The primary purpose of using a 75% ethanol solution in Nosema field surveys is to act as an immediate fixative and preservative. Upon collection, the solution kills the honeybee specimen and halts its physiological processes. This stabilizes the sample, preventing biological decay during the time between the apiary visit and laboratory analysis.
By effectively hitting "pause" on biological degradation, 75% ethanol ensures that Nosema spores remain structurally intact for weeks. This allows researchers to transport samples over long distances without refrigeration while maintaining the accuracy of subsequent microscopic or molecular diagnostics.
The Mechanics of Sample Preservation
Immediate Physiological Fixation
When honeybees are collected for a survey, the internal environment of the bee begins to change immediately upon death. 75% ethanol fixes the physiological state of the honeybee at the exact moment of collection.
This rapid fixation is critical because it stops metabolic processes that could otherwise alter the sample. It ensures the specimen analyzed in the lab is biologically identical to the bee in the field.
Inhibiting Bacterial Growth
Without a preservative, a dead honeybee is susceptible to rapid decomposition. The ethanol solution inhibits bacterial growth and prevents rotting.
This is essential because bacterial overgrowth can obscure microscopic views or degrade the specific DNA markers needed for molecular detection.
Ensuring Data Integrity During Transport
Protecting Spore Structure
The accuracy of a Nosema survey relies entirely on the ability to count or detect spores. Ethanol ensures that Nosema spores remain structurally intact.
If the spores were to degrade or break down due to poor preservation, laboratory counts would be artificially low, leading to a false diagnosis of colony health.
Extending the Analysis Window
Field surveys often take place in remote locations far from diagnostic facilities. The use of ethanol allows the samples to remain stable for several weeks during transport.
This flexibility removes the immediate need for cold chain logistics (like dry ice or portable freezers) right at the collection site.
Operational Considerations
The Importance of Concentration
It is vital to maintain the solution at the 75% concentration level.
A lower concentration may fail to inhibit bacterial growth effectively, leading to sample degradation. Conversely, while higher concentrations exist, the 75% standard is specifically optimized to balance penetration of tissues with preservation of the spores.
Container Integrity
While ethanol is effective, it is volatile. As noted in broader sampling protocols (such as those for Varroa mites), using sealed glass jars is recommended to maintain chemical stability.
If the container is not airtight, the ethanol may evaporate or leak, rendering the preservation ineffective and leading to sample loss.
Ensuring Reliable Laboratory Results
To ensure your Nosema field survey yields accurate actionable data, follow these guidelines based on your logistical needs:
- If your primary focus is Sample Integrity: Ensure the bees are fully submerged in the 75% ethanol solution immediately upon collection to prevent any initial decomposition.
- If your primary focus is Transport Logistics: Use high-quality, sealed containers to prevent leakage or evaporation of the ethanol during the weeks of transit.
- If your primary focus is Diagnostic Accuracy: Rely on this method for both microscopic counting and molecular detection, as the ethanol preserves the necessary biological structures for both.
Proper chemical preservation is the single most important step in translating field conditions into accurate laboratory data.
Summary Table:
| Feature | Function in Nosema Surveys | Benefit to Researchers |
|---|---|---|
| Immediate Fixation | Halts metabolic and physiological processes | Ensures specimen matches field condition |
| Bacterial Inhibition | Prevents decomposition and rotting | Maintains clear microscopic/DNA markers |
| Spore Preservation | Keeps spore structures intact | Prevents false-negative or low counts |
| Stability | Allows for weeks of storage without refrigeration | Simplifies transport from remote apiaries |
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References
- Shane S. Klassen, Ernesto Guzmán‐Novoa. Nosema ceranae Infections in Honey Bees (Apis mellifera) Treated with Pre/Probiotics and Impacts on Colonies in the Field. DOI: 10.3390/vetsci8060107
This article is also based on technical information from HonestBee Knowledge Base .
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