접촉 슬라이드 분석 및 현미경을 통한 토양 미생물 시각화
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Overview
출처: 이안 페퍼 박사와 찰스 게르바 박사의 연구소 – 애리조나 대학교
시연 저자: 브래들리 슈미츠
토양은 생명에 기여하는 생체 및 무생물 요인을 포함하는 지구 표면의 얇은 층을 포함한다. 상기 비생물부는 토양의 질감을 결정하는 크기와 모양에 이르는 무기 입자를 포함한다. 바이오틱 부분은 식물 잔류물, 뿌리, 유기물 및 미생물을 통합합니다. 토양 미생물의 풍부와 다양성은 토양 1 그램이 포함으로 광대 하다 107-8 박테리아, 106-8 actinomycetes, 105-6 곰팡이, 103 효모, 104-6 원생 조아, 103-4 조류, 53 선충. 함께, 생물학적 및 생물학적 요인은 토양 미생물에 유리한 조건을 제공하는 뿌리 권구로 알려진 식물 뿌리 의 주위에 건축을 형성합니다.
생물학적 및 비생물적 요인은 토양의 삶을 촉진합니다. 그러나, 그(것)들은 또한 미생물을 제한하는 스트레스역학을 기여합니다. 생물학적 스트레스는 환경 조건에서 적응하고 살아남기 위해 삶 간의 경쟁을 수반합니다. 예를 들면, 미생물은 이웃 미생물을 해치기 위하여 억제 물 또는 독성 물질을 분비할 수 있습니다. 페니실륨 노타텀은 인간이 약제페니실린을 만들기 위해 수확하는 항균체를 생산하여 영양소에 대한 경쟁을 감소시키기 때문에 악명 높은 곰팡이입니다. 비생물학적 스트레스는 빛, 수분, 온도, pH, 영양소 및 질감과 같은 미생물 생존을 제한하는 물리적 또는 화학적 특성에서 발생합니다.
Principles
Procedure
Results
Fungi display thick, filamentous hyphae (Figure 2). Actinomycetes display thin, filamentous hyphae. Bacteria display small cocci or rod shapes. They’re often found in clumps, on soil particles, or lining fungal hyphae. Soil particles display irregular, dark shapes (Figure 3).
Figure 2. Contact slide image using 100X objective lens.
Photo courtesy W.H. Fuller.
Figure 3. Contact slide image using 100X objective lens.
Photo courtesy W.H. Fuller.
Applications and Summary
The contact slide assay, also referred to as the buried-slide, is a simple technique utilized to qualitatively observe soil biota. This assay qualitatively shows the spatial interactions between fungal hyphae, actinomycete filaments, bacteria, and soil particles. Individuals or industry can employ this assay to gather knowledge on a particular soil’s health in regards to agriculture, gardening, composting, teaching, and studying. However, this technique does not quantify soil micro biota, as it only encompasses a small portrait of a larger heterogeneous environment.
Soil organism relationships can be observed by performing the contact slide assay and viewing the results through 100X oil immersion microscopy (Figures 2 and 3). The simplicity and ease to performing this assay makes it a great starting technique for those who have never been exposed to microbiology and may be viewing microorganisms through a microscope for the first time.
References
- Pepper, I. L., & Gerba, C P. 'Contact Slide Assay.' Environmental Microbiology A Laboratory Manual. 2nd ed. Elsevier 19-25 (2004).
- Pepper, I. L., Gerba, C. P., & Gentry, T. J. 'Earth Environments.' Environmental Microbiology. 3rd ed. Elsevier 59-88 (2014).
- Rossi, G., Ricardo, S., Gesue, G., Stanganelli, M., and Want, T.K. Direct Microscopic and bacteriological investigations of the soil. Soil Science. 41, 52 – 66 (1936).
Transcript
The relationships between the various organisms and inorganic components in soil are vital to understanding soil changes and environmental stresses, but cannot be elucidated without direct visualization.
Soil, an extremely complex system, is a habitat for millions of diverse organisms. The region of soil directly around plant roots in particular, called the rhizosphere, contains a unique array of organisms that are directly influenced by the plant roots.
The abiotic, or non-biological, component of the rhizosphere includes inorganic particles ranging in size and shape that contribute to the soil’s texture. The biotic, or biological, portion includes plant residues, roots, organic matter, and microorganisms.
This video will demonstrate the direct visualization of the biotic and abiotic components of rhizosphere soil, in order to understand factors affecting soil changes and to predict environmental stresses.
Microscopic organisms tend to reside in the water located within soil pores. Bacteria are among the simplest and most plentiful organisms present in soil, and are found in many morphologies including spheres called cocci, rods called bacilli, and filamentous forms.
Fungal species, such as yeast and molds, are the second most abundant organisms in soil. They work to decompose and recycle dead organic matter. Microscopic filamentous fungi visually differ from other microorganisms, as they possess long and branched hyphae that release spores.
