BIOL 153L General Biology II Lab
Black Hills State University
Lab 1: Diversity of Photosynthetic Organisms I
Photosynthesis is a process by which organisms use light to produce food in the form of complex
organic compounds. This is achieved by complicated biochemical machinery and uses light energy,
water, and trace nutrients (such as nitrogen) from the external environment. Organisms that make
their own food—using the energy of light or inorganic molecules—are called autotrophs. Organisms
that cannot generate their own food, but must instead eat other organisms, are called heterotrophs.
Organisms that will make their own food and eat other organisms are called mixotrophs.
Photosynthetic Organisms:
People most often associate photosynthesis with true plants such as moss, ferns, conifers, and
angiosperms (flowering plants). However, some bacteria, protists, and fungi utilize photosynthesis;
in the past, many of these organisms were classified as “plants" and studied in botany courses. The
purpose of Labs 1 & 2 is to introduce the diversity of photosynthetic organisms (as well as plant-like
organisms traditionally studied as part of botany courses) while highlighting the major structures,
functions, and ecological characteristics of these species (Chapter 12, pp. 248-250).
Photosynthesis on the Tree of Life:
Biologists' understanding of organismal classification has changed greatly over the past 150 years.
Early conceptions of the Tree of Life recognized two kingdoms, animal and plant, the latter including
bacteria, algae, many protists, and fungi in addition to true plants. A three kingdom system separated
protists from plants while a four kingdom system had prokaryotes as distinct from eukaryotes. In
the 1960s, a five kingdom system was developed and remained in favor for several decades—this
recognized Animalia, Plantae, Protista, Fungi, and Monera (prokaryotes). Concepts of organismal
diversity have changed radically since the 1990s, as DNA sequences have provided insights especially
for small organisms that lack easily measured phenotypic traits. Take notes on your instructor's
description of the current placement(s) of photosynthetic organisms on the Tree of Life.
In the space below, sketch the 'Tree of Life' presented by your instructor at the start of lab.
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SCIENTIFIC EQUIPMENT: TOOLS OF BIOLOGICAL RESEARCH
Microscope history:
For thousands of years, people have been creating simple magnifiers. Ancient Greeks and Romans
looked through drops of water, ancient Romans developed simple magnifying lenses, and technology
existed for making eyeglasses as early as the 13th century. However, it wasn’t until the 16th century
that “real” microscopes were developed. Crude microscopes with poor glass quality and limited
magnification (<10x) were introduced in the 1590s, allowing people to view previously unseen—and
unknown—things. In 1665, Robert Hooke published the first scientific bestseller, Micrographia,
which contained many drawings made of microscope images. Hooke called the honeycomb shapes
he saw when viewing plant tissues "cells," which was the first usage of that word. Highly-magnified
views were not seen until Anton van Leeuwenhoek discovered that glass beads, carefully polished
and placed into a handheld device, provided cleanly magnified images. He obtained magnifications
in the range of 250-500x, comparable to modern microscopes. These high magnifications allowed
van Leeuwenhoek discover ultra small living things—single-celled “animacules” swimming in pond
water, bacteria from dental plaque, mobile sperm cells, and so forth.
Although effective, each of van Leeuwenhoek’s microscopes had only one magnification—to reduce
or increase magnification, he needed a different instrument. By the end of his life, van Leeuwenhoek
had hundreds microscopes, only nine of which are still in existence. In contrast, modern microscopes
have several magnifications, and they achieve high magnification using two lenses rather than glass
beads. The magnifying lens closest to the viewer’s eyes is called the ocular lens, and generally has
10-15x magnifying power. The magnifying lenses closest to the object are called objective lenses.
There are 3-4 objective lenses that range in power from 5-100x. The actual image magnification is
the product of the ocular and objective magnifications. Thus, when the 5x objective lens is combined
with the 10x ocular lens, the total magnification is 50x. (Super-powered objective lenses require oil
to function and are not present on our microscopes; you may use oil lenses in upper-level courses.)
In BIOL 153L, we will primarily be using compound light microscopes. These are able to give high
magnifications but only very thin objects can be viewed. Normally, these are placed on slides for
viewing. Occasionally, we will use dissecting microscopes. Although the magnification of these
scopes is lower, these may be used to explore larger objects that are not mounted on a slide.
Microscope parts:
Ocular lens (eyepiece): The lens the viewer looks through. The ocular lenses on BIOL 153L
microscopes are 10x power.
Diopter adjustment: Used to change focus on one ocular lens to adjust for differences in vision
between the viewer’s two eyes.
