Hygienic behavior is the measurable ability of a honey bee colony to find and remove sick, dead, or damaged brood quickly.
This colony-level performance links directly to lower disease pressure and greater resistance to Varroa mites, chalkbrood, and American foulbrood.
Two validated field assays are widely used: a freeze-killed brood insert and a liquid nitrogen field assay. Both use a defined patch of capped brood and a strict 95% removal threshold after a set time.
Repeat assessment matters. A colony must score above 95% on two consecutive runs to earn a hygienic classification and avoid day-to-day variability.
Because the trait is heritable, reliable results guide queen selection and can reduce reliance on chemical controls for mites and other diseases. Practical, consistent methods let U.S. beekeepers strengthen long-term colony health.
For a detailed review of methods and chemical cues linked to removed brood, see this research summary.
Key Takeaways
- Hygienic behavior means workers remove compromised brood quickly.
- Two assays—freeze-killed insert and LN2 method—use a 95% removal benchmark.
- Run tests twice; only consecutive >95% scores classify a colony as hygienic.
- Heritability lets beekeepers select queens for lasting resistance to varroa and diseases.
- Consistent timing and method are critical for accurate, actionable results.
Prepare your colony and gear for accurate, safe testing
A careful pre-check of queen timing, brood placement, and equipment prevents wasted effort. Confirm that the current queen has been laying for at least seven weeks or about six to eight weeks after requeening. Younger workers (1–3 weeks old) do most removal work, so timing affects results.
Choose a frame with a dense capped brood area. Aim for roughly 100 cells inside a two-inch ring or about 160 cells for a 3-inch cylinder. Avoid spots with many open cells; they skew counts and under-freeze test areas.
Assemble gear: a Dewar with liquid nitrogen, a polystyrene foam cup for measuring, two 2-inch pastry rings or a 3-inch PVC cylinder, cryogenic gloves, safety glasses, and a marker or pin. Dip ring edges in honey to seal the edge and reduce leakage before pouring liquid nitrogen.
- Mark the exact test location on the frame and record nest location.
- Wear protective gear and follow supplier MSDS guidance for handling nitrogen for worker and bee safety.
- Bring a mild bleach solution for sanitizing rings between colonies.
| Item | Purpose | Recommended | Notes |
|---|---|---|---|
| Dewar | Store liquid nitrogen | 5–10 L | Follow MSDS handling |
| Rings / PVC | Define test area | 2″ rings or 3″ cylinder | Dip in honey for seal |
| Foam cup | Measure LN2 | Polystyrene | One cup per session |
| PPE & markers | Protection & tracking | Gloves, glasses, pin | Sanitize between hives |
For more background on methods and readings, see this beekeeping resources and books for practical guides and references.
How to test for hygienic traits in colonies
Standardized field assays give reliable measures of how quickly workers remove dead brood from a defined brood area.
Method A: Freeze-killed brood insert
Cut a 2 × 2.5 inch comb section of sealed brood (about 100 cells per side) and freeze it for 24 hours at very low temperature. Insert the frozen section into a matching frame and place that frame centrally in the brood nest.
Return after 48 hours and count sealed cells remaining. A removal rate above 95% in 48 hours indicates strong hygienic behavior.
Method B: Liquid nitrogen field assay
Use two 2-inch pastry rings or a 3-inch PVC cylinder (≥4 inches long). For two rings, pour about 35 ml of liquid nitrogen to pre-cool, then add ~40 ml to complete the freeze. For a 3-inch cylinder, pour at least 10 oz of nitrogen to freeze ~160 cells.
Dip ring edges in honey, twist into the comb to the midrib, wait 3–5 minutes for thaw, then remove hardware and replace the frame in the brood nest.
Marking, placement, timing, and scoring
Mark the exact location with a pin or paint and note the time. Return at 24 or 48 hours (protocol dependent) and tally remaining sealed cells in the area.
Calculate the score as: (total cells − sealed remaining) ÷ total cells. Two 2-inch rings approximate 200 cells; if 8 sealed cells remain at 24 hours, then 192 ÷ 200 = 96%.
Repeat tests and control variables
Run tests twice under similar conditions. Only classify a colony as hygienic after two consecutive results ≥95%. Keep records of frame, location, and number of sealed cells for reliable comparisons.
- Handle the cup and pour liquid carefully; wear cryogenic gloves and eye protection.
- Avoid spots with many open cells; they skew counts.
- Use consistent timing and the same test frame for repeat assays.
Interpreting results, common pitfalls, and applying findings to breeding and mite resistance
Meaningful selection decisions rest on consistent removal rates, not single observations. Treat a ≥95% score as provisional until you confirm it on a second run. Two consecutive successes are the standard for labeling a colony as reliably hygienic.
Variation between runs is normal. Day-to-day differences arise from worker age, weather, and comb placement. If a colony slips below threshold, review your method before concluding the result reflects the colony’s true ability.
Common sources of error and what to check
- Under-freezing from too little liquid nitrogen or uneven freezing in the area.
- Leakage past rings because the edge was not sealed with honey.
- Counting outside the defined circle or using an area with many open cells.
- Not returning at the scheduled time, which skews results.
From assay to action: selecting queens and reducing treatments
Use repeated, validated tests when choosing breeders. Prioritize queens from colonies that clear dead brood consistently; this trait often correlates with varroa-sensitive hygiene and lower mites loads.
| Issue | Check | Action |
|---|---|---|
| Low repeat score | Frame had open brood or wrong area | Retest same frame or choose dense capped brood |
| Partial freezing | Insufficient LN2 volume or short contact time | Increase LN2 or extend freeze interval |
| Inflated sealed count | Ring leakage or miscounting outside ring | Seal ring with honey and recount defined cells |
| Conflicting signs | Debris shows mite damage or uncapped pupae | Use debris and behavior as supporting evidence for breeding |
Document the frame, comb location, pin marks, method, and exact times. Good records help you compare colonies across seasons and build a breeding program that increases varroa resistance and reduces chemical treatments.
Conclusion
Consistent execution of freeze protocols and accurate cell counts yields scores you can trust.
Keep counts tight by marking the exact frame area, recording the number of cells, and returning the marked spot the agreed hours later. Use the right liquid nitrogen volume for your ring or cylinder and protect against cold contact with proper safety gear.
Run a second test on the same test frame. Only two consecutive ≥95% removal scores qualify a colony for breeding moves. Careful records of brood location, queen timing, and results let you compare yards and seasons.
For practical guidance on testing hygienic behavior, methods, and thresholds, follow the linked resource. Rigorous testing supports breeding choices that lower mites and diseases while strengthening honey bees.
