How to Count Nosema

Summary

This training from Washington State University's Honeybees and Pollinators Program provides a comprehensive guide on how to quantify Nosema Apis (NOA) infection in honeybees. It covers the necessary tools, including a compound microscope and disposable hemocytometer, and demonstrates how to prepare and crush bee samples for spore counting. The video compares counting NOA spores in whole bees versus just the abdomens, highlighting the advantages of each method. Detailed steps and tips for using a clicker counter and microscope adjustments are also shared to ensure accurate results, with a clear example showing how to calculate spore density.

Takeaways

• 😀 A compound microscope is necessary to quantify NOA (Nosema Apis) infection in honeybees.
• 😀 The sample of bees should be preserved in alcohol before processing.
• 😀 Whole bees can be crushed in a Ziploc bag with a rolling pin, or the abdomen can be separated for a cleaner sample.
• 😀 A clicker counter is recommended to track how many bees or abdomens are used in the sample.
• 😀 In the training, 50 whole bees or 50 abdomens are used as the sample for quantifying NOA.
• 😀 After preparing the bees or abdomens, add 1 mL of water per bee or abdomen for the crushing process.
• 😀 A disposable hemocytometer is used to load and analyze the sample under the microscope.
• 😀 The hemocytometer is placed under a compound microscope at 4X magnification to locate the sample and then adjusted to 100X or 400X magnification for better clarity.
• 😀 The goal is to count the number of NOA spores in five specific squares of the hemocytometer grid, including the four corners and the center square.
• 😀 The spores are counted using a serpentine pattern in the grid squares to avoid counting the same spores multiple times.
• 😀 NOA spores are very small, oval-shaped, with smooth edges, resembling Tic Tac candies under the microscope.
• 😀 The number of spores is calculated by multiplying the total number counted in the five squares by 50,000 to estimate the total spores per bee.
• 😀 When comparing whole bee samples to abdomen-only samples, the whole bee sample tends to have more debris, making it harder to identify NOA spores.

Q & A

What equipment is necessary to quantify NOA infection in honeybees?

To quantify NOA infection, you will need a compound microscope, a sample of bees preserved in alcohol, forceps, scissors, a rolling pin, a Ziploc bag, a graduated cylinder, a hemocytometer, and a clicker counter.

What is the difference between using whole bees and abdomens only for quantifying NOA?

The script demonstrates a direct comparison between using whole bees versus just the abdomens. Whole bees contain more debris (e.g., wings, legs, antennae) which can make it harder to detect NOA spores, while using only the abdomens might give a cleaner sample for analysis.

How do you prepare the bee sample for NOA quantification?

The bee sample can be prepared by crushing whole bees in a Ziploc bag with a rolling pin, or by detaching the abdomens using forceps and scissors. You should collect 50 bees or 50 abdomens and add 1 mL of water per bee or abdomen before sealing the bag tightly.

What is the purpose of using a hemocytometer in the NOA quantification process?

A hemocytometer is used to measure the number of NOA spores in the sample. The hemocytometer allows for precise counting of spores in a defined grid area under the microscope.

At what magnification should the hemocytometer be viewed when counting NOA spores?

You should first use 4X magnification to locate the hemocytometer, then adjust to 100X magnification for a clearer view, and finally use 400X magnification for counting the NOA spores.

How should you count the NOA spores in the hemocytometer?

You should count the spores in five of the smaller squares of the grid: the four corner squares and the one in the middle. Use a serpentine pattern to scan each square from right to left to ensure accuracy and consistency in your count.

How can you calculate the number of NOA spores per bee?

To calculate the number of spores per bee, multiply the total number of spores counted in the five squares by 50,000. For example, if you counted 31 spores, the calculation would be 31 × 50,000, resulting in approximately 1.5 million spores per bee.

What role does the clicker counter play in the quantification process?

The clicker counter helps keep track of the number of spores counted as you go through the grid, ensuring accuracy and making it easier to monitor your progress.

What are some challenges when using whole bees for NOA quantification?

When using whole bees, the field of view under the microscope contains more debris such as wings, legs, and antennae, which can make it difficult to focus on and count the NOA spores accurately.

Why is it important to focus carefully on the hemocytometer when using the microscope?

Proper focus is essential to ensure you are counting the spores accurately. A blurry field of view can lead to missed spores and inaccurate results. Use the fine focus knob to achieve a sharp, clear image at the highest magnification.