Post on 06-Jan-2018
description
Lab 5
Specimen PreparationStaining
Aerotolerance
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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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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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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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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