Microscopy And Staining 12a5b

Microscopy and Staining

Unit of Measure • Bacteria are VERY small, that’s why this is micro biology • The standard unit of measure in microbiology is the MICROMETER (µm) • A micrometer is 10-6 or .000001M • To see something this small you need to use a microscope and also color (stain) the cells to see them

Microscope • Because bacteria are so small a microscope is the essential tool in microbiology • Light microscope uses visible light to observe bacteria

Total Magnification Ocular (Eye Piece) 10X

Objective

Total Magnification

4X

40X

10X

10X

100X

10X

40X

400X

10X

100X (oil)

1000X

Resolution • Ability of the lens to distinguish fine detail • How close together can you distinguish two points as separate? • Because bacteria are so small good resolution is important • Resolution is DIRECTLY related to light in the following way: The SHORTER the wavelength of light the GREATER the resolution

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 (µm)

Visible Spectrum

Spectrum • Average wavelength of visible light is .55µm • Red light wavelength is .68µm, violet light is .42µm, blue light is .48µm • Which light is best to use? The one with shortest wavelength • Using a shorter wavelength of light in the blue range give better resolution

Size Matters

Why Use Oil?

Smears and Staining • Bacteria must be stained (dyed) so they can be seen with the microscope • Before staining a smear must be made • A smear is just a film of bacteria on a glass 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

Stains • Stains are dyes • Stains carry either a positive charge (basic dyes) or a negative charge (acidic dyes) • Bacteria typically carry a slight negative charge on 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 not stain the cell but rather the background

Staining - Increases Contrast Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

• Most microbes are… • Colorless • Very Small • Difficult to see • Staining increases contrast • Increases size sometimes

(a)

Simple Stains

Crystal violet stain of Escherichia coli

Methylene blue stain of Corynebacterium a1: © Kathy Park Talaro; a2: © Harold J. Benson

Staining

• Positive staining: the dye sticks to the specimen to give it color

Staining • Negative staining: The dye does not stick to the specimen, instead settles around its boundaries, creating a silhouette.

Staining Techniques • Simple Stain • Uses only one basic dye • Provides basic information about cell shape and arrangement

• Differential Stain • Uses more than one dye • These procedures react differently with different kinds of bacteria • Helps distinguish between different kinds of bacteria • Most common and important differential stain is the GRAM STAIN

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

Differential Stains • Use two differently colored dyes, the primary dye and the counterstain – Distinguishes between cell types or parts – Examples include Gram, acid-fast, and endospore stains

Gram Staining • The most universal diagnostic staining technique for bacteria • Differentiation of microbes as gram positive (purple) or gram negative (red)

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

Gram Stain

Gram stain

Acid-Fast Staining • Important diagnostic stain • Differentiates acidfast bacteria (pink) from non-acid-fast bacteria (blue) • Important in medical microbiology – Mycobacterium

Endospore Stain • Dye is forced by heat into resistant bodies called spores or endospores • Distinguishes between the stores and the cells they come from (the vegetative cells) • Significant in medical microbiology

Special Stains • Used to emphasize certain cell parts that aren’t revealed by conventional staining methods • Examples: capsule staining, flagellar staining