Week 4 Lab: Diversity Safari

Lab Objectives

· Learn how to use a compound microscope in order to observe living organisms belonging to different
eukaryotic groups

· Identify eukaryotic microorganisms common in freshwater samples

· Assess nutrition (autotroph vs. heterotroph) and locomotion in identified organisms

· Calculate and compare biodiversity measurements

Learning outcomes from class:

o Recognize the major supergroups of the Eukarya domain

o Distinguish between eukaryotic organisms by their common cell structure, specialization, and
nutritional mode, connecting structure to various ecological roles

o Compare differences in biodiversity values between two seasons

Vocab words from class:

Opisthokonts, Amoebozoans, Archaeplastids, Stramenopiles, Alveolates, Rhizarians, Excavates,

Autotrophic, Heterotrophic, Cilia, Flagella, Protists, Protozoans, Plankton, Phytoplankton, Algae,
Zooplankton

Useful resources for this lab:

1. How to use a compound microscope (Oregon State University)

https://www.youtube.com/watch?v=uEgM3gk8n6k

2. Phytoplankton identification guide (UCSC Ocean Data Center)

http://oceandatacenter.ucsc.edu/PhytoGallery/phytolist.html

3. Pond Life Identification kit (Microscopy UK)

http://www.microscopy-uk.org.uk/pond/
Introduction
Ponds are standing bodies of water that are, like lakes, lentic environments (in contrast to lotic
environments, or flowing water systems such as streams and rivers). The goal of this lab is to give you an
opportunity to explore the many organisms that live in lentic habitats. At first glance, a pond may appear
to be a homogeneous environment, but in fact, ponds and lakes have distinct zones with different physical
features. Each organism is adapted for life in the zone in which it lives, and some organisms will occupy
more than one zone during their lifetime.

Plankton is the broad term (it is not a taxonomic group) we use to refer to all the organisms found in
water whose movement is driven by the movement of water itself. This means either that the organisms
are unable to move or that they are motile but their size is too small to swim against the water current.
Plankton can be single-celled and/or multicellular organisms. Plankton include microscopic multicellular
animals (zooplankton), unicellular heterotrophs (protozoa), and unicellular plants (phytoplankton aka
algae), differentiated by the way they obtain nutrition. Autotrophic organisms (phytoplankton) produce
their own food from inorganic compounds and the energy of light (photosynthesis). Heterotrophic
organisms (zooplankton or protozoa) consume organic matter from other organisms, from dead, or
decaying material (detritus), or wastes. Autotrophic organisms will be generally recognized in this lab by
the presence of chloroplasts with photosynthetic pigments (chlorophyll and others) that will look
generally green, brown, or gold under the microscope. In fact, the color of these pigments will be a
relevant clue to help the identification. Heterotrophic organisms in this lab will be more or less
transparent. Organisms like the phytoplankton and protozoa are included in another broad grouping called
protists. Protists are a “junk drawer” of eukaryotic organisms that are neither true animals, plants, nor
fungi.

Example of a rotifer Example of a

(zooplankton) stramenopile
(phytoplankton)

Water samples containing a mixture of phytoplankton, zooplankton, and protists were collected from 5
local lentic waterbodies using a plankton net. When you pull a plankton net out of the surface waters, the
water drains from the net and concentrates the amount of plankton, as plankton are retained by the net. By
using fresh water samples, you can see the movement and true colors of the organisms. You will have
some samples to view that were collected from the Augustana Slough, Lake George, a backwater pond
from the Mississippi River, and/or purchased cultures. Today, using compound microscopes we will
count and identify (as you are able!) the protozoa, phytoplankton, and zooplankton from different seasons
using compound microscopes. The goal is for you to gain an appreciation of the diversity of planktonic
organisms and to learn about biodiversity measurements.

PART 1- Learn and the parts and methods to using a compound microscope
Part 1 should take you about 10 minutes to complete

Basic Rules for Protection of the Compound Microscope

Before you examine material with the compound microscope, you should take time to familiarize yourself
with the parts of this instrument. Since you will be using a microscope in many of the lab exercises, it is
important that you become comfortable and confident with its proper use and care. Microscopes are
delicate and expensive instruments that should be handled with care--

1. Always carry the microscope in an upright position, with its arm grasped by one hand and its base
resting on your other hand. Never tilt a microscope to one side, or turn it upside down; the eyepiece might
fall out.

