Lab Practical 1 Microscopy/staining Techniques

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Microbes

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are tiny and measured in um( micrometers)

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1 um /1m

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= 10^-6 meters/ 3.38 ft 10^2 cm 10^3mm 10^6um 10^9nm 10^10 A

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Light Microscopy

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Visualize cellular specimens (EUK & PROK)
Use visible light to observe specimen
total magnification 1000x- 2000x
Resolution = 2 um

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Bright field Microscope

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Most common
specimens dead and stained to contrast with medium
look at light passing through specimen

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Phase-Contrast Microscopy

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-Used to observe live specimen without stain
-look at light passing through and reflecting off specimen
-good resolution

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Differential Interference contrast DIC

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-Similar to phase contrast but used two light beams
-specimens appear 3D

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Fluorescence Microscopy

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-Detects substances that absorb UV energy and emit visible light
-Specimen is stained with fluorescent dyes that tag specific features of the cell.

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Confocal Microscopy

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computer controlled lasers illuminate one focal plane at a time through the whole depth of the specimen.
sliced into computer based 2d 3d images

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Electron Microscopy

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visualize organelles and viruses
uses electron beams to visualize specimen
total mag= 10,000- 100,000 x
res- 2.5 um
2 types
TEM / SEM

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Transmission electron microscopy

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specimens must be cut into ultra thin sections view internal features
different parts of specimen absorb differing amounts of electrons resulting in black and white shades of gray density

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Scanning electron Microscopy SEM

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Visualize molecules
uses probes traces surface topology
total mag = almost infinite

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scanned probe microscopy

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can visualize molecules
uses tiny probes to trace surface topology
total mag= infinite
total res 10^-12

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Positive Staining Basic Dyes

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Cations in dye attracted to negative cell surface.

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Negative staining acidic dyes

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anions repelled by most cells, adhere to glass slide instead of cell

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Simple stains

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single basic dye that stains whole cell
crystal violet

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Differential Stains

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stain that reacts diff with diff types cells
distinguish one type of bacteria from another Acid fast stain

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Special Stain

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stain specific feature of cell
Endospore stain