Method for transmission electron microscope observation of phytoseiulus persimilis reproductive system

A technology of Phytoseiid mite and reproductive system in Chile, which is applied in material analysis using radiation, material analysis using wave/particle radiation, sampling, etc. It can solve the problems of broken body, inability to accurately locate the reproductive system, full of body fluids, etc.

Inactive Publication Date: 2018-02-02

SAFE BEST & NECESSARY BEIJING BIOTECH CO LTD

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AI-Extracted Technical Summary

Problems solved by technology

[0004] However, due to the small size and fragility of Phytoseius militaris, the body is filled with opaque body fluids, it is impossible to accurately locate its reproductive system, and a little carelessness will lead to fragmentation of the worm body. There is...

Abstract

The invention relates to a method for transmission electron microscope observation of a phytoseiulus persimilis reproductive system. The method is characterized in that the phytoseiulus persimilis reproductive system is obtained prior to preparation of a transmission sample. The method specifically includes steps: acquiring male and female phytoseiulus persimilis in mating, and performing lactic acid soaking, dehydrating and drying treatment to finally obtain the phytoseiulus persimilis reproductive system; preparing the transmission sample of the reproductive system by seven steps including fixing, dehydrating, embedding, curing, slicing, dyeing and observing in sequence. By adoption of the simple and ingenious method, the phytoseiulus persimilis reproductive system is obtained for the first time, transmission electron microscope observation of an ultrastructure of the reproductive system is realized for the first time, and acquisition of the reproductive system provides raw materialsfor transmission electron microscope observation of the structure of the reproductive system, and transmission electron microscope observation results are beneficial to researching of reproductive mechanisms.

Application Domain

Preparing sample for investigationMaterial analysis by transmitting radiation

Technology Topic

UltrastructureObservation method +6

Examples

Experimental program(1)

Example Embodiment

[0022] Example Transmission electron microscope observation method of the reproductive system of Phytoseius chilei

[0023] The present invention is a method for first obtaining the reproductive system of Chilean phytoseiid mite, and then preparing its transmission sample. The specific operation steps are:

[0024] (1) Acquisition of the reproductive system of the Chilean plant seiid: firstly obtain the female and male mites of the Chilean plant seiid mites in mating, and then through lactic acid soaking, dehydration, and drying treatment, and finally obtain the reproductive system of the Chilean plant seiid mites. The operation is:

[0025] 1) Acquisition of female and male mites during mating: Under the microscope, pick out eggs from the Chilean plant seiid mites breeding population and raise them individually to adult mites, then pair the male and female mites. According to different needs, the male and female individuals in the mating process at different times Put it together with the breeding device in an ultra-low temperature refrigerator (-80℃) or liquid nitrogen to obtain female and male mites at different mating periods;

[0026] 2) Lactic acid soaking: After taking out the female and male mites of Phytoseiia chilei in mating, place the male and female mites on the slide with a fine brush, and then separate the male and female individuals with a 0-size insect needle and a fine brush, and finally pick the male and female mites separately Put it into a finger tube filled with lactic acid and leave it at room temperature for 3-4 days;

[0027] 3) Dehydration: Take out the female mites or male mites from the finger tube or put the two together in 70% alcohol, and then perform gradient dehydration. The alcohol concentration is 80%, 90%, 100%, and the specific operation is First use a plastic dropper to suck out the upper layer of alcohol, and then add the next gradient of alcohol, and repeat until 100% alcohol;

[0028] 4) Drying: Place the samples of female mites and male mites on the sample table, and dry them with a vacuum dryer for 2 to 3 hours;

[0029] 5) Reproductive system acquisition: The dried insect body can be clearly seen under the stereo microscope that the surrounding part of the sample of the female and male mites of Chilean phytoseiid mite is transparent, leaving only an orange part in the middle This is the reproductive system of Chilean Phytoseiid mite. Finally, under a microscope, the back plate of the mite body is gently removed with a No. 0 insect needle, and the orange part is taken out to successfully isolate the reproductive system of Chilean Phytoseiid mite;

[0030] (2) For the preparation of transmission samples of the reproductive system, the sequence of operations includes 7 steps: fixation, dehydration, embedding, curing, sectioning, staining, and observation, specifically:

[0031] 1) Fixation: Put the removed reproductive system into a fixative with a concentration of 3.5% glutaraldehyde. To make the fixation effect better, add Tween 100 (1L:600uL) and sodium chloride to glutaraldehyde (1L:0.9g), fix for 48 hours, then rinse with phosphate buffer (10 minutes/time) with a concentration of 0.1 mol/l and pH 7.2 for 3 hours, then fix with osmium acid for 2 hours, and finally use Wash phosphate buffer solution 3-4 times;

[0032] 2) Dehydration: After washing, the reproductive system needs to be dehydrated in gradients, which are respectively placed in 30%, 50%, 70%, 80%, 90%, and 100% alcohol for dehydration. Each gradient is dehydrated for 5 minutes, and then used Acetone dehydration for 15 minutes, 4-5 times;

[0033] 3) Embedding: Leave a little acetone solution, add resin solution, fully mix the sample and resin, soak for 12 hours, and then put the reproductive system neatly into the resin mold and straighten out the air bubbles in the resin;

[0034] 4) Curing: After removing the bubbles, put the mold in a 35 degree oven and bake for 2-3 weeks;

[0035] 5) Slicing: Reuse the ultra-thin microtome to slice 50-60nm;

[0036] 6) Dyeing: dye with uranyl acetate and lead citrate for about 1 hour;

[0037] 7) Observation: Observe and photograph the slices under a transmission electron microscope.

PUM

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