Lab Introduction Lab Introduction

Lab safetyLab safety

Lab #3 - microscopesLab #3 - microscopes

Safety infoSafety info

Read the pampletRead the pamplet Sign the blue sheetSign the blue sheet Under 18? Please see me.Under 18? Please see me.

Lab safetyLab safety

First aid kit at the front First aid kit at the front Fire extinguisher at the backFire extinguisher at the back Eye wash at sinkEye wash at sink Showers in the backShowers in the back

Lab SafetyLab Safety

No eating or drinking in labNo eating or drinking in lab Clean up rule:Clean up rule:

– It is your responsibility to clean up It is your responsibility to clean up after yourself and put everything after yourself and put everything back like you found itback like you found it

Lab clean upLab clean up

Put models back, like you found Put models back, like you found themthem

Put microscopes backPut microscopes back Put slides back Put slides back in the proper in the proper

boxbox

Lab clean upLab clean up

Dispose of other items as follows:Dispose of other items as follows:– Paper towels go in the trashPaper towels go in the trash– Broken glass and slide cover slips go Broken glass and slide cover slips go

in “broken glass”in “broken glass”– Biological waste goes in red bucketBiological waste goes in red bucket– Empty slides get rinsed, dried, and Empty slides get rinsed, dried, and

returned to boxreturned to box– Dissection tools: put in washDissection tools: put in wash– Wipe down your bench area when Wipe down your bench area when

donedone

Lab ManualLab Manual

Lab assignments will require you to Lab assignments will require you to turn in the “Review Sheet” at the end turn in the “Review Sheet” at the end of each lab exerciseof each lab exercise

This This mustmust be ripped out of your lab be ripped out of your lab manual, not photocopiedmanual, not photocopied

Sometimes you will also be asked to Sometimes you will also be asked to turn in separate drawings or dataturn in separate drawings or data

Due Wed of the week following the labDue Wed of the week following the lab

Lab 3: Lab 3: Intro to Intro to microscopesmicroscopes

Microscopic anatomy:Microscopic anatomy:– CellsCells– TissuesTissues

Who cares?Who cares?

Medically, microscopes are used Medically, microscopes are used for for pathologypathology, the study of , the study of disease.disease.– If a disease is suspected, a tissue If a disease is suspected, a tissue

sample is taken (biopsy, blood) or a sample is taken (biopsy, blood) or a cell sample (pap test)cell sample (pap test)

– Pathologist or technician looks for Pathologist or technician looks for abnormal cells, etc.abnormal cells, etc.

What about us?What about us?

Scientifically, microscopes are used to Scientifically, microscopes are used to study cells and tissues for all kinds of study cells and tissues for all kinds of reasons (e.g. CSI).reasons (e.g. CSI).

Educationally, microscopes are the Educationally, microscopes are the only way to truly learn about the only way to truly learn about the microscopic organizations of cells and microscopic organizations of cells and tissues.tissues.

Knowing how to use a microscope is a Knowing how to use a microscope is a great skill to have, no matter what great skill to have, no matter what medical or biology subfield you are medical or biology subfield you are involved in.involved in.

Compound microscopeCompound microscope

Uses multiple lenses to achieve Uses multiple lenses to achieve larger magnificationlarger magnification

Compound microscopeCompound microscope

Key parts: Key parts: – Eyepieces Eyepieces

(ocular (ocular objectives)objectives)

– ArmArm– BaseBase– ObjectivesObjectives– Stage (and Stage (and

stage clips)stage clips)

– Stage controlsStage controls– Coarse and Coarse and

Fine focusFine focus– CondenserCondenser– Iris diaphragmIris diaphragm– LightLight

Microscope RulesMicroscope Rules

Carry a microscope with two Carry a microscope with two hands, one on arm, one on basehands, one on arm, one on base

Put your microscope back in the Put your microscope back in the numbered cubby it came fromnumbered cubby it came from

Clean lenses only with special Clean lenses only with special lens paperlens paper

Start by using low powers, then Start by using low powers, then go up to higher powersgo up to higher powers

PropertiesProperties

Compound: occulars = 10x, Compound: occulars = 10x, objectives = 4x, 10x, 40x. objectives = 4x, 10x, 40x. Multiply to get total magnificationMultiply to get total magnification

ParafocalParafocal Inverted opticsInverted optics Field of viewField of view

How to use a How to use a compound scopecompound scope Start on lowest power (condenser up)Start on lowest power (condenser up) Load slid into clipLoad slid into clip Look thorough oculars, adjust (one image)Look thorough oculars, adjust (one image) Focus (coarse)Focus (coarse) Find region of interest, go to higher powerFind region of interest, go to higher power Focus (fine only because it’s parafocal)Focus (fine only because it’s parafocal)

