Ch 3:

Observing

Microorganisms

Through a

Microscope

SLOs

Review the metric units of measurement

Define total magnification and resolution

Explain how electron and light microscopy differ

Differentiate between acidic and basic dyes

Compare simple, differential, negative, and special stains

List the steps in preparing a Gram stain. Describe the appearance of Gram-positive and Gram-negative cells after each step

Compare and contrast Gram stain and acid-fast stain

Explain why endospore and capsule stains are used

SLOs cont.: Check Your Understanding

• If a microbe measures 10 μm in length, how long is it in

nanometers?

• What does it mean when a microscope has a resolution of

0.2 nm?

• Why do electron microscopes have greater resolution

than light microscopes?

• Why doesn’t a negative stain color a cell?

• Why is the Gram stain so useful?

• Which stain would be used to identify microbes in the

genera Mycobacterium and Nocardia?

• How do unstained endospores appear? Stained

endospores?

Units of Measurement

Review Table 3.1

• 1 µm = ______ m = ______ mm

• 1 nm = ______ m = ______ mm

• 1000 nm = ______ µm

• 0.001 µm = ______ nm

Figure 3.2 Microscopes and Magnification.

Tick

Actual size

Red blood cells

E. coli bacteria

T-even bacteriophages

(viruses)

DNA double helix

Unaided eye

≥ 200 m

Light microscope

200 nm – 10 mm

Scanning

electron

microscope

10 nm – 1 mm

Transmission

electron

microscope

10 pm – 100 m

Atomic force

microscope

0.1 nm – 10nm

Foundation Fig 3.2

Sizes Among Microorganisms

• Protozoa: 100 µm

• Yeasts: 8 µm

• Bacteria: 1 - 5 µm (some much longer than

wide)

• Rickettsia: 0.4 µm = _________ nm

• Chlamydia and Mycoplasma: 0.25 µm

• Viruses: 20 – 250 nm

Cells Alive –

How big is a . . .?

Principles of the Compound Light

Microscope

Fig 3.1

Magnification: Ocular and objective

lenses of compound microscope (total

mag.?)

Resolution: Ability of lens to . . .

Maximum resolving power depends

on . . .

For light microscope: ___ m

Contrast: Stains change refractive

index contrast between bacteria

and surrounding medium

Refractive Index

• Measures light-bending

ability of a medium

• Light may bend in air so

much that it misses the

small high-magnification

lens.

• Immersion oil is used to

keep light from bending.

Fig 3.3

Brightfield Microscopy

• Simplest of all the

optical microscopy

illumination. techniques

• Dark objects are visible

against a bright

background.

Darkfield Microscopy

• Light objects visible

against dark background.

• used to enhance the

contrast in unstained

samples.

• Instrument of choice for

spirochetes

Microscopy: The Instruments

Fig 3.4

Spirochetes (Treponema pallidum) viewed with darkfield microscope

Fluorescence Microscopy

• Uses UV light.

• Fluorescent substances absorb UV light and emit visible light.

• Cells may be stained with fluorescent chemicals (fluorochromes).

• Immunofluorescence

Fig 3.6; T. pallidum

Figure 3.6a

Fig 3.6

Principle of

Immunofluorescence

Electron Microscopy: Detailed Images of

Cell Parts

Uses electrons, electromagnetic lenses, and

fluorescent screens

Electron wavelength ~ 100,000 x smaller than

visible light wavelength

Specimens may be stained with heavy metal

salts

Two types of EMs:?

Bacterial division

Leaf surface

SEM or TEM?

Compare to Fig 3.10

10,000-100,000; resolution 2.5 nm.

?

Preparation of Specimens for Light

Microscopy

• Staining Techniques Provide Contrast

• Smear air-dry heat-fix

• Basic dyes: cationic chromophore

• Acidic dyes: anionic chromophore

negative staining (good for capsules)

• Three types of staining techniques:

Simple, differential, and special

Simple Stains

• Use a single basic

dye.

• A mordant may be

used to hold the

stain or coat the

specimen to

enlarge it.

React differently with

different bacteria

• Gram stain

• Acid fast stain

Differential Stains

Figure 3.12 Gram staining.

Application of crystal

violet (purple dye)

Application of iodine

(mordant)

Alcohol wash

(decolorization)

Application of

safranin (counterstain)

Gram-positive

Gram-negative

Cocci

(gram-positive)

Rod

(gram-negative)

Fig 3.12

Gram Stain

safranin

crystal violet

Compare to Fig 3.12

Gram Stains using Compound Light Microscope

Bacillus anthracis

Streptococcus mutans

Negative Stain

Observe cell shape and

size

Used for bacteria with

capsules

Fig 3.14

Acid Fast Stain • Cells that retain a basic stain in the presence of

acid-alcohol are called acid-fast.

• Non–acid-fast cells lose the primary stain when

rinsed with acid-alcohol, and are counterstained

with a different color basic stain

Fig 3.13

Special Stains

• Endospore stain: Heat is

required to drive a stain into the

endospore.

• Flagella staining: requires a

mordant to make the flagella wide

enough to see.

• Capsule stain uses basic stain

and negative stain

Compare to Fig 3.14

Clinical Case: Microscopic Mayhem