DO-IT-YOURSELF GUIDE

FOR MICROSCOPY OF AGRICULTURAL SOIL

Written by In collaboration with

Katelyn Solbakk, Janne Aalborg Nielsen,
Mikroliv Økologisk Landsforening

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”Do-it-yourself guide for microscopy of agricultural soil” is published by
Økologisk Landsforening 2020, for the project ”Styr på den økologiske
jordfrugtbarhed” (Managing organic soil fertility), which is supported by
“Fonden for Økologisk Landbrug”.

This publication may be downloaded freely from [Link], and may

be copied and distributed for non-commercial use. For citations and
reproduction in other literature, please provide the source: ”Do-
it-yourself guide for microscopy of agricultural soil”, published by
Økologisk Landsforening 2020.

Photos and illustrations: Katelyn Solbakk, Mikroliv.

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 CONTENTS
Introduction 4

Part 1
Collecting and preparing samples 5

Part 2
What can you see?
Bacteria 11
Fungi 12
Protozoa 13
Microscopic animals 14
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Part 3
Evaluation and next steps 20

Quick reference table 24

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 INTRODUCTION
The soil ecosystem is a highly complex web, and each organism has a unique role
to play. Science has barely begun to untangle the connections and functions of
the extremely diverse array of life in soil, which is why it is so important to protect
biodiversity in the ecosystem as a whole, and ensure that as many different
organisms as possible are able to thrive and ultimately support the growth of
healthy plants.

Some key functions of a healthy soil ecosystem

include:

• Good water retention and drainage

• Healthy structure and resistance to erosion
• Nutrient cycling
• Improved plant health
• Carbon storage
• Resilience against pest and disease outbreaks

Many of these functions are the direct result of microbial activity in soil.
Microscope analysis offers you a unique window into the life of your soil, giving
you the chance to directly check in on your own soil ecosystem using simple
equipment. There are even attachments that enable you to use your smartphone
as a microscope!

It does take some practice to learn how to identify organisms, but this guide
will help you get started and perform a basic evaluation of your own soil, and
introduce you to some of the organisms that may be living there.

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 Part one

COLLECTING
AND PREPARING
SAMPLES

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Step one Collect samples

1
Clear any vegetation from the sample area.

2
Collect soil 0-5 cm deep.

3
Repeat several times around the test area
and mix the soil to create one sample that
represents the whole plot.

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Step two Prepare samples

Materials required:
• Kitchen scale
• Glass jar(s) with lid
• Spoon
• Tape
• Marker
• Water (145 ml per sample)

1. Use tape and a marker to label jars with the sample

location/ID and collection date.

2. Mix 5 g of soil with 145 g of water in each jar.

3. Close the jars tightly, and shake gently for five

minutes.

4. Loosen the lid(s) and let the samples rest

undisturbed for two days.

Note: You can look at the sample immediately, but you will likely find more
organisms after the waiting period. This is because many protozoa will rest in
a dormant state called a “cyst” when conditions are dry. Allowing the sample to
rehydrate gives them a chance to “wake up”.

Tip: If you have more than one sample, remember to label jars before filling
them, and clean the spoon between samples.

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Step three Meet your microscope

Ocular lens
(Eyepiece)
Objective
Stage control

Stage
Coarse focus

Fine focus
Condensor

Image: [Link]

This is an example of a typical compound light microscope. There will be some

variation between different brands and types of microscopes, and you can even
use completely different types of microscopes, such as phone attachments.
Consult your user manual to learn about your specific microscope if it does not
match the image above.

Part Function
Objective Adjusts magnification
Coarse focus Moves the stage a large amount to roughly adjust focus
Fine focus Moves the stage a small amount to finely adjust focus

Ocular lens This is where you look

(Eyepiece)
Stage Where you put the sample. Use the metal clip to hold it in
place
Condensor Adjusts contrast
Stage control Use these controls to move the sample when viewing

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Step four Observe

Materials required:
• Microscope
• Glass slides
• Glass covers
• Pipette
• Rinse water
• Empty jar for waste water
• Prepared samples

Close the lid tightly and gently shake the sample.

Use the pipette to draw up some of the solution.
1 Allow the heaviest particles to settle and drip out
from the pipette (large debris will not fit properly
under the cover slip).

2 Place one drop of the sample on the glass.

Hold the cover glass gently by the edges (beware:

cover glass is very thin, sharp, and fragile!). Place
3 one edge on the glass next to the drop and slide it
over so the solution spreads along the glass edge
(see photo).

Carefully set the cover glass down so the sample is

4 spread evenly.

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Step four Observe

Place the sample on the microscope stage, using

5 the metal clip to hold the slide in place.

Begin with low magnification and use the focusing

knobs to make the image sharp, then switch to a
6 higher power. Use the stage controls to explore the
slide, and see what you can find!

Tips:
• Do not use the coarse focusing knob on high magnification! It moves
the stage too much and can crush the slide against the objective.

• When taking a drop with the pipette, avoid taking material from the very
bottom of the jar as this will be too dense to look at.

