Do you want to make this class easier?

Do yourself a favor. Use the…

Virtual Microbiology Classroom (VMC) !

The VMC is full of resources to help you succeed, including:

• practice test questions• review questions• study guides and learning objectives• photos of bacterial colonies, stains, media and

other lab-related material

You can access the VMC through Moodle, or by going to www.ScienceProfOnline.com

Laboratory 1

Microscopy

Your Light Microscope

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. I may randomly check scopes to see if they are put away correctly. If yours is not, I may subtract points from your lab grade.

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 if it is oily, 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?

Resolution:• What is it?

Contrast:• What is it?

AT THIS POINT, GO GET A SCOPE AND SIGN IT OUT. CORDS are in the drawer.

Bright-field Compound Microscope

Microscopy – Light Microscopes

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?

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

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

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

Onion Cells

Elodea

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