2. Before you return the compound microscope to the cabinet, be certain that you have removed the last
microscope slide that you observed and that the lowest power objective lens is in place.

3. When using the compound microscope, first examine the material with a low-power (4X) objective,
never with a high-power (40X) objective. Once an object is in focus under a lowpower lens, you should
be able to switch to the next higher power (10X) without changing focus. You should be able to get the
object into focus with a very slight turn of the fine adjustment knob.

4. Never use the coarse-adjustment to focus downward with the high-power objective in place.

5. Always cover moist materials with a coverslip. When focusing, also be sure to maintain a safe distance
between the coverslip and the objective to avoid damage to the lenses.

6. Clean lenses only with lens paper, never with coarse materials such as handkerchiefs, facial tissue, or
cheesecloth, because they will scratch the lenses. If the lenses are very dirty, first moisten the lens paper
with distilled water.

7. If you cannot obtain a clear focus or good lighting--if your microscope seems to be malfunctioning—
immediately notify your instructor, because the instrument may require repair.
Parts of the compound microscope

Your instructor will walk you through the different parts of the microscope. The parts of the compound
scope are described below. After locating each part on the diagram, identify that part on your own
microscope.

Light source. On your microscopes, the light source is built into the base with a lens that focuses lightonto
the lower condenser lens.

Condenser. Contains a system of lenses that focuses light on the material or specimen. The condenser is
located beneath the stage of the microscope. Locate the condenser knob that raises and lowers the
condenser. The purpose of the condenser lenses is to provide a cone of light of sufficient angle to fill the
aperture (diameter of the opening) of the objective lens.

Iris diaphragm or aperture disk. Look beneath the stage of the microscope for these parts. They are used
to control the contrast and definition (image quality) of the material being viewed. In addition, they can
be used to increase or decrease the intensity of illumination; however, light control is the least important
function of the iris diaphragm or aperture disk. The size of the opening in the iris diaphragm can be
adjusted by moving the lever attached to it. The aperture disk contains several holes of different sizes.
The hole of the desired size can be placed in position by rotating the disk.

Objective lenses.
Revolving nosepiece, or turret. Most newer microscopes are parfocal, meaning that when an object is in
focus with one objective lens, the objective lenses can be changed without completely losing focus. The
power magnification of the lens is indicated on the side of the objective The numerical aperture is often
engraved on each objective lens. In general, the higher the value of the, the better the resolving power, or
resolution (that is, the ability to see as distinct two objects that are very close together) of the objective.
To a point, as magnification increases, so does resolving power. Magnification without increased
resolution is not advantageous for studying material.

Eyepieces or oculars. The lens(es) you look through (usually 10X). Microscopes will have either one or
two oculars; that is, it will be either a monocular or binocular microscope. Your microscopes are
binocular.

Stage. Holds the slide to be viewed.

Stage clips or mechanical stage The stage clips hold the slide in place. Instead of stage clips your
microscope may be equipped with a mechanical stage. A pair of knobs for controlling its movement will
be located along the stage.

Coarse-adjustment and Fine-adjustment knobs are located at different places on different types of
microscopes, they are used for focusing on material. Coarse adjustment is used for initial focusing at low
power. Fine adjustment is used to make very slight changes, allowing precision focusing at high power.

Base and Arm

PART 2- practice using the microscope to find and identify organisms

Part 2 should take you about 30 minutes to complete

Now that you have familiarized yourself with the compound microscopes, you will have a selection of
different cultured and freshwater samples to examine. Draw and describe some of the organisms you have
observed in the cultures and fresh samples. State the magnification you are using, and if possible identify
organisms (superkingdom at least!).

Sample: Sample:
Sample: Sample:

Sample: Sample:

PART 3- calculate different diversity measurements using sample data

Part 3 should take you about 10 minutes to complete

After you are comfortable finding and focusing in on the organisms you will use preserved samples to
compare the seasonal differences in plankton communities. We will consider three ways to measure
diversity:

1) Species Richness is a measure of the number of species in a community and is calculated by simply
counting the number of different species present in the community.

