Ch 3:

Observing

Microorganisms

Through a

Microscope

Q&A

Acid-fast staining of a

patient’s sputum is a

rapid, reliable, and

inexpensive method to

diagnose tuberculosis.

What color would

bacterial cells appear if

the patient has

tuberculosis?

Objectives

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, 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

Units of Measurement

Review Table 3.1

• 1 µm = ______ m = ______ mm

• 1 nm = ______ m = ______ mm

• 1000 nm = ______ µm

• 0.001 µm = ______ nm

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 ___ 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

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 3.6b

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?

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

?

Rod-shaped Mycobacterium avium

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

Important Staining Reactions in Microbiology

Review of different staining techniques

For Gram stain

technique compare

to Fig 3-12

Gram Stain

safranin

crystal violet

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

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

See Fig 3.14