipping tape (Fig 25).

30. Place the mailing label (UMD Honey Bee Lab, College Park, MD) over the now sealed hole on the 

top of the live bee shipping box and secure it with tape (Fig 26). Write “FROM” and your return 

address on the upper left corner of the box. 

Note: Please ensure that the sample label is clearly visible on the live bee box and is not 

covered by tape or postage.

Figure 24: Secure the hole in the lid of the live bee shipping box with clear binding tape.

Figure 25: Secure the lid of the live bee shipping box to the bottom of the box using the clear tape. 

Figure 26: Place pre-addressed shipping label (UMD Honey Bee Lab, College Park, MD) on the box. 

Clearly print "FROM" and your return address on the top left corner of the box. Make sure the 

identification number label is legible and securely attached to box. Mail the live bee shipping box, 

containing collected live bees, at the nearest Post Office ASAP.

31. Mail the live bee shipping box, containing collected samples, at the nearest Post Office or 

other shipping facility ASAP.

32. Collect and clean sampling equipment (pans, hive tools, funnels, and 1⁄4 cup scoop) with 10% 

bleach and water solution. You may also sanitize hive tools by scorching them in your smoker.

33. Email the UMD Survey Coordinator (nhbs@umd.edu) within 24 hours of shipment to notify the 

UMD lab that a live bee shipment is expected. Please also email Rachel Fahey within 24 hours 

after shipping alcohol and filtered samples.
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Post Sampling Checklist

 Scan data sheets and email them to the UMD Survey Coordinator (nhbs@umd.edu)

 Put paper copies of data sheets into a Ziploc bag. Please do not put them in the Ziploc bag with 

the ethanol preserved samples as the samples may leak while in shipment and destroy the 

writing on the sheets.

 Take live bee sample to a United States Post Office or other shipping facility within 24 hours and 

mail it to the UMD Lab. Email UMD Survey Coordinator (nhbs@umd.edu) to notify UMD that a 

live sample is expected. 

 If you are not planning on shipping the ethanol preserved samples right away, store them in a 

refrigerator until your sampling trip has concluded. Please do not hold them for an extended 

time period because it delays the lab’s examination for invasive threats to honey bees. 

 When ready to ship samples, double check all lids are securely fastened, put the sample bottles 

in Ziplock bags and add packing material (e.g., newspaper), if necessary, so bottles are not 

damaged during shipment. Add sealed Ziplock bag with data sheets and seal box with binding 

tape. Again, notify nhbs@umd.edu when you are mailing samples to the lab.

Important: We request that all Tropilaelaps frame bump 

samples be sent in as soon as possible for us to perform our 

screening diagnostic. If you are sampling multiple apiaries over 

the course of a week, sending all the Tropilaelaps samples 

together would be optimal.