Contact Slides
Question of Interest:
What are the relationships between microflora in sediment communities and how are those relationship altered by changes in physical and chemical parameters?
Hypothesis:
If changes are made in physical and chemical parameters in sediment, then microflora and their relationships will be altered.
Materials:
Sediment samples
400 ml beakers, 1/2 pint mason type jars, or 9-oz. disposable tumblers
Aluminum foil or jar lids
Sediment amendments: paper, simple sugars, leaves, nutrients, and other
available materials (be creative)
Microscope slides (one end frosted)
Analytical balance
Acetic acid (40% solution)
Phenolic rose bengal: Rose bengal, 1.0 g; CaCl2 0.03 g in 100 ml 5% aqueous
phenol
Staining racks
Water bath
Microscope
Procedure:
1. Measure out a 1:1 sandy loam: compost sediment sample (around 200 g
total) and mix the sample thoroughly. Add this mixture to the beaker labeled
control.
2. Gently insert 2-4 clean microscope slides vertically in each container leaving at least 2 cm (3/4 in) protruding above the soil surface.
3. After inserting all the slides, carefully add enough water around the slides to bring the soil to approximately 50-60% of water holding capacity (about 20-25 ml for 200 g of sediment depending on how moist the soil is initially). Do not water log the soil.
4. Cover the container with aluminum foil and incubate at room temperature (or really whatever temperature you want). Be sure to label the container and the slides in it.
5. Repeat steps 1-4 at least twice more labeling the containers treatment 1 and treatment 2. Amend the sediment sample by choosing something new to add to the sediment in step one and mixing well.
6. After ~ 7 days of incubation, remove one of the slides from each
beaker. Remove the other slides after 21 days. To remove the slides, especially
from sediments of high clay content, carefully remove the sediment from behind
the slide then gently push the slide away from the intact soil. Remove the
slide so as to leave this side undisturbed for staining. Wipe the other side
of the slide with a damp paper towel and allow to air dry. When dry, tap the
slide on the
table top to remove any large particles. Treat the slide with acetic acid (40%)
for 1-3 minutes. Wash off any excess acid with distilled water.
7. Stain the slides over a boiling water bath. Keep the slides covered with phenolic rose bengal for 5-10 minutes and do not let the slides become dry. The slides can be stained without steaming, however, a minimum of 10 minutes must be allowed.
8. Wash the slides to remove excess dye, allow to dry, and examine under the microscope.
This is a slide from a Winogradsky column our
lab mentor made as a demonstration. It has not yet been fixed or stained.
Results:
Results may vary depending on the environments in which the slides are placed.
The magnification and resolution of most microscopes equipped with an
oil-immersion lens should be sufficient to allow observation of bacteria,
actinomycetes, and fungal
hyphae that appear on the slides, as well as any microscopic
eukaryotes such as nematodes. Such a microscope is usually not
sufficient, however, to identify bacteria to species. Instead, note the
abundance of each type of organism and any relationships of the organisms to one
another and to particles of organic matter. Look for evidence of colony
formation, cross feeding, cooperation, etc.
Conclusions:
The different treatments applied to the sediments may cause differences in the
microenvironments, resulting in succession of different microbial
populations and in different relationships among the resident organisms.
Modifications:
for K-8 modifications
Extensions:
1. Give slides to students to embed in environments at home such as
flower pots.
2. Simulate acid rain and its effects on an environment by preparing an acid solution and using it to moisten the soil instead of the water.
3. Add materials to the sediment to simulate pollution and waste materials to see what effect they would have on the microflora.
4. Prepare serial dilutions of household materials that are considered antimicrobial and substitute for the water added to the sediment to see how effective the antimicrobial substance is.
5. Use a sheet cake pan set up with a slight incline. Embed the slides at varying intervals throughout the pan. Put a substance such as motor oil at the top of the inclined part of the pan and allow it to ooze throughout the sediment. Examine the microflora by staining and observing the slides.
6. Flame a pair of tweezers and remove a pinch of soil from an area that you sampled and observed. Put the soil on an agar plate and add 5 drops of distilled water. Swirl this mixture around the plate to distribute it and incubate at conditions you choose. You may choose to vary light or temperature conditions.
7. Perform the original experiment and vary conditions of light or temperature during incubation of the sediment.