This document is available on the MCC public P: drive.
(when logged in through the school network
P:\Biol1000\Hay-Infusion\ )
Please also use the “Guide to Fresh-water Biology”, three “Pond Life Posters”, and other handouts provided in lab to identify your organisms.
Hay Infusion Experiment Handout
Introduction
The hay infusion cultures are extractions of nutrients from hay by boiling (2g/L hay in water, with sodium phosphate and
sodium sulfate buffers). A small amount of fresh hay was added to each culture to provide continuing nutrient addition
as the hay slowly decays. Room lighting plus closely placed incandescent bulbs provides the energy source for producers
at the base of the food web. All infusions were inoculated with organisms from a mature hay infusion culture. This
mature culture provided dozens of different types of organisms from all levels of the food web.
The four hay infusion Experimental Treatments are as follows:
Control Treatment
Acidic Treatment
All cultures started at pH7, room
temperature, no additional
chemicals or nutrients
Sulfuric Acid and Nitric Acid
added until the pH dropped
from pH7 to pH5 (100x more
acidic)
Enriched Treatment
Inorganic fertilizer, “Miracle
Grow” added
High Temperature
Treatment
Culture kept at 37°C
The Control Treatment simulates an unpolluted
environment.
H2SO4 and HNO3 are the two components of
acid rain that form in the atmosphere from
common pollutants.
Compounds of nitrogen, phosphorous,
potassium, and various micronutrients, are
common components of fertilizer runoff.
Thermal pollution is common with removal and
replacement of vegetation with hard, heat
absorbing surfaces, and from industrial and
power plant sources. Few if any eukaryotes
survive temperatures above 40°C.
You will be provided with samples from all the cultures, and access to the original cultures. Please take care that the
students do not contaminate the samples or especially the cultures with any of the chemicals and cleaners we keep in
the labs. Also, do not cross contaminate the cultures: keep the pipettes separate for each treatment.
Basic Procedures
Take color, odor, pH, temperature, turbidity, and organism observations, from the samples, but allow your students to
observe the original cultures to enhance their observations of odor, color, and general differences among the
treatments.
Color: Describe the color of the cultures. Green colors probably mean abundant photosynthesizer growth, while
browns indicate more decay and less photosynthesis. The culture’s original color was a light yellow, much like
the dry hay color.
Odor: Sniff the cultures and describe what you smell. Active biological growth will produce a variety of odors that are
not necessarily foul, while decay will produce foul, rotten, or moldy, unpleasant odors. The original extraction
smells like fresh hay.
pH: Use a dropper to place one or two drops from each treatment onto the pH paper, and read pH immediately. pH of
the cultures changes over time as respiration, decomposition, photosynthesis, organism growth and waste
production, all affect the chemicals present in the cultures.
Observe with the naked eye and with the microscope. Take samples from the top, bottom, and side.
Temperature: Record the temperature of the culture samples. All cultures except the High Temperature culture were
kept at room temperature. Record the High Temperature culture temperature as 37°C regardless of the
temperature of the sample.
Turbidity: Cloudiness – (not to be confused with color). Rank your samples on a six point scale with a “1” as clear as
water, and a “6” as completely opaque (cannot see through the culture water). The more turbid the water the
more growth and/or decay is occurring, and the more rich or eutrophic the culture is. Clearness generally
indicates less biological activity. The cultures began with slight turbidity, or rank 1.5.
Organism Observations
Make wet mounts from each culture sample to identify and count organisms.
Larger organisms: use the depression slides;
smaller organisms, use regular slides.

Observe first with the naked eye both the samples and the original cultures. Some of the organisms are
macroscopic. These may be captured individually with droppers, placed into depression slides, and
observed with dissecting scopes (or compound scopes at scanning power).

Keep in mind that these complex cultures will be full of non-living organic material and dead parts of
organisms called debris. Bits of hay, biofilms from bacteria, and other clumped organic matter may
accumulate on the bottom or float in the water. Don’t mistake the debris for living organisms.
 Take samples with a dropper from the top, bottom, and sides of each culture. Include a
small amount of the debris from the bottom as the organisms often collect in the debris.

