Lab 5 Specimen Preparation Staining Aerotolerance

2 Specimen Preparation for Optical Microscopes Preparation of slide depends on 1. Condition of specimen 2. Aims of examiner 3. Type of microscopy available 8/18/12MDufilho

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. 8/18/12MDufilho

Spread culture in thin film over slide Pass slide through flame to fix it Air dry Figure 4.15 Preparing a specimen for staining 8/18/12MDufilho4

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 8/18/12MDufilho

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 8/18/12MDufilho

Staining reactions of dyes 78/18/12MDufilho

Microbiological stains 88/18/12MDufilho

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 8/18/12MDufilho9

Figure 6.3 Oxygen requirements of organisms-overview