Direct observation of the relationships between these organisms is challenging, but can be achieved using a contact slide assay. This method is performed by submerging a glass slide into soil for several days and allowing the organisms and soil particles to adsorb to the slide surface.
The slide is then removed at an angle to prevent smearing of the surface. The microbes are fixed with acetic acid, and stained with Rose Bengal stain to enable visualization via light microscopy.
Now that you understand the principles behind the contact slide assay technique, lets take a look at the process in the laboratory.
First, collect surface garden soil and transfer the soil into the lab. Weigh 150 g of soil into the 2 separate containers. One container should be labeled as the treatment sample, which will be modified with nutrients to encourage rapid proliferation of organisms. Label the other as the control, which will be unchanged.
Calculate the water content in the soil, using the technique shown in this collection’s Determination of Moisture Content in Soil video. Based on this calculation, determine the amount of water in the soil on a dry weight basis. Now calculate the amount of water that needs to be added to give a 15% soil moisture content. This brings the moisture to field capacity, optimal for microorganism growth.
Measure the calculated amount of distilled water using a graduated cylinder. Pour the calculated volume of water into each container. Based on the previously determined dry weight of the soil, calculate the amount of glucose needed to achieve a final soil glucose concentration of 1% by mass, using the dry weight basis. Weigh this amount of glucose and add it to the treatment container only.
Weigh 200 mg of ammonium nitrate, then add it to the treatment container only. The ammonium nitrate serves as the nitrogenous nutrient source for the soil microbes. Mix the soil, glucose, and ammonium nitrate mixture in the container.
Next, label 4 clean microscope slides: two as treatment, and two as control. Insert the two treatment slides into the treatment soil container. Leave a section of each slide exposed above the soil surface, and ensure that there is a gap between the two slides.
Insert the two control slides into the control soil container in the same way. Cover the cups with plastic wrap, and secure with a rubber band. Puncture the plastic wrap several times to allow air transfer, but still prevent excessive evaporation.
Finally, weigh both cups, record their weight, and incubate them in a designated area at room temperature for seven days.
After the seven-day incubation, calculate the soil moisture content by weighing the soil cups. Determine if weight has been lost due to water evaporation, and replace the water if needed.
Remove the plastic wrap from the container, and remove the two slides from the soil by pressing each slide to an inclined position, and withdrawing so that the upper face of the slide is undisturbed.
Gently tap the slides to remove large soil particles. Using a damp paper towel, clean the lower face of the slides. Allow them to dry at room temperature in a fume hood. Once dry, pick up a slide with forceps, and immerse it into acetic acid for 1 to 3 min.
Rinse the top of the slide with a gentle stream of distilled water to remove excess acid. Repeat these steps for all slides. Allow the slides to air dry.
Support the slide on a staining rack over a container to catch excess dye. Using a dropper, gently cover the surface of each slide with phenolic Rose Bengal dye. Allow the slides to stain for 5 to 10 min, taking care to add more dye as needed to keep the slides wet. Gently rinse the slides with water to remove excess stain, and allow the slides to dry at room temperature.
Examine the slides on a light microscope, using an oil immersion objective. The treated soil will have more soil microbes.
The spatial interactions between fungal and bacterial organisms in typical soil samples can easily be visualized. Soil particles display dark irregular shapes.
Fungal organisms display thick filamentous hyphae, while actinomycetes display thin filamentous hyphae.
Bacteria are found as small cocci or rod shapes, typically in clumps, on soil particles or lining fungal hyphae.
The direct isolation of organisms from soil is important to the understanding of soil and environment characteristics.
Entomopathogenic nematodes are microscopic round worms that parasitize insects. While they are not visualized in the contact slide assay, they can be isolated from collected soil samples, as shown in this example.
First, the nematodes were baited in the soil using insects identified from visual examination. Nematodes were isolated from the dead insect bait, by placing the dead insects in a moist and dark environment and allowing the nematodes to migrate out into the surrounding water. The nematodes were then collected from the water, and analyzed.
Filamentous fungi are vital to soil health due to their role in nutrient recycling. The isolation and observation of filamentous fungi from soil was conducted in this example.
Soil samples were diluted with water, and added to separate sterile Rose Bengal streptomycin agar plates. The streptomycin prevented bacterial growth, and enabled fungal growth. Fungal colonies were counted and mounted to a glass slide using adhesive tape. The fungi were then imaged using a light microscope.
Soil microorganisms naturally break down components in soil, such as dead plants and organisms. Biodegradation and colonization of biodegradable plastic films was examined, as shown in this example.
Fungi were isolated from plastic films buried in soil for several months. The fungi were then tested individually for growth on plastic films. Plastic films were then incubated with the selected fungal strain with no growth media, in order to observe direct degradation of the plastic by the fungi.
You’ve just watched JoVE’s introduction to the contact slide assay for qualitative imaging of soil microbes. You should now understand how to prepare the contact slide, and visualize soil microbes. Thanks for watching!