Objective lenses: The lenses closest to the specimen. The Bio 153L microscopes have three
objective lenses (5x, 10x, and 40x). Be careful that the objective lens doesn’t crash into slides!
Stage: The flat platform where the slide is placed; stage clips hold the slide in position.
Stage control: This knob moves the stage to right or left, which allows proper positioning of the
slide under the objective lens.
Brightness adjustment: Used to adjust light intensity. Often, the brightest setting does not lead to
the best image viewing; different specimens sometimes require different levels of brightness.
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Coarse adjustment: Used to bring the specimen into “pretty good” focus. Because the stage moves
up and down quickly in response to the coarse adjustment knob, there is a risk of crashing into the
glass slide when using coarse adjustment. (The microscope has a “stop” to reduce the risk, but
crashes can still happen). Only use coarse adjustment on lower powers, and focus “up” when your
eyes are on ocular lenses. If you need to focus “down,” look at the slide.
Fine adjustment: Used to fine-tune the focus obtained with coarse adjustment.
Base: This supports the entire microscope.
Arm: This connects the base of the microscope to the rest of the body. It is also used as a “handle”
when carrying the microscope. Hold the microscope on the base AND the arm when carrying.
Generalized microscope diagram showing key parts.
Compare the photo above to the microscopes used by BIOL 153L. What parts are present on
the real microscope that aren’t shown above? Why do you think the microscope illustrated in
the photo lacks these particular components?
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Important rules to protect microscopes!
1. Don’t drop. Carry microscopes with two hands—one hand on the arm, one hand under the base.
If cords are dangling, wrap them around microscope so they aren’t a trip hazard.
2. Don’t scratch. Never clean microscope lenses with paper towels—only use special lens paper.
Before embarking on any microscope cleaning, consult with the instructor.
3. Don’t crash. Be careful not to crash the objective lens into the slide.
4. Store right. When putting the microscope away, put the lowest power objective lens—or empty
space—into position. Move stage to lowest position. Wrap cord around microscope and cover.
EXERCISE IN USE OF THE LIGHT MICROSCOPE
Familiarize yourself with the parts of the microscope listed above. Be prepared to use these
terms in conversation and for lab exams! You'll be using microscopes frequently in BIOL 153L.
1. Find the coarse and fine adjustment knobs and move the stage up and down. Use coarse
adjustment to put the stage into the fully down position (maximum distance between stage and
objective lens). This is the position from which you should always start viewing a slide.
2. Find the objective lenses, and carefully twist them into position.
a. What is the magnification of the three objective lenses?
__________
__________
__________
b. What is the magnification of the ocular lens? _____________
c. When using each objective lens on this microscope, what are total magnifications (multiply
the magnification of the ocular and objective lenses)?
first objective, low power
=>
x total magnification
second objective, medium power
=>
x total magnification
third objective, high power
=>
x total magnification
4. Find the light switch and turn on. Find the brightness control and dim and brighten the light.
5. View a prepared slide. Turn on light, confirm that the lowest power objective lens is in the
viewing position, and take a prepared slide from the front of the room. Place the slide on the stage,
make sure that the stage clips hold the slide in the proper position. Use the stage control to move the
slide into position—the light beam should shine on the image to be viewed. Note that the “barrels”
that hold the ocular lenses can be moved in and out to adjust for different spacing between people’s
eyes. Move these so that the ocular lenses are properly spaced for your eyes.
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Use the coarse-adjustment knob to bring image into decent focus. If necessary, use fine-adjustment
knob to bring image into sharp focus. When eyes are on objective lenses, only go up with the
coarse-adjustment knob. If you need to focus down, watch the slide so that you don’t crash into
the slide with the objective lens. Now turn the medium power objective lens into position—keep
the slide in the same position! The image will probably be blurry, so use focusing knobs to bring
into sharp focus. Next, turn the highest power objective lens into position. At high power, only use
fine adjustment control to bring into focus. If you completely “mess up” the focus, use a lower
power objective to get the focus back. It is quite difficult to start focusing “from scratch” using
the highest power objective lens, and thus more efficient to move to the lower power lens.
6. Investigate diopter adjustments. For most people, vision is not equally sharp in both eyes. The
diopter allows you to make changes so that both eyes can view the image with maximum sharpness.
Notice that one objective lens has “+” and “-” markings; the top of that objective lens can be turned
slightly clockwise or counterclockwise.
