CN104777025A - Method for preparing scanning electron microscope sample of silage corn leaf - Google Patents

Abstract

The invention discloses a method for preparing a scanning electron microscope sample of a silage corn leaf. According to the method, the prepared scanning electron microscope sample is made to be complete in structure through the processes of washing, dehydration, de-ethanol, freezing, drying, gold plating and the like, when scanning electron microscope observation is carried out, the surface structure of the leaf is clear and complete, the stereoscopic impression of an image is strong, observation and research for the ultra microstructure of the silage corn leaf after ruminal degradation of livestock are convenient to carry out, and the method has important significance for visually knowing and evaluating the degradation of rumen microbe of livestock to plant leaf fiber structures and improving the digestibility of straw fodder.

Description

A method for preparing scanning electron microscope samples of silage corn leaves

technical field

The invention relates to a method for preparing scanning electron microscope samples, in particular to a method for preparing livestock rumen-degraded silage corn leaf scanning electron microscope samples, and belongs to the technical field of microscopic observation samples.

Background technique

As a non-competitive resource, crop straw is a non-competitive resource. The annual output of crop straw in my country is nearly 600 million tons to 700 million tons. Most of the straw resources are not effectively utilized. Due to the rough stems and high crude fiber content of straw, it is difficult for animals to digest utilization, resulting in huge waste and environmental pollution. Through the analysis of straw fiber structure technology, it will bring huge social and economic benefits.

Silage corn does not refer to corn varieties, but in view of the purpose, fresh corn is stored in the silo (for silage) and fermented for a period of time to make livestock feed. The corn plants used for silage are tall and mainly produce fresh straw. The best harvest period for silage corn is from the late stage of milk ripening to the early stage of wax ripening. At this time, the yield is high and the nutritional value is also the best. Silage corn feed can be stored for a long time without seasonal restrictions, and a balanced supply of feed can be guaranteed throughout the year. However, due to its high fiber content and lignification degree, the digestion and utilization of silage corn by livestock is limited. As the main source of livestock feed, how to improve the digestibility of livestock silage corn stover fiber is very important. In addition to directly measuring the degradation rate of cellulose, the reflection of livestock's digestion and utilization rate of straw fiber can also be intuitively understood by observing the structural changes of straw fiber cell wall under the action of rumen microorganisms.

At present, the research technology on the microstructure of plant leaves after livestock rumen degradation is still immature. The samples obtained by the conventional method of preparing scanning electron microscope observation samples have incomplete fiber structure of plant leaves and incomplete images when observed under scanning electron microscope. clear question.

Contents of the invention

In order to solve the deficiencies in the prior art, the object of the present invention is to provide a method for preparing a scanning electron microscope sample of silage corn leaf after livestock rumen degradation, the preparation method can ensure that the structure of the scanning electron microscope sample is complete, and the surface structure of the blade in the scanning electron microscope observation room is clear , the image has a strong three-dimensional effect.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for preparing a scanning electron microscope sample of silage corn leaf, is characterized in that, comprises the following steps:

Step 1. Prepare fixative:

Take 25v% glutaraldehyde solution, 0.2M phosphate buffer and redistilled water, prepare in proportion to obtain 2.5v% glutaraldehyde fixative;

Step 2. Cleaning:

The silage corn leaves that have been degraded by the rumen of livestock are gently dialed one by one into a vial filled with 0.1mol/L pH 7.2 phosphate buffer solution for rinsing, and the buffer solution is replaced repeatedly until the surface of the leaves is clean;

Step 3, fix:

Under the condition of 4°C, fix the rinsed silage corn leaves with 2.5v% glutaraldehyde fixative solution for 1-3h;

Step 4, rinse:

Aspirate the fixative, re-add 0.1mol/L pH 7.2 phosphate buffer, and replace the buffer repeatedly until the leaf surface is thoroughly rinsed;

Step 5, dehydration:

Aspirate the buffer solution from the vial, and add different concentrations of ethanol dehydrating agents to dehydrate step by step;

