Tools of the Laboratory
I.
Methods of Culturing Microorganisms-Five I’s
A. Introduction
1. Microbes live in complex associations
2. Grown under artificial conditions
3. Contamination of cultures
B. Inoculation: Producing a Culture
C. Isolation: Separating one Species from Another
1. Colony
2. Streak plate method
3. Loop dilution or pour plate
4. Spread plate
D. Media: Providing Nutrients in the Laboratory
1. Introduction
a. Nutrient requirements vary widely
b. 500+ types of media
c. Media in test tubes, flasks, Petri dishes
d. Inoculate with loops, needles, pipettes, swabs
e. Cell cultures & host animals for viruses and some bacteria
f. Aseptic technique critical
2. Types of Media
a. Physical form
b. Chemical composition
c. Functional type
3. Physical States of Media
a. Liquid media
b. Semisolid media
c. Solid media
1) Liquefiable – agar (solidifies at 42C and melts at 100C)
2) Nonliquefiable-don’t melt
4. Chemical Content of Media
a. Synthetic media
b. Complex or nonsynthetic media
5. Media to Suit Every Function
a. General purpose media
b. Enriched media
c. Selective media
d. Differential media
e. Miscellaneous media
E. Incubation, Inspection and Identification
1. Incubation
a. 20-40 C
b. oxygen or carbon dioxide content
c. day to weeks
II.
2. Culturing
a. Subculture to obtain pure culture
b. Mixed culture
c. Contaminated
3. Identification
a. Eukaryotes-by morphology
b. Prokaryotes- cellular metabolism by biochemical tests
c. DNA analysis
4. Maintenance and Disposal of Cultures
a. Autoclave
b. Incineration
The Microscope: Window on an Invisible Realm
A. Magnification and Microscope Design
1. Introduction
a. Magnification
b. Resolution
2. Principles of Light Microscopy
a. Objective lens
b. Ocular lens
c. Total magnification
3. Resolution: Distinguishing Magnified Objects Clearly
a. Capacity to distinguish 2 adjacent points
b. Shorter wavelengths provide better resolution
c. Limitation to resolution
B. Variations on the Optical Microscope
1. Bright-field Microscopy
2. Dark-field Microscopy
3. Phase contrast Microscopy
4. Fluorescence Microscopy
C. Electron Microscopy
1. Introduction
a. Early 1930’s
b. Wavelength of electron is 100,000 shorter than visible light
c. Resolving power is 0.5 nm
d. Two lens system, condensing lens, specimen holder,
focusing apparatus
e. Specimens are dead
2. Transmission Electron Microscope
a. Thin slices of specimen
b. Detailed structures of cells and viruses
c. Resolution 0.5 nm & magnification 1,000,000x
3. Scanning Electron Microscope
a. Realistic 3-D view
b. Resolution 10 nm & magnification 1000.000x
D. Preparing Specimens for optical Microscopes
1. Fresh, living preparations
a. Wet mount
b. Hanging drop
2. Fixed, stained smears
a. Heat fixing or chemical fixing
b. Basic (cationic) dyes have + charge
c. Acidic (anionic) dyes have – charge
d. Negative versus Positive Staining
e. Simple versus Differential Staining
1) Simple uses one dye
2) Differential uses two dyes
a) Gram stain
b) Acid fast stain
c) Endospore stain
d) Flagellar stain