Observe the wet mounts at scanning power first, as some of the organisms are large and active. Have
patience with the observations under low and high power, as some of the organisms (like amoebas)
move very slowly.
o Use Detain to slow the fast organisms, if you wish. I recommend making a second wet mount
that includes Detain, after your first observation without Detain reveals the need.

Use the pictures provided on later pages here, and the books/pamphlets/posters provided in lab to
identify and classify the organisms you
find.

Scan for and count organisms by starting
at a corner of the cover slip, and
scanning across and back over the entire
width of the cover slip, moving the slide
over by one field-of-view at each pass.
o Count individual organisms when
possible. If organisms are too abundant to count, estimate their numbers in 100’s, 1,000,s, etc.
You could create a relative scale of abundance: one, few, several, many, abundant.
On the next several pages are a few of the organisms you may find in the hay infusion cultures. There are
likely to be many others. Use the “Pond Life” guide book, the Pond Life Posters, and your
textbook to help you identify others. Different groups of organisms, and different organisms within
these groups, will be more or less sensitive to the experimental treatments we have applied to the hay
infusion cultures. Use your observations and counts to see how these experimental treatments have affected
the abundance of the many types of organisms you find in these cultures.
Observe with the naked eye and with the microscope. Take samples from the top, bottom, and side.
Kingdom Protista – mostly unicellular (some filamentous
or colonial), phototrophs and heterotrophs
Left: Amoeba – phagocytic
heterotrophs, secondary consumer, eating other
protista, amoeboid crawling motion, move with
pseudopodia
Below: Blepharisma – Classic
ciliate, large, often pinkish, ciliate with an undulating
membrane, ingests other protista through food
vacuoles formed at base of oral groove, secondary
consumer – these often cannibalize each other when
other food is scarce
Vorticella – stalked ciliate, primary
consumer, stalk contracts in a spring-like manner
when disturbed, ingests small food in food vacuoles
Paramecium – Classic ciliate,
ingests small food through food vacuoles formed
at base of oral groove, primary consumer
Observe with the naked eye and with the microscope. Take samples from the top, bottom, and side.
Stentor - Classic ciliate, large spiral of cilia leads
food into oral grove, ingests food through food vacuoles
formed at base of oral groove, primary/secondary consumer
Euplotes &
Stylonychia – ciliates with cilia
reduced and grouped into large, hair-like
“legs” that these cells appear to walk upon.
Often seen “crawling” quickly on surfaces.
The ciliates and amoebae are Kingdom Protista, Phylum Ciliophora, and Phylum
Rhizopoda, respectively.
You will find many other species of Protists in these cultures.
Categorize them as flagellates if you see long flagella or they swim
with a vibrating movement. If they swim smoothly or crawl with
many short cilia, call them ciliates. If they crawl about by the
formation of pseudopodia, call them amoeboids.
Observe with the naked eye and with the microscope. Take samples from the top, bottom, and side.
Colpidium – common small ciliate with a slight Chilomonas – common small,
curve from the oral groove near the anterior end and
well-defined ciliary rows, primary consumer
heterotrophic flagellate – two flagella
Colpoda– common small ciliate with a slight curve from the oral groove near the middle of the
cell, primary consumer
Coleps– common small
ciliate with a barrel-shaped body
covered in small calcareous
plates, a predator on organisms
as large as rotifers, and a
scavenger, it is a fast swimmer.
It uses the calcium plates
around its mouth as teeth to drill
into other cells and feed upon
their cytoplasm.
Observe with the naked eye and with the microscope. Take samples from the top, bottom, and side.
These are multicellular animals in Kingdom Animalia, even
when they are not much larger than the protista
Rotifers – Testudinella, left, Philodina, right, Kingdom Animalia, Phylum Rotifera.
Rotifers have a
“crown” of cilia on their heads, used for swimming and to bring food to their mouths. The Testudinella
name comes from its shell or “test”, and it is sometimes called the turtle rotifer. Philodina can swim
with its cilia or crawl like a leech, using sticky pads on its “toes”
Right: Planaria – flatworms, Kingdom Animalia, Phylum