Try it! Use the medium power objective lens to bring a prepared slide focus. Close your right eye,
and use the adjustment knobs to bring the slide into sharp focus for that eye. Now close your left eye
and view the image with your right eye. If the image is still in sharp focus, you do not need to adjust
the diopter. If the image is blurry, turn the diopter knob to bring it into sharp focus for that eye. Now
if you look through the ocular lenses, the focus should be perfect for both eyes. Note the position of
the adjustment: in the future, you can turn the diopter to the correct adjustment before you start.
There are 'types' of slide preparations!
Whole mount (w.m.): the entire organism is mounted (e.g., very small organisms such as will be
viewed in Lab 2).
Epidermal mount (sometimes considered whole mount): the outer layer of an organism is
mounted (e.g., stomatal peels such as will be viewed in Lab 3).
Cross section (c.s. AKA transverse section): sliced perpendicular to the main axis
Longitudinal section (l.s.): sliced parallel to the main axis
1. One cross section and longitudinal section are shown below for a pencil; this cross section
was taken from location “A.” Now sketch cross sections of the pencil from “B” and “C.”
c.s A
c.s B
c.s C
l.s
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2. Note that sections vary in appearance depending on sample location. What are the big
differences between sections A, B, & C?
View prepared slide of Venus Fly Trap (Dionaea muscipula, Droseraceae)
Let's practice use of the microscope today with a relatively large photosynthetic organism – the
Venus Fly Trap! These are cool carnivorous plants that are threatened by over-collection (one should
be wary of purchasing Fly Traps, as it likely was illegally collected). The podcast series Criminal,
Episode 5, gives some background and for sure is worth a listen outside of class.
Watch Fly Trap video: http://www.discovery.com/tv-shows/life/videos/venus-flytrap-catches-flies/
Link to Podcast: http://thisiscriminal.com/episode-five-dropping-like-flies-4-24-2014/
1. Pick up the Venus Fly Trap slides, including the whole mount (slide #13) and cross section (slide
#14), from the table organized by the instructor. Look at these slides and draw the structures you
observe in the space below. Please label and indicate magnification for your sketches.
2. On the drawing of a Venus Fly Trap below, indicate where the whole mount and cross
section were collected. In this class, always compare what is being viewed on a slide to the
whole organism! Be sure to label your sketches.
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3. Using information presented in the video and textbook, interpret the structures you drew above.
What are these called and what functional purpose(s) do they serve for the plant?
We'll be talking more about carnivorous plants in later labs; in the meantime, you can learn more about
the Venus Fly trap, if you'd like, from your textbook (chapter 28, pp. 678-680; chapter 29, p.29).
SURVEY OF "SMALL" PHOTOSYNTHETIC ORGANISMS
The focus of the remaining lab section will be on tiny photosynthetic organisms; we'll start
with what is generally the very smallest – the photosynthetic bacteria.
1. Cyanobacteria. One of the clearest divisions of life is between prokaryotes (without nuclei) and
eukaryotes (with nuclei); cyanobacteria are photosynthetic prokaryotes. Historically called bluegreen algae, biologists now prefer to reserve the term algae for certain photosynthetic eukaryotes.
Cyanobacteria do not have nuclei nor chloroplasts in which to conduct photosynthesis; rather, they
have chlorophyll pigments in their cells. The chloroplasts of true plants originated as a free-living
ancestor of modern day cyanobacteria, which are related to both heterotrophic and chemo-autotrophic
eubacteria but more distantly related to archaebacteria; see textbook, chapter 13, pp. 263-266.
Cyanobacteria are found in fresh and ocean water, in soil and rocks, and even in the fur of sloths!
Some cyanobacteria fix atmospheric nitrogen into a chemical form that is usable by true plants.
Cyanobacteria can also be poisonous. During summer months, surface water may experience massive
growth of cyanobacteria, sometimes called “toxic algae blooms.” Animals—including mammals,
birds, and potentially fish and insects—that consume or even swim in such waters can become ill. In
summer 2014, a large cyanobacteria bloom in Lake Erie polluted water sources in Toledo, OH, leaving
1000s of residents without drinking water. Follow these links to read more about toxic algae blooms.
http://www.nbcnews.com/science/environment/toxic-algae-blooms-persist-lake-erie-experts-say-n172466
http://www.dec.ny.gov/chemical/77118.html
Based on this information, describe some factors that may cause toxic algae blooms.
Explain why the phrase “toxic algae bloom” is not botanically accurate (hint, focus on “algae”).
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New Method: Preparing a Wet Mount:
Take a clear glass slide and a cover slip from the front of the room. Use paper towels to clean
fingerprints or watermarks off slides. Reuse paper towels to reduce waste. It is okay to use paper
towels on slides, but never use paper towels on microscope lenses.
