BIO1140 Lab 3:
Cellular processes in
Amoeba proteus
Objectives of the lab
• Identify, photograph and measure organelles
found within Amoeba proteus.
• Observe cell processes such as: amoeboid
movement, contractile vacuole cycle and
endocytosis.
• Measure the diameter of the contractile vacuole
at selected intervals throughout its cycle.
• Use these measurements to plot a graph showing
the variation of the vacuole volume during time.
Method (setup)
• Carefully connect compound microscope
camera to your computer .
• Clean optical surfaces of microscope (oculars,
objectives, top of condenser and top of lamp
housing) using kimwipes.
Microscopic observations
• Amoebas are sensitive to light and heat:
• Keep the light intensity low during
observations.
Part I: Amoeba anatomy
Identify and know the function of structures and
organelles listed in the file: ‘Amoeba
structures’ (Lab web site).
• Locate an amoeba on your slide using the 4x
objective. Then, switch to the 10x and 40x
objectives.
• There will be questions about Amoeba
anatomy and movement in the final exam.
granular endoplasm
plasmalemma
hyalin cap
food vacuole
uroid
ectoplasm
contractile
vacuole
nucleus
dinner
Part II: Amoeboid movement
• Your task: Under the 10X observe the formation
and elongation of pseudopodia
• Switch to the 40x objective to observe the
movement of granular endoplasm.
• See animation on Digizoo Site
• Take several pictures during amoeboid movement
• Advice: Take notes, draw sketches and label all
structures you observed. Represent the stream of
fluid endoplasm using arrows.
Part II: Amoeboid movement
TIPS:
• Locate an amoeba on the slide using the 4X
objective
• Adjust the aperture diaphragm (closed = darker
with more contrast / open = brighter with less
contrast).
Part III: Contractile vacuole cycle
Your task: measure the contractile vacuole (CV)
diameter throughout its cycle.
Contractile vacuole:
• Function: osmoregulation and waste removal
• Location in the cell: variable
• Duration: about 5 minutes at 20°C (cycle
duration increases with temperature).
• Cycle components:
– Diastole (coalescence and continual growth)
– Systole (release of CV contents to exterior).
Part III: Contractile vacuole cycle
Part III: Contractile vacuole cycle
t=0
Systole
t=5-8 minutes (300-480s*)
*stop
measurements
after 480s
t=30 s
No vacuole visible yet
(diameter=0)
t=60-120 s
Small vacuoles
start to aggregate and fuse
Contractile Vacuole: Methods
• Locate the contractile vacuole (CV) using the
10x objective.
• THEN: Once you’ve found a CV switch to 40x
objective
• Observe at least one full cycle before starting
the measurements.
• Time is 0 when systole occurs (=vacuole
disappears).
Contractile Vacuole: Methods
• Take picture of CV every 30 seconds during 2 full cycles
(for up to 480 seconds each).
• Save your pictures in: K:/BIO1140/BIO1140XX as JPEG
files
• Warning: SAVE YOU PICTURES PROGRESSIVELY or you’ll
lose them.
• Once done, measure the diameter of the contractile
vacuole (in µm) on each pictures.
while measuring, do NOT trace lines on computer
screen (-10% immediately)
• Pictures must be taken at the 40x objective in order
to get a precise measurement of the diameter
Contractile Vacuole: Methods
• Enter your data into the excel spreadsheet(s)
opened on the designated computers
• Diameters will be converted into volumes, and
class average and standard error will be
calculated automatically on the spreadsheet.
• Data will be available on the Lab3 page of the
lab website (NOT Bblearn) tomorrow.
Part IV: Endocytosis
• Unmount the Amoeba slide - DO NOT THROW AWAY THE SLIDE (tech. skills!)
• Dispose of coverslips in one of the 2 beakers located a the back of
the lab
• Go to the endocytosis station
• Get a few Amoebas from the flask and transfer them into watch
glass
• Add 1-2 drops of inducing agent (1% BSA + alcian blue)
• Wait 1 minute
• Transfer amoebas to the “amoeba slide” with very little liquid (Ask
TA)
• Add coverslip
• Observe shape changes and formation of endocytic canals under
microscope
Time Budget
• Lab3 Quiz – 10 minutes
• TA Prelab Talk - 15 min.
• Amoeboid Observation – locomotion &
anatomy – 45 minutes
• Amoeba contractile vacuole cycle - 75 min.
• Endocytosis – 10 min.
• Questions to TAs and cleaning of bench 10
min
Lab 3 Report: Title page + Graph
Contractile vacuole Graph
• Plot CV volume (average +/- standard error) over time
(0 to 480 seconds = one cycle) using the class data.
• Graph (dot plot) must be done BY HAND (no computer
generated graph) on millimeter paper.
• Include a caption located below the graph (see lab
manual appendix)
• Read instruction file on the Lab3 page of lab web
• Due date: In one week
• Data will be available on lab web site tomorrow.
Reminders
• Clean up or lose technical skill marks
– Rinse Amoeba slide and put it back on the tray where
you took it.
– Dispose of cover slips in the broken glass beaker.
– Delete all files your saved
– clean up work bench
– clean optical surfaces of your microscope
– proper microscope return verified by TA before you
leave
– NEXT LAB: MITOSIS with prelab quiz about lab4
Available documents
• Read lab3 documents on the lab website:
– List of Amoeba structures
– Anatomy scheme
– Instruction file for report