2.4: Visualizing Soil Microorganisms via the Contact Slide Assay and Microscopy

1. Soil Slide Microcosm Preparation

1. Collect garden soil from the surface (0-6” depth), and weigh 150 g soil into two separate cups.
   1. If soil has high density of organic matter, weigh 100 g.
2. Label one cup “Treatment” and the other “Control.”
3. Calculate amount of water needed to alter moisture content.
   1. Moisture content is often close to field capacity.
      
4. Measure amount of distilled water with a graduated cylinder.
5. Pour amount of distilled water into two vials.
6. Label one vial “Treatment” and the other “Control.”
7. Amend the water in the “Treatment” vial with enough glucose for a final soil glucose concentration of 1%, according to a dry weight basis in the “Treatment” soil.
8. Add 200 mg NH₄NO₃ into the “Treatment” vial and stir to dissolve the amendments. The nitrate serves as a nitrogen source of nutrients for the soil microbes.
9. Do not amend the “Control” vial.
10. In small aliquots of approximately 50 mg, mix the contents of the “Treatment” vial into the “Treatment” cup. Stir with a spatula after each aliquot addition.
11. In small aliquots, mix the contents of the “Control” vial into the “Control” cup. Stir with a spatula after each aliquot addition.
12. Label four clean microscope slides: two “Treatment” and two “Control” slides.
13. Insert the two “Treatment” slides into the “Treatment” soil cup, and insert the two “Control” slides into the “Control” soil cup. Leave 2 cm of each slide projecting above the soil surface, and be sure to leave a gap between the two slides.
14. Cover the cups with plastic wrap and secure it with a rubber band.
15. Puncture the wrap several times to allow air, but still prevent excessive evaporation.
16. Record the weight of both cups.
17. Incubate the soil-filled cups at room temperature in a designated area/incubator for 7 days.

2. Slide Staining and Microscopy

1. Record the weight of both cups.
2. Calculate the soil moisture at the time of slide removal.
3. Remove the two slides from each cup by pressing each slide to an inclined position and withdrawing so the upper face of the slide is not disturbed.
4. Identify and mark the side of the slide to be stained and observed.
5. Gently tap the slides on the bench top to remove large soil particles.
6. Using a damp paper towel, clean the lower face of the slides. Dry the slides at room temperature.
7. Wearing protective goggles and holding each slide with forceps, immerse the slides into 40% (v/v) acetic acid for 1-3 min under a fume hood.
8. Wash off the excess acid under a gentle stream of water.
9. Using a dropper bottle, cover the surface of the slides with phenolic Rose Bengal. Support the slide on a staining rack over a container to catch the excess stain. Be careful, do not wash with force that may remove microorganisms from the surface of the slides.
10. Stain the slides for 5-10 min. Do not let the slide become dry. Add more stain as needed.
11. Gently wash the slides to remove excess stain.
12. Let the slides dry at room temperature.
13. Using the oil immersion objective, observe the slide using a microscope (Figure 1).

Figure 1. A slide under a microscope.

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!