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 1
Microscopy &
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.
ARM
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: BASE
• 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.
 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
Microscopy – Light Microscopes

Bright-field
Compound
Microscope

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. Ocular lenses

Commonly used to view stained cells.

Simple microscopes have single magnifying lens

Objective
(like a magnifying glass). Lenses

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
 Objective Lenses
Scanning 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 Lenses
Low 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 Lenses
High 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!!!
Microscopy – Electron Microscopes
Two types:
Both huge, expensive
machines.

Transmission Electron
Microscope (TEM):
2-D image: Transmission
Electron Micrograph
A transmission electron micrograph of Escherichia coli (E.coli).

Scanning Electron
Microscope (SEM):
3-D image: Scanning
Electron Micrograph

SEM AIDs virus attacking T4 lymphocyte

 Procedure
Onion Cells
Learning to use the
compound light
microscope
1. How to make a wet mount

2. Letter “e” Elodea

- 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
Cheek Cells
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!

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

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.

a. b.

Put a couple drops of sterile water in

Draw a circle with wax pencil the circle.

c.
d.

Inoculate the circle of water with After heat fixing on top of

microincinerator, stain with crystal
small sample of bacteria.
violet, rinse, then look at with oil
immersion lens.

Raise your hand when you are ready to view your smear under scope.
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
Onion Cells
Learning to use the
compound light
microscope
1. How to make a wet mount Elodea
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 Protozoans Cheek Cells
- 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!
 Wrap-up
When 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.