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?

ObjectivesReview the metric units of measurementDefine total magnification and resolution Explain how electron and light microscopy differDifferentiate 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 stainExplain 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 3.6bPrinciple 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 MicrobiologyReview 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