Platyhelminthes. These are free-living scavengers and predators, but
many of their relatives are parasites such as flukes and tapeworms.
They have an arrow-shaped head covered in sensory organs,
including two “eye-spots” which can detect the direction of light but
cannot form an image. They have a sack-type digestive system with
only one opening.
Nematodes
– roundworms are recognized by their snake-like undulations. Kingdom Animalia,
Phylum Nematoda, these organisms molt their cuticle like the Arthropods molt their exoskeletons.
Annelid worms – segmented worms, aquatic relatives of the earthworms.
Left: Head and entire organism of Aeolosoma. They are found swimming in the water or among debris.
Right: Black Worms, Lumbriculus variegatus. They are found in the sediment at the bottom.
Both are in Kingdom Animalia, Phylum Annelida
Observe with the naked eye and with the microscope. Take samples from the top, bottom, and side.
Producers – photosynthetic Protista and higher plants
in Kingdom Plantae
Euglenoids – Kingdom Protista, Phylum
Euglenozoa. Euglena (right) is a ubiquitous photosynthetic
flagellate, noted for its vibrating swimming movement (due to
the whip-like actions of its long flagellum which pulls the
organism through the water) and for euglenoid motion – an
undulating dance-like movement of the entire cell
Green Algae – Kingdom Protista, Phylum Chlorophyta. These Chlamydomonas
(below) are single-celled relatives of filamentous Spirogyra, and colonial Volvox. They are
photosynthetic, and swim about with two short equal length flagella.
To the right is the green algae Closterium, members of the
group known as the desmids. They consist of two semi-cells
attached “head-to-head”. In some desmids, very conspicuous
vacuoles at the ends of the cell can be seen, with CaSO4
(Gypsum) crystals "dancing" around within them.
Higher Plants, Kingdom Plantae – Don’t miss these tiny “foot-ball”
shaped clumps of cells floating on the surface of some of the cultures. These are Wolffia, or
watermeal, the world’s smallest flowering plant. Kingdom Plantae, Phylum Anthophyta. These tiny
plants are actually more closely related to the large plants you are familiar with that have flowers and
fruits, rather than the tiny protistan algae shown above. These were collected from the pond in the
South Campus Environmental Education Center.
Observe with the naked eye and with the microscope. Take samples from the top, bottom, and side.
Higher Plants – You may also see a few Duckweeds (Lemna minor). They are also
flowering plants. Kingdom Plantae, Phylum Anthophyta. These were collected from the pond in the
South Campus Environmental Education Center.
The larger floating plants are Water ferns (Salvinia minima). They are in Kingdom Plantae, Phylum
Pterophyta – the ferns. Ferns have vascular tissue (veins of xylem and phloem) but do not produce
seeds, flowers or fruits. These water ferns produce leaves in whorls of three: two leaves float, are
covered in hairs that repel water, and look like leaves, while the third leaf is modified into what looks
and functions like a root, being submerged and covered with root-like extensions. This plant is a
native of Florida. The picture below left also contains some duckweed.
Observe with the naked eye and with the microscope. Take samples from the top, bottom, and side.
Larger Animals: These are multicellular animals in the Kingdom Animalia,
Phylum Arthropoda, Class Crustacea – crustaceans – relatives of crabs,
lobsters, and more distantly to the spiders, and insects
Copepods – This one is Cyclops. They are common
predators, and it is easy to see where they got their name. The
female below is carrying two egg sacks. They swim using two
long antennae.
Ostracods – These crustaceans live inside their clam-like shells, crawling or swimming with
legs and antennae that stick out of the “shell”. They are sometimes called seed shrimp. Many are
scavengers. Your cultures may contain both a large species with a mottled shell and a smaller species
with a very “hairy” shell.
Amphipods – These large
crustaceans are important food for many
larger animals like fish. They are laterally
compressed and have several types of
specialized legs (hence the name amphipod
“different footed” - unlike their cousins the isopods or
“roll-up bugs” that are dorsoventrally flattened and
have legs that are all the same). They are mostly
detritivores and scavengers. At far right is a
mating pair, male on top. The females only
lay eggs when they molt, so the male grabs
on waiting for his one opportunity to fertilize
the eggs when she finally releases them.