Ch 3: Observing Microorganisms Through a Microscope

Q&A
Acid-fast staining of a patients 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 m = ______ m = ______ mm
1 nm = ______ m = ______ mm

1000 nm = ______ m
0.001 m = ______ nm

Sizes Among Microorganisms

Protozoa: 100 m

Yeasts: 8 m

Cells Alive How big is a . . .?

Bacteria: 1 - 5 m (some much longer than wide) Rickettsia: 0.4 m = _________ nm

Chlamydia and Mycoplasma: 0.25 m

Viruses: 20 250 nm

Principles of the Compound Light Microscope

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

Fig 3.1

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

Microscopy: The Instruments

Brightfield Microscopy

Darkfield Microscopy
Light objects visible against dark background. used to enhance the contrast in unstained samples. Instrument of choice for spirochetes

Simplest of all the optical microscopy illumination. techniques Dark objects are visible against a bright background.

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:?

SEM or TEM?

Bacterial division

Leaf surface

?
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.

Differential Stains
React differently with different bacteria

Gram stain
Acid fast stain

Review of different staining techniques

Important Staining Reactions in Microbiology

For Gram stain technique compare to Fig 3-12

Gram Stain

crystal violet

safranin

Acid Fast Stain

Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast. Nonacid-fast cells lose the primary stain when rinsed with acid-alcohol, and are counterstained with a different color basic stain

Special Stains

See Fig 3.14

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