Step 6, replacing ethanol:

Aspirate ethanol, add different concentrations of tert-butanol to remove ethanol;

Step 7, freeze drying:

Put the de-ethanolized sample into the sample cup, freeze it at -20°C for 15-30min, then dry it in a freeze dryer until it is drained;

Step 8, the sample is sprayed with gold coating:

Paste the conductive double-sided tape on the sample stage, lightly clamp the side of the dried sample with tweezers, ensure that the observation surface is upward, stick it firmly on the tape in order, and spray a layer of gold film on the surface of the sample with an ion gold sprayer.

The aforementioned method for preparing a SEM sample of silage corn leaves is characterized in that, in step 3, the aforementioned silage corn leaves are completely submerged in the fixative solution.

The aforementioned method for preparing silage corn leaf scanning electron microscope samples is characterized in that, in step 5, the volume concentration of ethanol of the aforementioned dehydrating agent is 30%, 50%, 70%, 80%, 90%, 95%, 100% %, where dehydration is carried out at 4°C when using dehydrating agents with ethanol volume concentrations of 30%, 50%, and 70%, and at room temperature when using dehydrating agents with ethanol volume concentrations of 80%, 90%, and 95%. Dehydration is carried out. When using a dehydrating agent with a volume concentration of ethanol of 100%, dehydration is performed twice at room temperature for 15-30 minutes at each concentration.

The aforementioned method for preparing silage corn leaf scanning electron microscope samples is characterized in that, in step 6, the volume concentration of the aforementioned tert-butanol is sequentially 20%-40%-60%-80%-85%-90%-95% -100%, each stage removes ethanol for 5-10 minutes.

The benefits of the present invention are: the preparation method of the present invention is simple and convenient, low in cost, the prepared scanning electron microscope sample has a clear structure, the surface structure of the leaves in the scanning electron microscope observation room is clear, and the image has a strong three-dimensional effect, which is conducive to the development of digested silage corn leaves. Observation and study of ultrastructure, which is of great significance for qualitative and quantitative understanding and evaluation of sheep rumen microorganisms to decompose the fiber structure of silage corn leaves and improve the digestibility of livestock straw.

Description of drawings

Fig. 1 is the scanning electron microscope image of a sample that adopts preparation method of the present invention to obtain;

Fig. 2 is the scanning electron microscope image of another sample that adopts preparation method of the present invention to obtain;

Fig. 3 is the image after the SEM image in Fig. 1 is further enlarged;

FIG. 4 is a further magnified image of the SEM image in FIG. 2 .

Detailed ways

The present invention will be specifically introduced below in conjunction with the accompanying drawings and specific embodiments.

The experimental methods that do not indicate the specific conditions in the following examples are usually in accordance with the conventional conditions or the conditions suggested by the manufacturer.

Preparation Process:

Step 1. Prepare fixative

Take 25v% glutaraldehyde solution, 0.2M phosphate buffer and redistilled water, prepare in proportion to obtain 2.5v% glutaraldehyde fixative.

Step 2. Cleaning:

The silage corn leaves degraded by the rumen of domestic animals (sheep were used in this embodiment) were gently dialed one by one with tweezers into the penicillin vial filled with 0.1mol/L pH 7.2 phosphate buffer for rinsing, and the buffer was repeatedly changed until The blade surface is clean, if it is not cleaned, it will affect the follow-up photographing effect. Generally, it is rinsed for 1-1.5 hours, and the solution is changed 3-4 times. In this embodiment, the solution is rinsed for 1 hour, and the solution is changed 3 times.

Step 3. Fix

Under the condition of 4°C, the rinsed silage corn leaves were fixed with 2.5v% glutaraldehyde fixative solution, generally the fixation time was 1-3h, and the fixation time was controlled at 3h in this embodiment.

During the fixation process, the silage corn leaves should be completely submerged in the fixative solution. If there is a sample floating on the surface of the fixative, the sample should be completely submerged in the fixative by vacuuming.

