Welcome to Lab!• Stash backpacks in cubbies on right

side of room.

• Choose your seat.

• Put on your “lab coat” and set goggles on bench next to you.

• Come get the following handouts: - “Lab Cards” that you will be using each lab for sign in and sign out.

- Lab Exercise 1 and blank Lab Report

- Signature page.

- IMPORTANT!!!! Bring BOTH Lab #2 and Lab #3 next week!!!

Laboratory 1Microscopy & Simple Stains

Your Light Microscope

CORDS are in the drawer.

TAKE CARE OF YOUR SCOPE: Your responsibility to take care of your scope and learn to use it properly.

MICROSCOPE LOG:Every time that you get your scope out, you will make an entry in the microscope log.

GETTING YOUR SCOPE OUT:When transporting your scope, always hold it with one hand under the base, and one hand around the arm.

PUTTING SCOPE AWAY:Whenever your are getting ready to put your scope away:

• Use alcohol swab to clean stage and lens paper to clean lenses.

• Shortest lens (the one with the red band) should be facing down toward stage.

• Use course focus to position stage as low as it can go.

BASE

ARM

Microscopy

General Principles

Magnification:• What is it?• Apparent increase in size of an object.• Indicated by a number and “X”, which is read “times”.

Resolution:• What is it?• The ability to distinguish between objects that are close together. • The optometrist’s eye chart is a test of resolution at a distance of 20

feet.• Limits maximum magnification

Contrast:• What is it?• Difference between the object and the background• Easiest ways to improve contrast are to use dye and/or manipulate

light

Bright-field Compound Microscope

Microscopy – Light Microscopes

AT THIS POINT, GO GET A SCOPE AND SIGN IT OUT.

Microscopy – Light Microscopes

Bright-Field Microscope

Light microscope produces a dark image against brighter background.

Commonly used to view stained cells.

Simple microscopes have single magnifying lens (like a magnifying glass).

Compound microscopes have two sets of lenses for magnification.

Lens closer to the eye = ocular lens (magnifying power of 10x).

Lenses closer to the object being viewed = objective lens. (Most light microscopes used in biology have three or four objective lenses).

OBSERVATION OF MICROORGANISMS

Ocular lenses

Objective Lenses

Objective LensesScanning Objective Lens

Has red band around it.

Magnifies objects 4x.

Total magnification = 40xTM

This lens is of no use to us in looking at bacterial stains.

Objective LensesLow Power Objective Lens

Has yellow band around it.

Magnifies objects 10x.

Total magnification = 100xTM

Start with this lens to get your bacterial smear into crisp focus.

You will not see individual bacteria with this lens, you are just using it to focus so that you can move up to the next magnification.

Remember the term parfocal?

Objective LensesHigh Dry Objective Lens

Has blue band around it.

Magnifies objects 40x.

Total magnification = 400xTM

Move up to this lens after focusing your smear at 100xTM.

You will not be able to clearly see individual bacteria with this lens. Just get the image in focus as much as possible.

High Dry Objective Lens

After you focus the image at 400xTM, you need to cover this lens with a finger cot so that it does not get oil on it.

Do not move the focus knob or the stage when placing the finger cot on the high dry lens or you will take the image out of focus!

NEVER use coarse focus with high dry or oil immersion lenses!!!

Oil Immersion Objective Lens

Has black and a white band around it.

Magnifies objects 100x.

Total magnification = 1000xTM

Move up to this lens after focusing your smear at 400xTM and covering the 400xTM lens with a finger cot.

NEVER use coarse focus with high dry or oil immersion lenses!!!

Two types:

Both huge, expensive machines.

Transmission Electron Microscope (TEM): 2-D image: Transmission Electron Micrograph

Scanning Electron Microscope (SEM):

3-D image: Scanning Electron Micrograph

Microscopy – Electron Microscopes

SEM AIDs virus attacking T4 lymphocyte

A transmission electron micrograph of Escherichia coli (E.coli).

Procedure Learning to use thecompound light microscope

1. How to make a wet mount

2. Letter “e”- Wet mount- What happens to the “e” when you

look at it through the lens?

3a. Onion- Wet mount, use stain- Note nucleus and cell wall

or

3b. Elodea - Wet mount NO stain- Depth of field- 2 layers of cells- Note cell wall, chloroplasts streaming

4. Cheek cell- Wet mount using NaCl- Try to view, then add stain.- Contrast!

Onion Cells

Elodea

Cheek Cells

Before you get started, I want you to prepare a slide with a bacterial sample.

Preparing Bacterial Smear

Draw a circle with wax pencilPut a couple drops of sterile water in the circle.

Inoculate the circle of water with

small sample of bacteria.

After heat fixing on top of microincinerator, stain with crystal violet, rinse, then look at with oil immersion lens.

a. b.

c. d.

Raise your hand when you are ready to view your smear under scope.

STAINING: You saw how much easier it was to view the check cell after it had been stained.

TO VIEW BACTERIA:

1. First we make a smear, by putting a drop of water on a slide and then mixing in a little bit of bacteria that we have been growing in a petri dish.

A smear is a thin film of organisms on a slide.

Preparing Bacterial Smear

TO VIEW BACTERIA:

2. Heat Fixing the Smear…

The smear is attached, or fixed to a slide using either heat or chemicals.

Drying and fixation:- kill the microorganisms- attach them firmly to the slide (so they don’t wash away during staining)- and generally preserve their shape and size

3. Simple Stain…

Composed of a single basic dye, such as crystal violet.

OBSERVATION OF MICROORGANISMS

Viewing bacteria under oil immersion

• Don’t EVER use coarse focus when working with high dry or oil immersion.

• Remember PARFOCAL!!

• Using oil immersion:– View bacteria with med power then

high dry (cant see much, but at least get them in your sights)

– Protection for your high dry (finger cot)

– Place drop of oil directly on smear

– Switch to oil immersion lens

– ONLY USE FINE FOCIS ADJUSTMENT!!!

– When done, use alcohol wipes to clean up your lenses and stage.

Procedure Learning to use thecompound light microscope

1. How to make a wet mount

2. Letter “e”- Wet mount- What happens to the “e” when you look at it through the

lens?

3a. Onion- Wet mount, use stain- Note nucleus and cell wall

or

3b. Elodea - Wet mount NO stain- Depth of field- 2 layers of cells- Note cell wall, chloroplasts streaming

4. Cheek cell- Wet mount using NaCl- Try to view, then add stain.- Contrast!

Onion Cells

Elodea

Protozoans Cheek Cells

Wrap-upWhen putting away scope:

- Make sure lenses are clean (wipe with alcohol swab)

- Have scanning power lens in position (4x)

- Make sure stage is in lowest position

- Put away the cord and cover the scope

- Return scope to its proper “address” in cabinet

IMPORTANT!!!! Bring BOTH Lab #2 and Lab #3 next week!!!

Lab 1 Lab Report due next time we meet for lab.