MICROSCOPY AND STAININGCHAPTER 3

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Metric Units

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Light Properties

Wavelength

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polarity

Light is a wave

Filters can block waves in off axis planes

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Waves can be added

+ +

= =

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Light Properties

Resolution

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Wavelength/Resolution Interaction

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Light Properties

Reflection

Transmission

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Light Properties

Absorption

Refractionbending

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Light Microscopy Types

Compound

Bright Field

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Oil immersion

With oilwithout

Some info lost

Oil with intermediaterefractive index

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Microscopy — Dark Field

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Microscopy — Phase Contrast

Dual Beam

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Phase contrast Phase-contrast microscopy was invented in 1936 by Frits

Zernike, a Dutch mathematical physicist. It is based on the principle that cells differ in refractive index (a factor by which light is slowed as it passes through a material) from their surroundings. Light passing through a cell thus differs in phase from light passing through its surroundings. This subtle difference is amplified by a device in the objective lens of the phase-contrast microscope called the phase ring, resulting in a dark image on a light background (Figure 2.5b). The ring consists of a phase plate—the key discovery of Zernike—that amplifies the minute variation in phase. Zernike’s discovery of differences in contrast between cells and their background stimulated other innovations in microscopy, such as fluorescence and confocal microscopy (discussed below). For his invention of phase-contrast microscopy, Zernike was awarded the 1953 Nobel Prize in Physics.

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Microscopy — DIC

Differential Interference Contrast

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DIC “differential interference contrast”

Similar to phase contrast, but input light is polarized

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Microscopy — Fluorescence Ultraviolet light

flourescein

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Advantages of fluorescence

Can use specialized chemical probes that target specific features and then tag with fluorescent dyes

Downside: must use expensive filters and excitory frequencies

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Microscopy — Confocal

ConfocalAllows 3 dimensional viewing

Allows multiple dyes to be overlaid

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Confocal microscopy

Allows 3 dimensions

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Combined confocal and fluorescence

Antibody labeling

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Microscopy Imaging

Digital

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Fig. 2-15

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Electron Microscopy Transmission

(TEM)

Scanning (SEM)

Scanning Tunneling (STM)

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TEM

Most popular for bacteria. Allows imaging internal features, but requires heavy metalstaining.

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Electron Microscopy Images

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Microscopy Techniques

Wet Mounts Smears Staining

Fig. 2-3

100Slide

Dry in air

Flood slide with stain;rinse and dry

Place drop of oil onslide; examine with 100objective lens

Spread culture in thinfilm over slide

Pass slide throughflame to heat fix

Oil

I. Preparing a smear

III. Microscopy

II. Heat fixing and staining

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Staining cells - increases contrast

Simple stain - one dye - shows size, shape, and arrangement

Methylene blue -

yeast

Cheek cell

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Common stains

Safranin (*basic, + charge)

Crystal violet

red

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Differential stains

Use multiple dyes or dyes that interact with organisms differently.

Primary stain / counterstain

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Gram Stain

Gram Stain The single most important stain in microbiology. Set the initial taxonomy of bacteria.

Crystal violet *(basic stain)

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Gram Stain

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Acid Fast Stain

Carbol-fuchsin stains acid fast organisms

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Acid Fast

The Ziehl-Neelsen stain, also known as the acid-fast stain, was first described by two German doctors; Franz Ziehl (1859 to 1926), a bacteriologist and Friedrich Neelsen (1854 to 1894), a pathologist. It is a special bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. Mycobacterium tuberculosis is the most important of this group, as it is responsible for the disease called tuberculosis (TB). It is helpful in diagnosing Mycobacterium tuberculosis since its lipid rich cell wall makes it resistant to Gram stain. It can also be used to stain few other bacteria like Nocardia. The reagents used are Ziehl-Neelsen carbolfuchsin, acid alcohol and methylene blue.

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Acid Fast of Mycobacterium tuberculosis

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Negative Stain

Sometimes referred to as capsular stainIndia ink or nigrosin

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Flagellar StainSalmonella typhimurium

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Endospore Stain

Used on spore forming bacteria such as Bacillus sp.

Malachite greenstains spores