**Adjust light level and iris diaphragm as **Adjust light level and iris diaphragm as needed (less light at low power)needed (less light at low power)

CarefulCareful

When using higher power When using higher power objectives, it is easy to break objectives, it is easy to break slides because the working slides because the working distance is small (lens is close to distance is small (lens is close to slide)slide)

Activity 1Activity 1

ID scope partsID scope parts

Activity 2Activity 2

Get a letter e slideGet a letter e slide Follow instructions and fill in table Follow instructions and fill in table

on p.30on p.30 Total mags = ocular x objectiveTotal mags = ocular x objective Measure working distances w/ rulerMeasure working distances w/ ruler Draw what you see Draw what you see Don’t do the 100x (oil) objectiveDon’t do the 100x (oil) objective

Activity 3Activity 3

Get a grid slide and Get a grid slide and measuremeasure the the field diameter using low power field diameter using low power (4x) objective by (4x) objective by countingcounting the the number of grid squares that you number of grid squares that you can see across the field of view (1 can see across the field of view (1 sq = 1mm)sq = 1mm)

CalculateCalculate the field diameters for the field diameters for the other lenses (not oil)the other lenses (not oil)

Field diameter formula Field diameter formula (p.32)(p.32)F.D. lens B = F.D. lens B = F.D. lens A x total magnification lens F.D. lens A x total magnification lens

AA total magnification lens Btotal magnification lens B

Example: say you count 10 squares across using Example: say you count 10 squares across using the 4xthe 4x

F.D. (10x obj) = F.D. (10x obj) = F.D. at low power x total mag @ low powerF.D. at low power x total mag @ low power

total magnification med powertotal magnification med power

= = 10mm10mm X 40x X 40x 100x100x

= 4mm= 4mm

Activity 4 - depthActivity 4 - depth

Get a thread slide (has three threads Get a thread slide (has three threads that cross each other)that cross each other)

Focus Focus downdown with the coarse focus with the coarse focus until all the threads are just out of until all the threads are just out of focusfocus

Then, slowly Then, slowly focusfocus up, taking note of up, taking note of which thread comes into focus first which thread comes into focus first (top), then second (middle), and last (top), then second (middle), and last (bottom)(bottom)

Activity 5Activity 5

Practice making a wet mount Practice making a wet mount slide slide – Put drop on slidePut drop on slide– Put on over slip at an anglePut on over slip at an angle

Take an epithelial cell slide and Take an epithelial cell slide and estimate the diameter of one cell estimate the diameter of one cell based on your field diameter based on your field diameter measurements. (e.g., if it takes measurements. (e.g., if it takes up half the field and the filed = up half the field and the filed = 10 mm then it’s 5 mm)10 mm then it’s 5 mm)

Clean upClean up

Cover slips in lab glass containerCover slips in lab glass container Wash/dry fresh mount slides, put backWash/dry fresh mount slides, put back Put prep’d slides back into Put prep’d slides back into correct boxcorrect box Clean off lens with lens paper and lens Clean off lens with lens paper and lens

cleaner ONLYcleaner ONLY Clean off stage and return scopeClean off stage and return scope Clean up your areaClean up your area

ActivitiesActivities1.1. Get scope and set up, I.D. partsGet scope and set up, I.D. parts2.2. Letter “e” slides (prepared). Letter “e” slides (prepared). 3.3. Grid slide: count and calculate – Grid slide: count and calculate –

measure at lowest power, use this measure at lowest power, use this figure to calculate answers at figure to calculate answers at higher power (table on page 30). higher power (table on page 30).

4.4. Thread slides (Careful, they’re Thread slides (Careful, they’re thick). Determine order of thick). Determine order of threads.threads.

5.5. Make pond water wet mount slide Make pond water wet mount slide and observe (practice focusing)and observe (practice focusing)

Clean up Clean up

Turn inTurn in

Lab 3 report (review sheet) due next Lab 3 report (review sheet) due next Thurs (rip out). Thurs (rip out).

Turn in page 30 with completed Turn in page 30 with completed summary table (except oil objective)summary table (except oil objective)

Write the correct order of the threads Write the correct order of the threads on the bottom of page 38 like this:on the bottom of page 38 like this:Top___________Top___________Middle____________Middle____________Bottom____________Bottom____________