Presentation on theme: "Tetrahymena as a model system to study phagocytosis"— Presentation transcript:
1 Tetrahymena as a model system to study phagocytosis Module 2 – Week 1
2 Before we start this week… We have assigned new 4-person lab groups for everyoneYou will be doing much of the work in pairs todayHalf of class will start exercise #1: pipettingOther half will start exercise #2: microscopy
3 Goals1) 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.
4 Gilson Pipetman Identity of pipet on top button P20 and P200 use yellow tipsP1000 uses blue tipsTwo stop positions on piston1st: to fill2nd: to expel remaining liquid during dispensingThe numbers on the dial depends on the pipet in use
5 Pipetting exerciseP-1000 pipettor set to 500 μl (water weight = 500 mg = 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.
7 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.
8 Viewing Tetrahymena in the microscope Tetrahymena cell culture prepared for youThree 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 HOODObserve three Tetrahymena samples in the microscope
9 Glutaraldehyde Hazard Statements: H302: Harmful if swallowed H315: Causes skin irritationH317: May cause an allergic skin reactionH318: Causes serious eye damageH330: Fatal if inhaledH334: May cause allergy or asthma symptoms or breathing difficulties if inhaledH400: Very toxic to aquatic lifeIf you would like to review the complete MSDS, please consult with your TA
10 Proper Personal Protective Equipment (PPE) All Glutaraldehyde must be handled in the fume hoodEvery student must wear a lab coat, gloves, and safety glasses when working with 2.5% glutaraldehyde or samples treated with glutaraldehyde
11 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 hoodWipe down your bench before leaving and put paper towel in Biohazard BoxDon’t touch computers with gloved hands
12 Write up your work today Pipetting exerciseCalculate mean and S.D. for each pipet verificationDetermine whether pipetting technique was precise or impreciseInitial observations of TetrahymenaDrawing of typical cellDescribe swimming and eating behaviorsEffects of glutaraldehydeFor 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.
13 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