Download LAB 13 - Nitrogen Cycling

LAB 13 - Nitrogen Cycling
April 01-05, 2002
Purpose
Learning Goal
Students will gain an understanding of the significance of the nitrogen cycle in both human and ecological
contexts.
Overview (Strategy)
Students will follow a schematic diagram of the
nitrogen cycle as they complete activities that
address sources of input (ammonia, urea, uric acid,
fertilizers) and environmental response to varying
levels of nitrogen products.
Time
One Lab period
Level
Introductory non-majors BIOL101/102/105
Prerequisites and Concurrent Lectures
Human Nutrition and energy conversion
Primary producers
Agriculture and Sustainability
Background
The metabolic processes of animals produce
wastes that are harmful and must be removed. In
simple animals, this is accomplished by diffusion
alone, but more complex animals use special organs
or organ systems. The principal metabolic wastes
are carbon dioxide (CO2), formed by cellular
respiration, and nitrogenous wastes, produced by
the breakdown of amino acids
composing proteins. Most animals use respiratory
organs to expedite the removal of CO2 and have
special excretory organs to get rid of nitrogenous
wastes.
When amino acids are broken down, the amine
groups tend to form toxic ammonia. This substance
diffuses from small, simple animals, but diffusion is
inadequate for removal of amine groups in larger,
more complex forms. In aquatic vertebrates,
nitrogenous wastes, including ammonia are flushed
from the body with large amounts of water.
Terrestrial forms, which must conserve water,
convert ammonia into urea and uric acid. Urea, the
primary nitrogenous waste in amphibians and
mammals, is secreted in aqueous solution, but
uric acid, the primary nitrogenous waste in insects,
reptiles, and birds, is excreted as a semisolid mass
with relatively little water.
The process of urinary excretion not only
removes the nitrogenous waste products of
metabolism but also regulates the concentration of
ions, water, and other substances in body fluids.
The urinary systems of marine, freshwater, and
terrestrial vertebrates are each uniquely adapted to
perform these functions in radically different
environments.
What to Do and How to Do It (Students)
PART I
Nitrogen and the human perspective – Urinalysis
In humans, the composition of urine is commonly
used to assess the general functioning of the body.
Diet, exercise, and stress may cause variations in
composition and concentration, but significant
deviations usually result from malfunctions of the
body.
Materials Needed:
Biohazard bag
Collecting cup, plastic
Multi-test reagent strips
Simulated urine samples
Test tubes and holder
In this section you will use urinalysis dip sticks to
analyze several simulated urine samples to determine
if they are normal or if their characteristics suggest
Reformatted from Gunstream labs 22 and 39
Page 1
possible disease. Table 1 indicates normal values and
some abnormal characteristics. Study it before
proceeding.
4/8/2002
1.
2.
3.
4.
5.
Obtain six reagent strips (dipsticks) and a color chart that indicates how to read the results. Study the
color chart carefully to be sure that you understand the time requirements for reading the dipsticks and
the location and interpretation of each reagent band.
Obtain six numbered test tubes containing simulated urine samples. Place them in a test tube rack at
your workstation.
Analyze the urine samples one at a time by dipping a dipstick completely into the sample so that all
reagent bands are immersed. Remove the dipstick and place it on a paper towel with the reagent bands
facing upward. Read your results after one minute.
OPTIONAL - If you wish to test your own urine, obtain a plastic collecting cup and a reagent strip from
your TA and perform the test at home. Wash your hands with soap and water. Read your results.
Record your results on the assessment sheet, page 6.
Component
Normal
Abnormal (Not all possibilities are listed)
Color
Straw to amber
Turbidity
Clear to slightly turbid
pH
4.8 to 8.0 (average 6.0)
Specific Gravity
1.003 to 1.035
Blood or hemoglobin
Absent
Protein
Absent or trace
Glucose
Ketones
Absent or trace
Absent or trace
Bilirubin
Small amounts
Pink, red-brown, or smoky urine may indicate blood
in the urine. The higher the specific gravity, the
darker the color. Nearly colorless urine may result
from excessive fluid intake, alcohol ingestion,
diabetes insipidis, or chronic nephritis.
Excess and persistent turbidity may indicate pus or
blood in the urine.
Acid urine may result from a diet high in protein
(meats and cereals) or from a high fever; alkaline
urine results from a vegetarian diet or bacterial
infection of the urinary tract.
Low values result from a deficiency of antidiuretic
hormone or kidney damage that impairs water
reabsorption.
High values result from diabetes
mellitus or kidney disease, allowing proteins to enter
filtrate.
Presence of intact RBCs may result from lower
urinary tract infections, kidney disease allowing RBCs
to enter filtrate, lupus, or severe hypertension.
Presence of hemoglobin occurs in extensive burns and
trauma, hemolytic anemia, malaria, and incomplete
transfusions.
Proteins are present in kidney diseases that allow
proteins to enter the filtrate, and they may be present
in fever, trauma, anemia, leukemia, hypertension, and
other nonrenal disorders. They may occur due to
excessive exercise and high-protein diets.
