Chapter 3

Microorganisms

Measured in micrometers (μm) and nanometers (nm).

Total Magnification

Calculated by multiplying the magnification of the objective lens by the ocular lens.

Resolution

The ability to distinguish two points, with a maximum resolution of 0.2 μm for a compound light microscope.

Compound Microscope

A microscope with multiple lenses, commonly used in microbiology.

Brightfield Illumination

Used for observing stained smears of microorganisms.

Darkfield Microscopy

Shows a light silhouette of an organism against a dark background, useful for detecting small organisms.

Phase-Contrast Microscopy

Enhances contrast in unstained cells, allowing for better observation.

Scanning Acoustic Microscopy (SAM)

Uses sound waves to study living cells attached to surfaces like biofilms.

Electron Microscopy

Uses a beam of electrons instead of light for imaging specimens.

Transmission Electron Microscope (TEM)

Produces thin sections of organisms with magnification of 10,000–10,000,000× and resolution of 10 pm.

Scanning Electron Microscope (SEM)

Provides three-dimensional views of microorganism surfaces with magnification of 1000–500,000× and resolution of 10 nm.

Simple Staining

Involves coloring microorganisms with a single basic dye to enhance visibility.

Gram Stain

A differential stain that uses a purple stain, iodine, alcohol decolorizer, and red counterstain to differentiate bacteria.

Gram-Positive Bacteria

Retain purple color after decolorization in the Gram stain process.

Gram-Negative Bacteria

Appear pink after the Gram stain due to the counterstain.

Acid-Fast Stain

Differentiates acid-fast microbes, which appear red, from non-acid-fast microbes, which appear blue.

Capsule Stain

Used to visualize microbial capsules.

Endospore Stain

A special stain to visualize endospores in bacterial cells.

Flagella Stain

A special stain used to visualize flagella in bacterial cells.

Fixing

The process of using heat or alcohol to kill and attach microorganisms to a slide.

Explain the purpose of simple staining

• an aqueous or alcohol solution of a single basic dye

• make some structures more visible

Gram stain

uses crystal violet

Iodine is required as a mordant

gram positive cells appear as blue colored cells, while gram neg appear as red in color

Capsule stain

a neg staining used to make microbial capsules visible

used to determine organisms virclence

Acid Fast

primary stain is carbolfuchsin

mordant is not required

cells are red in color, non-acid fast cells appear blue

endospore/ flagella stain

special stains that color only certain parts of bacteria

endospore: used to differentiate the inclusions of stored material

flagella: used as diagnostic aids

Which of the following places the steps of the Gram stain in the correct order?
1-Alcohol acetone
2-Crystal violet
3-Safranin
4-Iodine

2-4-1-3

The purpose of a mordant in the Gram stain is to:

-make gram-negative cells visible
- make the flagella visible
-remove the simple stain
-prevent the crystal violent from leaving the cells

prevent the crystal violet from leaving the cells

Which of the following places the steps in the correct sequence?
1- Staining
2-Making a smear
3-Fixing

2-3-1

Simple staining is often necessary to improve contrast in which microscope?

electron microscope
compound light
fluorescence
phase-contrast

compound-light microscope

This microscope produces an image of light cell against a dark background.

compound
darkfield
electron
acustic

darkfield

Which step in the Gram stain is the critical step in differentiating gram-positive from gram-negative cells?

crystal violet
safranin
Iodine
Alcohol-acetone

alcohol-acetone

Which microscope is best used for observing the surfaces of intact cells and viruses?

brightfield microscope
phase-contrast microscope
scanning electron microscope
fluorescence microscope

scanning electron microscope

Bacterial smears are fixed before staining ti

accept stain
make cells visible
affix the cells to the slide
make their walls permeable

affix the cells to the slide