Lab equipment & procedures Chapter Ⅱ

Microscopes A. Light Microscopes 1. Single Lens 2. Compound microscopes eye pieceobjectiveTotal M Low P10X10X100X High P10X43X430X Oil 10X97X970X Resolving Power : 0.2 um ( bare eye resolving power : 0. 1mm)

B. U.V. Microscopes U.V. light Resolving power : 0.1 um C. Fluorescence Microscope Fluorescence dye + U.V. Light eg. TB. Use auramine (yellow dye) D. Phase Contrast Microscope For living material, viscosity differences are increased.

The light microscope

E. Electron Micoscope 1. Transmission EM use a beam of electrons ( very short wavelength) Resolving power : um Magnification : 100,000 X 2. Scanning EM electrons strike on the surface (3D)

The electron microscope

How to Prepare Slides 1.Living material Hanging drop technique (for movement) 2. Stain bacteria Basic dye : color on “+” charge eg. methylene blue Acidic dye : color on “-” charge eg. nigrosine

Staining Techniques a. Simple stain b. Gram stain1884 Christian Gram Crystal violet – primary dye Iodine – mordant 95% ETOH – decolorizing agent Safranin –counter stain c. Acid – fast stain for mycobacteria

The Gram stain (1)

The Gram stain (2)

Preparation of a Pure Culture Colonies : Pure culture techniques 1. Streaking methods 2. Liquid dilution methods Culture medium: carbon, nitrogen, minerals and water - defined medium - undefined medium

Method (cont.) Isolation-pure Culture (Discontinuous Streaking) 1. Sterilize wire loop by holding over flame until it turns bright orange 2. Wait a few seconds, cool loop by lightly touching NA 3. Take bacteria with loop from sample culture and streak on other plate. 4. Cover, label, and tape plates. 5. Put in incubator at 37°C

Method (cont.) Isolation: continuous streaking Do the same procedure are discontinuous streaking except follow a different pattern.

Dilutions

Special media: - carbohydrate media - selective medium : select a certain organisms and inhibit other organisms. eg. Bile salt select Salmonella and Shigella but no E. coli - differential medium eg. Blood agar - enrichment cultures eg. Isolate organisms from environment such as soil or pond water mineral medium + sodium benzoate select Pseudomonas.

Viable counting

Turibdimetric growth measurements (1)

Turibdimetric growth measurements (2)

Turibdimetric growth measurements (3)

Bacterial morphologies (1)

Bacterial morphologies (2)

Bacterial morphologies (3)

Bacterial morphologies (4)