Presence usually indicates diabetes mellitus.
Presence results from excessive fat metabolism, as in
diabetes mellitus and starvation.
Excess may indicate liver disease (hepatitis or
cirrhosis) or blockage of bile ducts.
Presence indicates a bacterial urinary tract infection.
Presence indicates a urinary tract infection.
Nitrite (bacterial)
Absent
Leukocytes (pus)
Absent
Table 1. Urine Components Evaluated by Urinalysis
PART II
Nitrogen and an environmental perspective
Aquatic microecosystems
Pollution often results from the by-products of human
activities accumulating in the environment at levels
that are harmful to living organisms. Sources of
pollution are varied. For example, air pollution is
usually caused by submicroscopic particles, sulfur
oxides and nitrogen oxides produced by the burning
Reformatted from Gunstream labs 22 and 39
Page 2
of coal or petroleum products. Lakes and rivers may
be polluted by pesticides and fertilizers in water
runoff from agricultural land, contaminated
wastewater from mining operations or chemical
plants, and sewage from cities. Whatever the type
and source of pollution, it overloads the natural
4/8/2002
processes of an ecosystem and damages its delicate
balance by altering one or more populations within
the community of organisms. Its effect may be
sudden and dramatic or gradual and subtle.
In this section you will analyze aquatic
microecosystems set up in the laboratory to simulate
three lake environments: (1) Normal, (2) polluted
with acid rain, and (3) polluted with organic
materials. Your task is to compare these ecosystems
to ascertain the effect of acid and organic pollution
on the diversity and density of organisms.
Acid rain is a side effect of air pollution. Sulfur
and nitrogen oxides react with water in the
atmosphere to form sulfuric acid and nitric acid,
respectively, which are returned to Earth in rain.
Rainwater with a pH of less than 5.6 is considered to
be acid rain. Lakes and ponds exposed to acid rain
have a lowered pH, which adversely alters their
biogeochemical cycles, decreasing the availability of
nutrients.
Lakes and ponds normally undergo a process
called eutrophication, which gradually increases the
available nutrients. Eutrophication over many years,
leads to changes in the community of organisms
within an ecosystem and, ultimately, leads to the
conversion of a pond to a meadow. However, the
process is so slow that it is seldom noticed within a
person’s lifetime. In contrast to the slow process of
eutrophication, organic pollution of lakes and ponds
by fertilizers or sewage causes a sudden increase in
the available nutrients, which alters the ecosystem by
favoring some members of the community while
harming others. A polluted aquatic ecosystem tends
to have a reduced concentration of oxygen, which
may kill off desirable species such as gamefish.
Materials Needed:
Aquatic ecosystems in small aquaria simulating (1) normal, (2) acid pollution, and (3) organic pollution.
Small beaker
Algae key
Pipettes
Slides with cover slips
1.
Observe
the
three
microecosystems
macroscopically to determine the relative
density of autotrophs. Do this by noting the
degree of clarity or greening color of the water.
Record your observations on the assessment
sheet, page 7.
2. Use figure 1 to learn the recognition
characteristics of the crustaceans; Gammarus,
Cyclops, and Daphnia. Gammarus is the largest
form and tends to stay near the bottom of the
container. Daphnia is intermediate in size and
swims in the water by pulsating beats of the
antennae,
as
it
engulfs
microscopic
photosynthetic organisms.
Cyclops is the
smallest and will be either swimming in the
water or attached to the filamentous algae or to
the sides of the container; all these crustaceans
can be located with the unaided eye or with a
hand lens.
3.
Locate the crustaceans in the microecosystem.
Note the type and relative density of the
crustaceans in each system. Record your
observations on the assessment sheet, page 7.
4.
Learn the recognition characteristics of the
autotrophs in the microsystems by studying the
diagrams and the demonstration slides provided.
5.
Determine
the
autotrophs
in
each
microecosystem by making slides of water from
Figure 1. Heterotrophs
Reformatted from Gunstream labs 22 and 39
Page 3
4/8/2002
each microecosystem and observing them with
your microscope. If filamentous algae are
present, remove a small amount with forceps and
mount it on a slide for observation. Most of the
autotrophs are very small. Use the 10X objective
to locate a specimen, then switch to the 40X
objective to identify it. Use reduced illumination
for best results. It may be necessary to male
many slides to locate specimens in the
microecosystem with the lowest density of
autotrophs.
Record your results on the
assessment sheet, page 7.
Figure 2. Autotrophs
Reformatted from Gunstream labs 22 and 39
Page 4
4/8/2002
PART III
Testing for ammonia, nitrates and nitrites
(Standardized test kits using specific reagents and color keys) Instructions will be provided in class.
