Observing Microorganisms Through a Microscope

7/27/2019 Observing Microorganisms Through a Microscope

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Unit of Measure

Bacteria are VERY small, thats why this

is micro biology

The standard unit of measure in

microbiology is the MICROMETER (m)

A micrometer is 10-6 or .000001M

To see something this small you need touse a microscope and also color (stain)

the cells to see them


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Microscope

Because bacteria are so small a

microscope is the essential tool in

microbiology

Light microscope uses visible light to

observe bacteria


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Total Magnification

Ocular

(Eye Piece)

Objective Total

Magnification

10X 4X 40X

10X 10X 100X

10X 40X 400X

10X 100X (oil) 1000X


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Resolution

Ability of the lens to distinguish fine detail

How close together can you distinguishtwo points as separate?

Because bacteria are so small goodresolution is important

Resolution is DIRECTLY related to light in

the following way: The SHORTER thewavelength of light the GREATER theresolution


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More Resolution

To get the best resolution, use the

SHORTEST wavelength of visible light

that you can

Our microscopes use blue wavelength

light to maximize resolution

Using blue light we can get a resolution of

about .9 micrometers (m)


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Visible Spectrum


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Spectrum

Average wavelength of visible light is

.55m

Red light wavelength is .68m, violet light

is .42m, blue light is .48m

Which light is best to use? The one with

shortest wavelength

Using a shorter wavelength of light in the

blue range give better resolution


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Size Matters


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Why Use Oil?


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Smears and Staining

Bacteria must be stained (dyed) so theycan be seen with the microscope

Before staining a smear must be made

A smear is just a film of bacteria on aglass slide

After the smear dries it is heat fixed, this

Kills the bacteria Helps adhere the cells to the slide

Makes the cells more receptive to the dye


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Stains

Stains are dyes

Stains carry either a positive charge (basic dyes)

or a negative charge (acidic dyes)

Bacteria typically carry a slight negative chargeon the cell surface so they attract a basic dye

Most of the stains used in the lab are basic dyes

A negative stain uses acidic dyes that do notstain the cell but rather the background


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Staining

Positive

staining: thedye sticks to thespecimen togive it color


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Staining Techniques

Simple Stain

Uses only one basic dye

Provides basic

information about cellshape and arrangement

Differential Stain

Uses more than one dye

These procedures react

differently with differentkinds of bacteria

Helps distinguish

between different kinds of

bacteria

Most common and

important differential stain

is the GRAM STAIN


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Simple Stains

Require only a

single dye

Examples include

malachite green,crystal violet, basic

fuchsin, and

safranin

All cells appear the

same color but can

reveal shape, size,

and arrangement


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Differential Stains

Use two differentlycolored dyes, the

primary dye and the

counterstain

Distinguishesbetween cell

types or parts

Examples

include Gram,

acid-fast, and

endospore stains


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Gram Staining

The most universal

diagnostic staining

technique for bacteria

Differentiation ofmicrobes as gram

positive (purple) or

gram negative (red)


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Gram Stain

Most important differential staining

technique

Differentiates all bacteria based on cell

wall composition

Bacteria are either Gram + and stain blue

or Gram- and stain red

Gram stain is usually the first step in

identifying an unknown bacteria


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Gram Stain


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Gram stain


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Endospore Stain

Dye is forced by heat

into resistant bodies

called spores or

endospores Distinguishes

between the stores

and the cells they

come from (thevegetative cells)

Significant in medical

microbiology


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Special Stains

Used to

emphasize

certain cell parts

that arentrevealed by

conventional

staining methods

Examples:capsule staining,

flagellar staining


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Documents

Observing Microorganisms Through a Microscope