• The microscope has a very narrow depth of field. Keep one hand on the
fine focusing knob and continuously adjust the focus “up and down” while
viewing samples.

• Analysis is typically performed at 400x, but 100x lets you see see larger
areas at once and get a better view of larger, fast moving organisms.

• If you get “lost” and can’t seem to bring the sample into focus, switch back
to the lowest magnification and use the coarse focusing knob until you can
see the sample again. Then switch back to higher power and try again.

• If you have difficulty finding something to focus with (ie. it’s just white), try
moving to the edge of the glass first and make that line sharp, then try
exploring the sample again.

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 Part two

WHAT CAN YOU

SEE?

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Group one Bacteria

Bacteria are very small; often just 1 μm in diameter (0.001 mm). The image below
demonstrates how tiny they are at 400x magnification in a microscope with a field
of view 500 μm (0.5 mm) across.

Bacteria are grouped into three broad categories based on the shape of the cells.
These are round (cocci), rod-shaped (bacilli) or spiral shaped (spirilla). The arrows
in the diagram above demonstrate the type of movement that is often seen with
these different types of cells. Cocci often just vibrate, or move with a roughly
straight motion. Bacilli sometimes move in a straight line as well but often have a
rocking or zig zag motion. They can sometimes be seen linked together in chains.
Spirilla have a characteristic corkscrew motion that is easy to spot.

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Group two Fungi

Healthy soil typically has robust networks of diverse fungal threads called
“hyphae”. In the microscope, these look like clear or brown strands, typically
between 2-6 μm in diameter. They can be small fragments or long strands that
cover large areas of the microscope slide. Soil with many robust fungal hyphae
typically has stronger structure and better aggregation. When disturbance is
minimal, fungal networks weave through soil, binding particles together into
strong aggregates, and they provide significant benefits to plants. Because fungi
grow slowly and are easily disturbed, they can be good indicators of ecological
succession. Agricultural soil often has low fungal populations and forest soil tends
to be very rich in fungal networks.

Below are some examples of fungal hyphae that you can compare with your soil
sample.

The fine horizontal line in the center of

this picture (left) is a very narrow, thin
fungal hypha. If you are seeing mostly
or only strands like this, it could be a
sign that conditions in the soil are not
optimal.

The brown threads in the images above are examples of robust fungal hyphae
fragments. Note the distinct smooth edges and clear segments. Hyphae like
this are considered a positive indicator of soil health.

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Group three Protozoa

Protozoa are incredibly diverse single-celled organisms. They are usually larger
than bacteria and easier to see in the microscope. They tend to be very active,
and sometimes will notice the movement of soil particles before you see the
organism itself.
When protozoa graze on bacteria, they free up nutrients that have been
immobilized in bacterial cells, and make them available to plants again. This
makes them incredibly important members of the soil ecosystem. They have
also been found to promote plant health and improve growth independently of
nutrients.
Protozoa are known to be picky eaters. Each species hunts for specific types
of bacteria. That means diversity in protozoa may be useful as an indicator of
bacterial diversity in the soil. The more unique types of protozoa you can find in
your soil, the better!
There are three main categories of protozoa, based on their physical
characteristics and movement:

Amoebas Flagellates Ciliates

10-50 μm 3-10 μm 10-80 μm

Amoebas are protozoa Flagellates have one Ciliates come in many

without a fixed shape. or two long, whip-like diverse shapes and sizes.
They move with a slow appendages that they use They have many smaller
oozing motion, extending to move. They often have hairs (called cilia), which
temporary “false feet” a twitching, spinning, or allow them to move much
or pseudopods to slide rocking motion. faster and often more
across surfaces or engulf smoothly than flagellates.
their food.

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Group three Protozoa

Amoebas are a type of protozoa without a fixed shape. They move with a slow
oozing motion, extending temporary “false feet” or pseudopods to slide across
surfaces or engulf their food. Amoebas are particularly voracious predators of
bacteria. Their flexibility makes it possible for them to reach bacteria in the tiniest
soil pores that other predators can’t access.
There are two main types of amoebas: testate (with a shell) or naked (without a
shell).
Testate amoebas are common and easy to identify in soil. The shell has a
distinct almond or balloon-like shape with an opening on one end. You can
sometimes see a subtle pattern of tiny scales on the shell.

Here are some examples of testate amoebas in the microscope:

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Group three Protozoa

Naked amoebas have no shell. They can be more difficult to spot than other
protozoa because their movement is often slow. They can be very tiny or quite
large, very active or barely moving at all.

Here are some examples of naked amoebas in the microscope:

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Group three Protozoa

Flagellates are the most abundant protozoa you will see in soil samples. They
are hardier and more pioneering than larger organisms, and are better able
to withstand tough conditions. They are usually smaller than ciliates. The main
difference between flagellates and ciliates is that flagellates have one or two long,
whip-like appendages that they use to move. They sometimes have a smooth
movement, but they most often have a twitching, spinning, or rocking motion and
they tend to be slower than ciliates It could be compared to paddling a boat with
one or two oars, versus many people paddling together in perfect unison.

