Endospores, also known as spores, are mainly derived from microsporidium parasitizing in the epithelial cells of the digestive tract of insects. Typical examples include microsporidia in the intestinal tract of bees and microsporidia in the silkworm. Microsporidiosis infection spreads quickly, and it is prone to population weakness or even death. Therefore, it is necessary to detect endospores in the insect intestines to achieve the prevention and control of microsporidiosis and determine the best treatment period.

At present, the laboratory can only count the spores in the intestines of bees through a microscope. The process is time consuming and laborious, and the diameter of the spores in the intestines of bees is small, only 2~3μm, and the results of microscopy between different personnel have large deviations. When the concentration is too high, it cannot be directly checked by microscopy. The customized version of JIMBIO CL cell counter can replace microscopy, and the detection concentration range is 1e5~5e6, which can improve counting efficiency and give accurate and objective counting results.

material:

Bees infected with microsporidiosis, PBS (1X)

Purification and preparation of spore suspension:

1. Take the abdomen of the bee and grind it in a mortar with distilled water and grinding powder

2. After grinding evenly, transfer to a 10ml centrifuge tube and centrifuge at 500rpm for 3min at low speed

3. Transfer the supernatant to a new 10ml centrifuge tube, discard the precipitate, and centrifuge at 2000rpm for 10min at high speed

4. Discard the supernatant, resuspend the pellet in clean water, and centrifuge at 4000 rpm for 10 minutes

5. Take the precipitate, dissolve the precipitate with 1ml PBS, store at 4°C for later use

Microscope inspection image:

Figure 1 Picture of endospores in bee intestine under 10x objective lens

Figure 2 A picture of the endospores in the intestine of a bee under a 20x objective lens

Results and analysis:

It can be seen from Figure 1 and Figure 2 that when the intestinal spores of bees are relatively small and the concentration is high, the samples are spread very densely on the hemocytometer and it is difficult to count under the microscope.

As can be seen from Table 1, the test results of the customized version of JIMBIO-CL are basically the same as the counting board statistics. Since the customized version of JIMBIO-CL counts 10 microliters of samples, the test results have less fluctuation and obvious stability. Better than imported hemocytometer.

Precautions:

1. Only use PBS (1X) to dilute the bee spore samples to be tested;

2. The sample to be tested must be thoroughly mixed before sample addition (different specifications of liquid guns can be selected for pipetting according to the total amount of samples);

3. To ensure the stability of the test results, the total amount of samples to be tested should not be less than 100 microliters.

Thanks:

Thanks to Guo Rui's research group and Huang Weifeng's research group from the Bee College of Fujian Agriculture and Forestry University for providing samples and jointly completing application development.