Tetrahymena as a model system

to study phagocytosis Module 2 – Week 1

Before we start this week…

• • • • We have assigned new 4-person lab groups for everyone You will be doing much of the work in pairs today Half of class will start exercise #1: pipetting Other half will start exercise #2: microscopy

Goals

1) Become competent in the use of micropipettors to deliver very small volumes of liquids.

2) Develop the ability to use the Olympus CH-2 brightfield microscope for studying single-celled organisms.

Next week…

3) Explore how polystyrene microbeads can be used to identify subcellular structures and organelles.

4) Develop an experimental strategy to test a hypothesis related to the phagocytotic mechanism of Tetrahymena.

Gilson Pipetman

• • • • • Identity of pipet on top button P20 and P200 use yellow tips P1000 uses blue tips Two stop positions on piston – 1 st : to fill – 2 nd : to expel remaining liquid during dispensing The numbers on the dial depends on the pipet in use

• • •

Pipetting exercise

P-1000 pipettor set to 500 μl (water weight = 500 mg = 0.500 g) P200 set to 200 μl (water weight = 200 mg) P20 set to 20 μl (water weight = 20 mg) • • • • • Empty 1.7 ml centrifuge tube on a 3-place pan balance. Tare (zero) the balance so the weight of the tube is zeroed out.

Pipette water into the tube. Weigh the tube and record the weight.

Repeat five times.

Repeat this exercise with all three pipets.

Using Excel, calculate the average and standard deviation of your three different pipettors.

Pipette

P-1000 P-200 P-20

Volume set (ul)

1000 500 200 100 20

Approx. wt.

(mg)

1000 500 200 100 20

Precision (%)

0.15

0.2

0.15

0.25

0.3

Precision range (mg)

998-1002 499-501 199.7-200.3

99.7-100.3

19.9-20.1

Olympus CH-2 microscope

• • • • • • • • • • •

Introduction to the microscope

Place sample slide with coverslip facing up on specimen holder.

Turn the light intensity dial to the zero position.

Turn the main power switch on and increase the light level.

Adjust specimen stage so that edge of glass is in beam.

Make sure the condenser is all the way down.

Rotate the 10x objective into position for viewing.

Turn the coarse focus knob to a position so that the specimen is basically in focus.

Adjust the distance between two eyepieces (interpupillary distance) so that you see through both eyes.

– Use the fine-focus knob to achieve optimum focus through the right ocular.

– Use the diopter adjustment ring to focus the image through the left ocular, so that both eyes see the image properly focused.

Adjust the aperture iris (using its lever) for optimum contrast. The iris aperture must be adjusted each time you switch to a different objective.

After focusing with the 10x objective, you can increase total magnification with the 40x objective. You will need to adjust the fine-focus each time you change.

Don’t let any objective lens contact the slide.

– Adjust the light intensity when you change objectives, and then re-adjust the iris aperture.

• • • • • • •

Viewing Tetrahymena in the microscope

Tetrahymena cell culture prepared for you Three 1.7 ml microtubes: A, B, and C.

A = 50 μl of Tetrahymena + 50 μl of 3 micron polystyrene beads. B = 50 μl of Tetrahymena + 50 μl of 0.2% glutaraldehyde. – PERFORM IN FUME HOOD; CAUTION: glutaraldehyde is highly toxic. Mix both tubes gently. C = 20 μl of cells from Tube “A” (~10 min bead exposure) + 20 μl of glutaraldehyde, then mix. Record the duration of ink exposure.

– PERFORM IN FUME HOOD Observe three Tetrahymena samples in the microscope

Glutaraldehyde

• Hazard Statements: • H302: Harmful if swallowed • H315: Causes skin irritation • H317: May cause an allergic skin reaction • H318: Causes serious eye damage • H330: Fatal if inhaled • H334: May cause allergy or asthma symptoms or breathing difficulties if inhaled • H400: Very toxic to aquatic life • If you would like to review the complete MSDS, please consult with your TA

Proper Personal Protective Equipment (PPE)

• • All Glutaraldehyde must be handled in the fume hood Every student must wear a lab coat, gloves, and safety glasses when working with 2.5% glutaraldehyde or samples treated with glutaraldehyde

Disposal of Tips, Slides, and Tubes

• • • • • • • Tips that have not come in contact with glutaraldehyde can be placed in the coffee can labeled “Tips Only” located on your lab bench.

Slides that have not been contaminated with glutaraldehyde can be put in the red “sharps” container on the side bench.

All tubes containing liquid with trace glutaraldehyde must be emptied into the container in the fume hood labeled “Liquid Waste.” Tips, tubes, and slides contaminated with glutaraldehyde must go in the container in the fume hood labeled “Solid Waste.” Gloves and kimwipes must go in the Biohazard Box located by the hood Wipe down your bench before leaving and put paper towel in Biohazard Box Don’t touch computers with gloved hands

Write up your work today

• • • Pipetting exercise – Calculate mean and S.D. for each pipet verification – Determine whether pipetting technique was precise or imprecise Initial observations of Tetrahymena – Drawing of typical cell – Describe swimming and eating behaviors • Effects of glutaraldehyde – For fixed cells exposed to beads, count number of beads per cell for ten cells. Mean and S.D.

Turn in your observations, results and conclusions for Exercise #1 and #2 next lab session.

Next week’s experiment?

• From your initial observations, consider exploring the following: – What do Tetrahymena consider food?

– What is the cellular mechanism by which food is ingested?

– ???

• Fill out the “My Proposed Experiment” form and turn in with summary of today’s work on flip side