2) Species Evenness is the comparison of the relative proportion (hint: this is just like allele or genotype
frequency!) of each species. For example, a community with 30 dandelions, 30 buttercups, and 30 daisies
is more even than a community with 60 dandelions, 15 buttercups, and 15 daisies. We will not directly
discuss evenness but you should understand the concept, as we will need it to calculate species diversity.

3) Species Diversity is a measure that considers both richness and evenness. There are multiple ways to
estimate diversity but we will use the Simpson’s Index (D). To calculate the Simpson's Index, you will
first need to compute the pi (relative proportions or species evenness) values for each species observed in
each sample in Table 1 below. The larger the value of D the more even your sample is. What does it mean
to be more even?

The equation for Simpson’s index is: D = 1/ SUM of pi2

Complete Table 1 for the pi and pi2 values. The first one has been done for you (5/20 = 0.25).
Then determine species richness and calculate the Simpson's Index (D) for each, using the
information from Table 1.
Season 1 Season 2

Species ni pi pi2 ni pi pi2

A 5 0.25 0

B 5 10

C 5 2

D 5 2

E 0 6

(N) Total
D= D=
Individuals 20 20

Table 1. Count data from equal-sized samples of two different aquatic seasons. Note that the total individuals (N) of
each sample is equal to 20. It is important to use the same sample size when comparing different communities.

Having considered both species richness and Simpson’s Index, do you learn more about diversity
by considering the two together or by using either alone? Why do you think this is?

PART 4- calculate and compare the diversity of 2 different seasons from

preserved samples
Part 4 should take you about 45 minutes to complete

In your groups of 4, split into pairs. Each pair will calculate the species diversity for one season from the
preserved samples. For each season—
1. add a drop of the sample from your season (warm season or cold season) to a clean slide and place a
cover slip gently down on top of it (tip: start with one edge of the coverslip and gently lower the other
side).

2. Using the microscope as described above, explore the sample at the 10x objective (magnification of
100x [10x from eyepiece x 40x from objective]) by scanning through fields of view (the circle you see
when you look into the microscope) and describing the organisms that you see. When you place a new
season, you will identify all of the organisms in a field of view until you reach 50 organisms counted. On
the spreadsheet on Moodle, record each species (column B) you see and how many there are of each
species (column C). Work together in your groups to identify the species to the best of your ability. To
identify these organisms, take pictures to refer back to, look online, and use the handouts provided. You
will need to use hypotheses about their mode of nutrition as well as their morphological features visible
under the microscope, such as color, shape, size, and motility to narrow down the identification. Once you
name an organism (even if it is “Species A” or “Green Circle”) make sure to name every organism of
similar morphology that as well. We will need to be very careful of ‘naming’ new species so we can get
an accurate biodiversity measurement.

3. On a scrap piece of paper or this handout, when you record the organism include the species name (or
what you have named it) it would be helpful to complete a drawing/picture of it, superkingdom (if you
know it!), and some notes about how you identified it. After you have identified 50 organisms for each
season, you will use the number of each species to calculate the diversity for your two seasons as a
group of 4.
Using the spreadsheet on Moodle, calculate the Species Richness (count up number of rows in
column B) and Simpson’s Index (D) for your two seasons. Answer the questions below— These are
also available as a word document on Moodle that you can complete and submit 1 per group.

1. What is the Species Richness and Simpson’s Index for each season? Which one had a greater richness?
Greater diversity?

2. What environmental factors do you think could lead to the differences (or lack thereof) you see in
Species Richness and/or Simpson’s Index values between the two seasons? Do you think the organisms’
size/volume could play a role in their response? Why or why not?

3. What species concept were you relying on for your diversity calculations? How could you optimize this
procedure to get a more accurate view of what determines each species?

4. Some of these organisms are heterotrophs, autrotrophs, multicellular, unicellular, motile, nonmotile….
Highly diverse. What allows for this great diversity observed in these eukaryotes? Why have these
organisms not all become complex multicellular beings?