The Nitrogen Cycle
Vocabulary and Glossary
Acidic
Cellular respiration
Alkaline
Diffusion
Ammonia
Diuretic
Autotroph
Eutrophication
Bilirubin
Filtrate
Heterotroph
Ketones
Metabolic
Nitrate
Nitrite
Reagent
Turbidity
Urea
Uric acid
Suggested Reading
Campbell Chapter 20: Pages 460-470
References and Resources
Gunstream, Stanley E. 1996. Explorations in Basic Biology. Prentice Hall, New Jersey.
Harmful Algal Blooms Event around the globe
http://www.redtide.whoi.edu/hab/notedevents/notedevents.html
Marine Notes Uncommon Blooms: The Nitrogen Factor
ftp://ftp.mdsg.umd.edu/pub/MDSG/MarNotes/MN16_3.PDF
Red Tide Maps
http://www.redtide.whoi.edu/hab/
Reformatted from Gunstream labs 22 and 39
Page 5
4/8/2002
Lab 13 – Nitrogen Cycling
Assessment Sheets
Name ______________________
Section _____________________
PART I - Urinalysis
Record the results of your analysis of the simulated urine samples in the table below. Use an X to indicate the
presence of a component and record the color and specific gravity.
Tubes
Characteristic
1
2
3
4
5
6
Glucose
Bilirubin
Ketone
Specific Gravity
Blood
PH
Protein
Nitrite
Color
Indicate for each urine sample the health condition of the “patient” based on your urinalysis. Select the health
condition from the list that follows:
Tube 1 ____________________________________________________________________
Tube 2 ____________________________________________________________________
Tube 3 ____________________________________________________________________
Tube 4 ____________________________________________________________________
Tube 5 ____________________________________________________________________
Tube 6 ____________________________________________________________________
Diabetes insipidis
This disorder is caused by a deficiency of antidiuretic hormone, which results in the inability of kidneys to
reabsorb water. Symptoms are constant thirst, weight loss, weakness, and production of 4-10 liters of urine
each day. The urine is very dilute (a low specific gravity) and is nearly colorless.
Diabetes mellitus
This disorder is caused by a deficiency of insulin, which results in a decreased ability for glucose to enter cells.
Therefore, glucose accumulates in the blood and fats are used excessively in cellular respiration, producing
ketones as a waste product. Symptoms include weakness, fatigue, weight loss, and excessive blood glucose
levels. In uncontrolled diabetes, a urine sample contains excess glucose and ketones and has a low pH.
Hepatitis
Hepatitis is usually caused by a viral infection. In type A hepatitis, symptoms may include fatigue, fever,
generalized aching, abdominal pain, and jaundice (yellowing tinge to the whites of the eyes due to excessive
blood levels of bilirubin). Urine is dark amber in color due to excessive bilirubin.
Glomerulonephritis
In this kidney disease, excessive permeability allows proteins and red blood cells to enter the filtrate. They are
present in the urine since they cannot be reabsorbed.
Hemolytic anemia
A number of conditions cause the destruction of red blood cells, thereby releasing hemoglobin into the blood
plasma. Such conditions include malaria and incompatible blood transfusions. The urine of such patients
contains hemoglobin and may be red-brown or smoky in color.
Reformatted from Gunstream labs 22 and 39
Page 6
4/8/2002
Normal
Concentrations of dissolved substances in normal urine (mg/100ml)
Urea
2000
Uric Acid
50
Inorganic salts
1500
Protein
0
Amino Acids
0
Glucose
0
Strenuous exercise and a high-protein diet
Athletes in excellent health may produce urine with an acid pH, a small amount of protein, and an elevated
specific gravity due to the presence of proteins.
Urinary tract infection
Bacterial urinary tract infections may involve structures of the urinary tract including the kidneys. Production
of alkaline urine promotes the growth of infectious bacteria. Urethritis (infection of the urethra) often causes a
burning pain during urination. Symptoms may include a low-grade fever and discomfort of the affected
region. Urine will test positive for nitrite because bacteria in the urine convert nitrate, a normal component,
into nitrite. Severe infections may also cause the urine to contain blood and pus (leukocytes), producing
cloudy urine.
PART II - Aquatic Microecosystems
Based on macroscopic observations, which microecosystem seems to have:
The greatest density of autotrophs? (darkest green) _____________________________________________
The lowest density of autotrophs? (lightest green) ______________________________________________
The greatest density of heterotrophs? ________________________________________________________
The lowest density of heterotrophs? _________________________________________________________
Based on macroscopic observations, record the relative abundance of each kind of crustacean in the
microecosystems in the table below. Use this scale: 0=none; 5=most abundant. Based on examination of your
slides, record the presence of autotrophs in the Microsystems by placing an “x” in the appropriate spaces in the
table below. Compare your data with the class data.
Organism
Acid Pollution
Normal
Organic Pollution
Cyanobacteria
Oscillatoria
Protista
Euglena
Peridinium
Green Algae
Microspora
Pandorina
Spirogyra
Crustacea
Cyclops
Daphnia
Gammarus
Reformatted from Gunstream labs 22 and 39
Page 7
4/8/2002