1. BIO1140 Lab 3: Cellular processes in Amoeba proteus

2. 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.

3. 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.

4. Microscopic observations
Amoebas are sensitive to light and heat:
Keep the light intensity low during observations.

5. 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.

6. plasmalemma
granular endoplasm
hyalin cap
pseudopodium
food vacuole
uroid
ectoplasm
contractile
vacuole
nucleus
dinner

7. 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.
There will be few extra slides containing Amoebas if needed + one flask per lab with more amoeba for the pinocytosis or replacement.
They don’t have to hand in the sketches.

8. 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).

9. 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).

10. Part III: Contractile vacuole cycle

11. Diastole Part III: Contractile vacuole cycle
Systole
t=5-8 minutes ( s*)
t=30 s
No vacuole visible yet
(diameter=0)
Diastole
t= s
Small vacuoles
start to aggregate and fuse
*stop measurements after 480s

12. 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).

13. 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
Students measure 2 cycles to increase the sample size – they plot only ONE CYCLE on the graph

14. 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.

15. 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

16. 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
For TA info only

17. 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.

18. 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

19. Available documents Read lab3 documents on the lab website:
List of Amoeba structures
Anatomy scheme
Instruction file for report