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.