Microbiology Lab 1A

What is microscopy?

The use of microscopes to view objects too small to be seen with the naked eye.

What type of microscope is used in this lab?

Compound bright-field (light) microscope

What does “bright-field” mean?

Specimen appears dark against a bright background

What is a compound microscope?

A microscope that uses multiple lenses to magnify an image.

What is a simple microscope?

A microscope that uses a single lens.

What types of organisms are viewed at low magnification?

Large eukaryotic microorganisms

What type of organisms require the highest magnification?

Small prokaryotic microorganisms (bacteria)

What is electromagnetic radiation (EMR)?

Energy that travels in waves

What type of EMR does a light microscope use?

Visible white light

What is the wavelength range of visible light?

400 nm to 750 nm

What is wavelength (λ)?

The distance between wave crests or troughs.

As wavelength decreases, what happens to energy?

Energy increases

As wavelength increases, what happens to energy?

Energy decreases

What types of EMR are more energetic than visible light?

UV, X-rays, gamma rays

What types of EMR are less energetic than visible light?

Infrared, microwaves, radio waves

What colors make up the visible light spectrum?

Red, Orange, Yellow, Green, Blue, Indigo, Violet

What mnemonic helps remember the visible spectrum?

ROY G BIV

Which color has the longest wavelength?

Red

Which color has the shortest wavelength?

Violet

Which color is most energetic?

Violet

Which color is least energetic?

Red

What is reflection?

Light bouncing off a surface

What is absorption?

Light being taken in by a material

What is refraction?

Bending of light as it passes between different media

Which property of light is most important for microscopy?

Refraction

Why is refraction important in microscopy?

It affects image clarity and resolution

How many objective lenses does the microscope have?

Four

What type of lenses do objectives contain?

Bi-convex lenses

What does parfocal mean?

Image remains nearly in focus when changing objectives.

What does parcentric mean?

The object stays centered when changing objectives

Why are parfocal microscopes important?

They reduce refocusing time and prevent damage.

Which objective lens requires immersion oil?

100X oil objective

Why is immersion oil used?

To reduce refraction and increase resolution.

What is special about immersion oil?

It has the same refractive index as glass.

What happens if oil is not used with the 100X objective?

Image will be blurry and unclear

What should the 100X objective look like when properly focused?

It is directly in the oil

What is the magnification of the scanning lens?

4X

What is the purpose of the 4X objective?

Scanning large specimens and locating areas of interest

Why can’t bacteria be seen at 4X?

Magnification is too low

What type of light is used for 4X?

Used with lowest amount of light, controlled by condenser diaphragm

What is the magnification of the low-power lens?

10X

Why is 10X important for bacteria viewing?

It is used to focus before switching to 100X oil

What type of light is used for 10X?

Used with lowest amount of light

What is the magnification of the high dry lens?

40X

Why is it called “high dry”?

High magnification without oil

What type of light is used for 40X?

Used with moderate amount of light

What is the magnification of the oil immersion lens?

100X

What focus knob is used at 100X?

Fine focus only

What type of light is used for 100X?

Used with highest amount of light, controlled by condenser diaphragm

What is the magnification of the ocular lens?

10X

How do you calculate total magnification?

Objective magnification × ocular magnification

What is total magnification using a 40X objective?

400X

What is total magnification using a 100X objective?

1000X

What is resolution?

The ability to distinguish two close objects.

What happens to resolution as magnification increases?

Resolution increases

What is the field of view?

The circular area visible through the microscope

What happens to field of view as magnification increases?

It decreases

Why should multiple fields of view be observed?

To get a complete view of the specimen

What is working distance?

Distance between the objective lens and the slide

What happens to working distance as magnification increases?

It decreases

At which magnification is working distance smallest?

100X oil

What is depth of field?

Thickness of specimen that remains in focus

What happens to depth of field as magnification increases?

It decreases

Why can bacteria not be seen if the slide is upside down at 100X?

Depth of field is too shallow

How does light requirement change with magnification?

Higher magnification needs more light

What controls the amount of light passing through the specimen?

Condenser diaphragm

What is the ocular lens?

Lens you look through

Why must oculars be adjusted?

To match the distance between your eyes

What is the stage?

Platform that holds the slide

What moves the slide?

Slide control knobs

What are objective lenses mounted on?

Revolving nosepiece

What is the condenser lens?

Focuses light onto the specimen

What is the diaphragm?

Controls the amount of light

What does the coarse focus knob do?

Moves stage quickly

When is coarse focus used?

Only at low magnification (4X, 10X)

What does the fine focus knob do?

Makes small focus adjustments

When is fine focus used?

At higher magnifications

What paper is used to clean lenses?

Lens paper only

What paper is used to clean the stage/body?

Blotting / bibulous paper

What objective must be used before switching to 100X oil?

10X

Why should the stage not be moved during oil immersion?

To maintain focus

What objective should never be dragged through oil?

40X

What does “directly proportional” mean?

Both variables increase or decrease together

What does “inversely proportional” mean?

One variable increases while the other decreases

What does “X” mean in magnification?

Times magnified

Magnification vs resolution

Directly proportional

Magnification vs objective length

Directly proportional

Magnification vs light required

Directly proportional

Magnification vs field of view

Inversely proportional

Magnification vs working distance

Inversely proportional

Magnification vs depth of field

Inversely proportional