Step 4. Rinse

Aspirate the fixative, re-add 0.1mol/L pH 7.2 phosphate buffer, and replace the buffer repeatedly until the leaf surface is thoroughly rinsed to reduce the reaction between the fixative and the later dehydrating agent. Rinse for 1-1.5 hours in general. The solution was changed 3-4 times. In this example, the solution was rinsed for 1 hour and the solution was changed 3 times.

Step 5. Dehydration

Dehydration is the process of completely removing free water in the tissue. The buffer solution is sucked out from the vial, and ethanol dehydrating agents of different concentrations are added to dehydrate step by step. If the dehydration is not complete, the image will be unstable when viewed under the electron microscope, which will easily damage the instrument.

The volume concentration of ethanol in the dehydrating agent is 30%, 50%, 70%, 80%, 90%, 95%, and 100% in sequence. The specific process of dehydration is:

(1) Use the dehydrating agent whose volume concentration of ethanol is 30%, 50%, 70% respectively, carry out dehydration at 4 ℃, generally dehydrate 15-30min under each concentration, and blade stays 15min in each stage in the present embodiment;

(2) using ethanol volume concentrations of 80%, 90%, and 95% dehydrating agents respectively, dehydrating at room temperature, generally dehydrating for 15-30min at each concentration, and the leaves stay at each level for 15min in this embodiment;

(3) Using a dehydrating agent with a volume concentration of ethanol of 100%, dehydration is performed twice at room temperature, usually for 15-30 minutes each time, and the leaves stay for 15 minutes in this embodiment.

Pay attention to the following during dehydration:

(1) Dehydration step by step according to the concentration gradient, not sharp dehydration;

(2) When replacing the solution, the action should be fast, especially do not let the tissue leave the solution, otherwise bubbles will be generated inside and outside the tissue, which will affect the structure observation;

(3) If you want to stay for a long time or overnight during the dehydration process, you should put it in 70% dehydrating agent and store it at 4°C.

Step 6, replacing ethanol

Aspirate ethanol, add different concentrations of tert-butanol to remove ethanol.

The volume concentration of tert-butanol is 20%-40%-60%-80%-85%-90%-95%-100% in turn, and the ethanol removal is generally 5-10min for each stage, and the replacement time for each stage in this embodiment is 10min .

Step 7. Freeze drying

Put the sample after deethanol into the sample cup, and freeze it at -20°C. Generally, the freezing time is 15-30min. In this example, the freezing time is 20min, and then put it into a freeze dryer (VFD-21S) dry. Before freezing, the switch of the freeze dryer should be turned on and the temperature should be lowered to 0°C.

Put the frozen sample into the freeze dryer, turn on the EVAC button until the sample is dry for 20 minutes (usually 15-20 minutes), then adjust the temperature mode to warm to 30°C, then adjust the temperature mode to stop, then turn off the EVAC button for 2 minutes Then remove the sample.

The freeze-drying process must be thorough, otherwise tert-butanol will easily remain, which will affect the observation effect and image clarity.

Step 8, the sample is sprayed with gold coating

Paste the conductive double-sided tape on the sample stage, lightly clamp the side of the dried sample with tweezers, ensure that the observation side is facing upward, and stick it firmly on the tape in order.

Fresh samples conduct electricity by themselves, while dried biological samples do not. When this kind of non-conductive sample is observed under SEM, it will generate charge accumulation and affect the stability of observation. Therefore, it is necessary to spray a layer of gold film on the dry sample surface with an ion spray gold instrument (msp-1s).

The current of the ion gold spraying instrument is 15mA, and the gold spraying time is 200s. The gold-plated samples can be observed under the scanning electron microscope.

Sample observation:

Observe the scanning electron microscope sample prepared after gold spraying with S3400N (HITACHI) scanning electron microscope, the image mode is SE, the accelerating voltage is 1.00KV-2.00KV, the inclination of the sample stage is 0, the brightness contrast is automatic plus manual, and the focus is manual. The magnification can be determined according to the structure you want to see, and the minimum can be 50 times.