Use provided pipette to put 1 drop of liquid at center of slide. Do not contaminate pipettes! They
are labeled for the sample for which they should be used. Carefully drop cover slip over droplet. If
lots of liquid oozes from edge of cover slip, there is a risk you will get liquid onto the objective
lens. Place a paper towel at the edge of the cover slip and remove some of the liquid.
Follow the steps used with prepared slides to view. Note that wet mounts may not be as flat as
professionally prepared slides so there is a greater risk of crashing the objective lens. It may
also be more difficult to get objects into focus, due to the greater depth of field. When finished, clean
slides and cover slips and return to designated area. Some slides may require a rinse under the faucet.
Others can simply be wiped clean with a paper towels. In this course, broken plastic cover slips may
be discarded in regular trash; broken glass slides cannot be discarded in the regular trash because they
could cut the custodians. Please place those into a broken glass container!
Wet mounts of cyanobacteria: Note that both species you will be viewing are filamentous.
However, cyanobacteria can also be unicellular, or exist in colonies of balls or sheets.
1.1. Anabaena is a filamentous cyanobacteria that can fix nitrogen via cells called heterocysts.
Sometimes Anabaena forms a symbiotic relationship with the roots of true plants, and rice
plantations often use it to fertilize plants. Anabaena is one of the species of cyanobacteria that can
produce neurotoxins that are harmful to animals.
Using a disposable pipette, make a wet-mount of Anabaena (see textbook, chapter 13, p. 265) and
observe it at increasing magnification up to 400x. In the space below, draw a single filament of
Anabaena that includes a heterocyst; heterocysts are more circular than other cells in the chain.
Thick-walled, large, and less circular akinetes, the resting stage for Anabaena may also be present.
Sketch the Anabaena filament. Draw it to scale, using field of view as a reference. Note the
magnification and label heterocysts and akinetes if you see them.
What does it mean that Anabaena forms a symbiotic relationship with other plants? How may
this benefit the other plant? How may this benefit Anabaena?
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1.2. Spirulina is a filamentous cyanobacteria that grows in tropical and subtropical waters, and is
widely sold as a dietary supplement. Although Spirulina itself is safe, there is a risk that cultures can
be contaminated by toxic cyanobacteria. Because food supplements are not well regulated, some
health experts advise avoiding Spirulina consumption. The carotenoid pigments in Spirulina, and
other cyanobacteria, give rise to the pink color of flamingoes and shrimp.
Sketch a Spirulina filament; draw to scale (use FoV as a reference) and note magnification.
Why do you think this cyanobacteria is called Spirulina?
2. Protists, which are eukaryotes, have long been the junk drawer on the Tree of Life. Organisms
that are not quite plant, animal, or bacteria have been placed here, even as biologists recognized that
the Kingdom Protista was not a natural grouping. Over the past 15 years, this group has undergone
massive re-organization based on molecular sequence data, but there still is not clear consensus about
how these organisms should be sorted. Thus, for this lab, we will stick to classical terminology and
simply refer to the group as "Protists." Note that protists can be heterotrophic, autotrophic,
mixotrophic and that some types of algae are included in the protists; see textbook, chapter 15.
2.1. Flagellates are often green (like plants) and mobile (like animals). The genus Euglena is a wellknown example of unicellular flagellate protists. Euglena are found in both fresh and salt water; some
members of the genus are non-photosynthetic while other members contain chloroplasts. Those with
chloroplasts may be autotrophic or mixotrophic. Euglena chloroplasts are different from those
found in true plants, and it is believed they originated via consumption of eukaryotic green algae.
Using a disposable pipette, make a wet-mount of Euglena (see textbook, chapter 15, p. 324). Spend
some time watching them move, then try to find one that is mostly stuck in one spot—it is your best
chance to see the internal parts. Euglena move via flagella (singular=flagellum); you cannot see the
flagellum on individuals that are actively swimming, but you may be able to see it on those resting in
one place. Euglena also have a distinctive red eyespot (=stigma) that senses light in the environment.
You may be able to see the nucleus, though this is often obscured by chloroplasts.
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Draw Euglena, using FoV as a reference and noting magnification. Label parts that you see.
For Euglena, what is the advantage of mixotrophy? How may the eyespot help Euglena?
3.1. Dinoflagellates are a diverse group of unicellular, biflagellated protists found in fresh and
marine waters. Due to the orientation of their flagella, dinoflagellates tend to spin like tops when
they move. Like Euglena, dinoflagellates include autotrophic, heterotrophic, and mixotrophic species.