Here are some examples of flagellates in the microscope:

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Group three Protozoa

Ciliates are less common in agricultural soil samples, but can be seen
occasionally. They are also the most fun to watch, because they are usually very
active and come in a huge variety of shapes and sizes. Remember that the main
difference between flagellates and ciliates is the number of “hairs” they have.
Ciliates have many smaller hairs (called cilia), but flagellates just have one or two
long ones (called flagella).
Here are some examples of different ciliates that you might find in soil samples:

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Group four Microscopic animals

Microscopic animals are highly diverse multicellular organisms. They have

organs including a complete digestive tract, but they are so small that a
microscope is needed to see them. In soil and compost samples, you may see
rotifers (left photo) and nematodes (right photo). Nematodes are well known as
plant parasites, but in fact they are extremely diverse and most soil nematodes
are actually beneficial. Both nematodes and rotifers, like protozoa, consume food
(mainly bacteria and fungi), and release nutrients that plants can use.
In forest samples, especially mosses, you may even find tardigrades, also known
as water bears or moss piglets, like the one illustrated below.

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 Part Three

EVALUATION AND
NEXT STEPS

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Evaluation Ecological succession

Soil progresses through stages of ecological succession just like other

ecosystems. In early stages the habitat tends to be relatively “simple” and
dominated by resilient pioneering organisms that reproduce and spread rapidly.
Soils in this stage are mainly populated by bacteria, and perhaps some small
flagellates and thin fungal hyphae. Early stage soils are often dominated by
plants we refer to as “weeds”. As succession progresses, the habitat becomes
more complex, allowing more diverse organisms with more nuanced functions to
thrive.
Agricultural activities have a direct influence on the ecological status of the soil.
When soil is disturbed (eg. by mechanical or chemical interference), it is pushed
back to an earlier stage of succession. If the soil is frequently reset to an early
ecological stage and dominated by pioneering organisms, it becomes more
vulnerable to erosion and there is a greater risk of pest, weed, and disease
outbreaks for crops. It is very common for agricultural soil to be in the pioneer
category, and this is something that should be addressed in order to achieve
better soil health and sustainability in agriculture.

Ecological succession in the microscope

Pioneer Climax
• Bacteria dominant • More diverse bacteria • Fewer visible bacteria
• Small flagellates • Bacteria and fungi in • Fungi dominant
• Highly mineral balance • Very high biodiversity
• Little organic matter • Greater biodiversity • More complex
• Few or no fungi • Microorganisms are organisms
• Low biodiversity more noticeable • Strong aggregation;
• Poor aggregation • Better aggregation the sample looks
“clean”

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Evaluation Ecological succession

You can use the following characteristics as a guide to evaluate the ecological
status of your soil samples.

Pioneer Middle succession Climax

There may be dense Bacteria and fungi are in Bacteria are less dense,
bacteria, but few signs of balance. It will likely take but with more types of
diverse cell types or active some experience before cells and they may be
movement. you can recognize this. more active.

Very few fungi. Mostly Fungi are more Fungi are clearly dominant
thin, pale or clear noticeable. Fungal hyphae compared to bacteria. It is
fragments. are larger, more robust, easy to find large, robust
more numerous, and networks of fungal hyphae
often darker in colour. of various colours and
sizes.

Large amount of loose The appearance is cleaner Physical structure is

material. Very cluttered and more “organized” dominated by large, dark
and messy, with few or no looking, with stronger, aggregates with plenty of
large aggregates. Much of darker aggregates clean, open space.
the non-living material is and some open space
pale or clear. between aggregates.

Few protozoa. Mostly Protozoa are easier to Protozoa should be easy

small flagellates with low find. They may be larger to find, active, and highly
diversity (eg. 1-4 types). and more active, and diverse.
there should be better
diversity (eg. 5-10 types).

Low overall biodiversity. Overall biodiversity is Overall biodiversity is

Need to search for signs moderate. It is easy to see high and there are many
of life. signs of life. obvious signs of life.

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Evaluation Next steps

How can we support soil life?

Here is a summary of the types of conditions that favour microbial activity in soil,
and some basic principles for how you can provide them.

Microbes need: You can provide it by:

Moisture Keeping the soil covered as much as possible.

Allowing natural structure to develop and avoiding

Oxygen
compaction.

Energy and nutrients Maintain cover with living plants and mulch, with as
much diversity as possible.

Shelter Keeping the soil covered as much as possible.

Reduced disturbance Minimizing tillage, driving, and chemical interference.

Earthworm activity* All of the above.

*Earthworms are known as “ecosystem engineers”. Their activity improves soil quality and creates
conditions that support beneficial microorganisms.

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Evaluation Quick reference table

Organism Example

Bacteria

Fungi

Testate amoeba

Naked amoeba

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Evaluation Quick reference table

Organism Example

Flagellate

Ciliate

Nematode

Rotifer

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