When taking pictures, you should follow the principle of high magnification focus and low magnification photography.

Observation results:

Figures 1 and 2 show the remaining fiber structure after digestion, that is, the vein part. The clear and complete structure can be seen from the scanning electron microscope, and the three-dimensional effect is strong.

Figure 3 and Figure 4 are further enlarged electron microscope images, the outline of the cells can still be clearly seen from the scanning electron microscope, and the stereoscopic effect is strong.

It can be seen that the method for preparing a scanning electron microscope observation sample of the present invention, through processes such as cleaning, dehydration, ethanol removal, freezing, drying, and gold plating, makes the prepared scanning electron microscope sample structure complete, and the surface structure of the blade in the scanning electron microscope observation room is clear and complete. , The image has a strong three-dimensional effect, which is conducive to the observation and research of the ultrastructure of silage corn leaves after livestock rumen degradation, and is of great significance for intuitively understanding and evaluating the degradation of plant leaf fiber structure by livestock rumen microorganisms and improving the digestibility of straw feed.

It should be noted that the above embodiments do not limit the present invention in any form, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims (4)

1. prepare a method for silage corn blade scanning electron microscope example, it is characterized in that, comprise the following steps:

Step 1, preparation immobile liquid:

Get 25v% glutaraldehyde solution, 0.2M phosphate buffer and redistilled water, preparation obtains 2.5v% glutaraldehyde immobile liquid in proportion;

Step 2, cleaning:

The silage corn blade of degrading through Rumen of livestock gently dial in rinsing in the bottle filling 0.1mol/L pH 7.2 phosphate buffer one by one, exchange buffering liquid repeatedly, until blade surface is clean;

Step 3, fixing:

Under the condition of 4 DEG C, the silage corn blade 2.5v% glutaraldehyde immobile liquid of rinsed clean is fixed 1-3h;

Step 4, rinsing:

Sucking-off immobile liquid, rejoins the phosphate buffer of 0.1mol/L pH 7.2, repeatedly exchange buffering liquid, until by thorough for blade surface rinsed clean;

Step 5, dehydration:

Sucking-off damping fluid from bottle, adds the ethanol dehydration agent serial dehydration step by step of variable concentrations;

Step 6, displacement ethanol:

Sucking-off ethanol, adds the variable concentrations tert-butyl alcohol and takes off ethanol;

Step 7, freeze drying:

Sample after de-ethanol is put into sample cup, and freezing 15-30min at inserting-20 DEG C, then put into freeze-dryer dry, till draining;

Step 8, sample metal spraying plated film:

Conductive double-sided tape is pasted on sample stage, gently press from both sides dried sample side with tweezers, ensure sightingpiston upwards, paste jail in order on adhesive tape, spray one deck gold film with ion gold spraying instrument at sample surfaces.

2. the method preparing silage corn blade scanning electron microscope example according to claim 1, is characterized in that, in step 3, described silage corn blade is immersed in immobile liquid completely.

3. the method preparing silage corn blade scanning electron microscope example according to claim 1, it is characterized in that, in steps of 5, the volumetric concentration of the ethanol of described dewatering agent is followed successively by 30%, 50%, 70%, 80%, 90%, 95%, 100%, wherein, the volumetric concentration using ethanol is 30%, 50%, 70% dewatering agent time dewater at 4 DEG C, the volumetric concentration using ethanol is 80%, 90%, 95% dewatering agent time at room temperature dewater, at room temperature twice dehydration is carried out when using the volumetric concentration of ethanol to be the dewatering agent of 100%, dewater under each concentration 15-30min.

4. the method preparing silage corn blade scanning electron microscope example according to claim 1, it is characterized in that, in step 6, the volumetric concentration of the described tert-butyl alcohol is followed successively by 20%-40%-60%-80%-85%-90%-95%-100%, every grade of de-ethanol 5-10min.