Photosynthetic dinoflagellates tend to be golden brown or red, and their choloroplasts probably
originated from other unicellular eukaryotic algae. Some dinoflagelates are endosymbiotic with
marine animals and are essential for coral reefs. Others are parasitic and can consume their host
from the inside. When dinoflagellate algae blooms occur, the water can turn red, an event referred to
as red tide. (Some dinoflagellate blooms are colorless, so the name “red tide” may be a misnomer.)
During blooms, toxic chemicals may be released and lead to paralytic shellfish poisoning: filterfeeding shellfish accumulate the toxins, and pass them along to predators (including humans!) higher
up on the food web. The old saying “don’t eat shell fish in months without an R” is due to the fact
that “red tides” are more common in hot summer months (chapter 15, pp. 323 & 327-330.
Using a disposable pipette, make a wet-mount of Peridinium. This critter can be a bit tricky to
locate. Start at the lowest magnification, and look for brown, moving spherical objects; you may need
several wet mounts before you find one. If you make a good wet mount, consider sharing it with
your neighbor! 100x magnification is probably the best for viewing Peridinium, but try 400x. Spend
some time watching it move. This is an armored dinoflagellate (note the plates on its outer surface).
Draw a Peridinium to scale, using field of view as a reference and noting the magnification.
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Some species of dinoflagellates are bioluminescent (see textbook, chapter 6, p. 117) and will emit
short bursts of blue-green light when startled. Because of this, dinoflagellates are sometimes called
“fire algae.” As shown in the video Bioluminescence Surfing XTreme Video (4:37), filmed in
California, dinoflagellates can produce very impressive light displays. (No need to watch the entire
video; the actual surfing starts at the 1:15 mark.)
Watch the fluorescent dinoflagellates video: http://youtu.be/uUbIWqiynBY
In the 1990s, the colorless species, Pfiesteria, was linked to fish kills and harm to human health;
fisherpeople and Pfiesteria researchers claimed to suffer neurological damage from exposure to its
toxins. Danger posed by Pfieseria is now debated. Watch the video Pfiesteria Update: An Enduring
Debate, MDSeaGrant (4:57) to see competing scientific viewpoints.
Watch the Pfiesteria Update video: http://youtu.be/IAFVC1T0KpU
Briefly explain the competing viewpoints about Pfiesteria toxicity.
Are researchers on both sides following the scientific method? Briefly explain your answer.
Why might Pfiesteria researchers obtain different results?
3.2. Diatoms are exceptionally common in fresh and marine waters, and also occur in soils and on
damp surfaces. They have huge ecological importance. It is estimated that nearly half of an ocean’s
primary production results from diatom photosynthesis. Thus, they provide much of the energy to
the marine food web and are also major players in ocean carbon sequestration. Diatom cell walls
are composed of intricate silica plates (i.e., glass) that are lovely to look at and resistant to decay.
Diatomaceous earth—used as a filters, insect repellant, food additive, and scouring powder—is
composed of the geologic remains of diatoms. Diatom cells are rich in lipids, or oils, which makes
them a high-energy food source. In fact, the “fishy” flavor in marine life originates in part from the
consumed diatom oils! Diatom chloroplast are generally yellow-brown or green, and likely originated
by secondary capture of eukaryotic red and green algae; see textbook, chapter 15, pp. 330-333.
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View a permanent slide of mixed diatoms (slide #225). Sketch some of the shapes that you
see. Draw to scale, using the field of view as a reference. Note the magnification.
South Dakota rivers are being invaded by the diatom Didymos (AKA “Rock Snot”). Unlike most
invasive algae, it requires clear, unpolluted rivers—which we have. They were found in Rapid Creek
in 2005, and have spread to other rivers. A photograph of solitary Didymos is shown below (note
the glass cell wall and the central line that separates the two halves). Unfortunately, this unicellular
organism forms colonies: severe infestations are slimy brown and have edges that kinda resemble
toilet paper. Watch Didymo: the invading alga to see an infestation (watch the 2:00 – 3:00 section;
note that this is a Spanish video with subtitles!).
Watch the Didymo video: http://y2u.be/Kk_Ti-y1OWk
Why do you think SD Game, Fish, & Parks is so concerned about the spread of Didymos into
local streams? In other words, how might it damage aquatic ecosystems?
How do you think the spread of “Rock Snot” may be slowed? Why is the dispersal of this
particular organism so difficult to control?
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