Lab 5

Specimen PreparationStaining

Aerotolerance

2

Specimen Preparation for Optical Microscopes

Preparation of slide depends on 1. Condition of specimen2. Aims of examiner3. Type of microscopy available

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3

Specimen Preparation for Optical Microscopes

• Wet mounts and hanging drop mounts – allow examination of characteristics of live cells: size, motility, shape, and arrangement

• Fixed mounts are made by drying and heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts.

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Spread culture inthin film over slide

Pass slide throughflame to fix it

Air dry

Figure 4.15 Preparing a specimen for staining

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5

Staining

Dyes create contrast by imparting a color to cells or cell parts

• Basic dyes – cationic, positively charged chromophore

• Acidic dyes – anionic, negatively charged chromophore

• Positive staining – surfaces of microbes are negatively charged and attract basic dyes

• Negative staining – microbe repels dye, the dye stains the background

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6

Staining• Simple stains – one dye is used; reveals

shape, size, and arrangement• Differential stains – use a primary stain

and a counterstain to distinguish cell types or parts (examples: Gram stain, acid-fast stain, and endospore stain)

• Structural stains – reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains

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Staining reactions of dyes

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Microbiological stains

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Growth Requirements

Oxygen requirements• Obligate Aerobes require oxygen as the final

electron acceptor of the electron transport chain• Obligate Anaerobes -cannot tolerate oxygen and

use an electron acceptor other than oxygen• Facultative anaerobes - maintain life via

fermentation or anaerobic respiration• Aerotolerant anaerobes - prefer anaerobic

conditions• Microaerophiles - require low levels of oxygen

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Figure 6.3 Oxygen requirements of organisms-overview