136k - UCLA.edu

Todd Herman
310-923-0986
[email protected]
Lab manual available at:
Course Reader Material
1137 Westwood Blvd.
443-3303
office hours:
Tue. 11am YH3336
Wed. 1pm
YH3340
Professor Robert Simons
3610B MSB
825-8890
[email protected]
Announcements
• purpose of these lectures/discussions
• outlines on web
• additional info (not covered in
lecture) on the 101L CD-ROM
• switching lecture sections
• the textbook
Madigan (10th ed.)
• same as text for 101
• reading assignments are online
• copies on reserve in Powell
Announcements
• the 101L CD-ROM
• integral to this class
• (readme)F04.txt file tells you how to run it
• syllabus and checklist on the CD (MS Word docs)
• lecture notes will include notices to look at
certain sections (as below)
• replacements
for week one:
see the Techniques module on the CD-ROM
-- “Aseptic Technique” (all four sections)
-- “Equipment & Methods” (first three sections)
-- “The Microscope” (first eleven sections)
see the General Media module (one section)
Announcements
• your lab manual
• work schedule
• the first 35 pages
• the last 35 pages
• readings listed with each expt. (optional)
• questions at the end of each expt.
• your approach to this class
• concepts, not recitation
• analysis of data
website
“syllabus” section:
• an outline of each lecture will be posted
on the web (before the weekend)
• password: _________
• course bulletin board
• other items… assigned readings for the quarter
http://www.lsic.ucla.edu/classes/mimg/fall_04/mimg101l/
today’s lecture
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background/review, morphology
selection, enrichment, and some terms
media
Expt. 1 -- Intro. to the microbial world
Expt. 3 -- Bacteria in the soil:
isolation from enrichment cultures
Expt. 2 -- Morphology
microscope preview
Background
Prokaryotes
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in this class, we’ll study primarily the eubacteria
• eukaryotes will be treated as contamination
(molds, fungi)
no nucleus or membrane-bound organelles
replicate by binary fission
transcription and translation occur together
one ds circular DNA
cell wall
Morphology
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bacillus
coccus
spirilium
amorphous, filamentous
Morphology
1000X
bacillus:
rod-shaped
Morphology
cocci:
spherical
“staphlococcus” -- clustered spheres
Staphlococcus aureus
Morphology
cocci:
spherical
Streptococcus
lactis
“streptococcus” -strung-out spheres
Morphology
cocci:
spherical
Streptococcus pneumoniae
“diplococcus” morphology
Morphology
1000X
cocci:
spherical
Staphlococcus
variety
Morphology
spirillium:
spirals
Leptospira interrogans
1000X
Morphology
filamentous
Streptomyces
Controlling the environment -- selection
selection: “controlling growth conditions to
favor organisms with a particular genotype”
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requires that we know the biochemical/genetic
background of the organism
(in our case, the type of bacteria)
Controlling the environment -- selection
methods of selection:
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stagnate the undesired cells & grow the desired cells
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kill the undesired cells & grow the desired cells
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stagnate the desired cells; grow up the undesired
cells to kill them (called counter selection)
e.g., penicillin only kills actively dividing cells
Controlling the environment -- selection
enrichment:
“the use of selective
media and growth
conditions to increase
the relative number of
a particular organism
in a population
(usually directly from
natural samples)”
Selective factors we can use for enrichment
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temperature
pH
carbon source
energy
nutrient dependency
oxygen
spore formation
source of inoculum
Media – liquid/solid; complex/simple
complex
rich, or undefined media
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predigested protein:
• tryptone (enzyme digest of caseine)
• peptone (enzyme digest of meat)
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yeast extract: lots of vitamins, biomolecules
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might add sugar(s)
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e.g., YTA, YTB, SYTA
Media – liquid/solid; complex/simple
simple
defined, synthetic, or minimal media
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exact amounts of ingredients known
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need N, P, S, K, Na, Ca, Mg, Fe, trace elements
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e.g., MSA, MSB
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might add C-source (e.g., GMA)
Media – selective/indicator
selective media
“controlling growth conditions to favor
organisms with a particular genotype”
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definition of selection:
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could just be simple media
selecting for
bacteria which are prototrophs (able to
synthesize all their biomolecules from simple
precursors)
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could be complex media, plus an antibiotic
• e.g., YTA + cycloheximide (targets 80S
ribosome, selecting against eukaryotes)
Media – selective/indicator
indicator
screening, or diagnostic media
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e.g., blood agar plates
• α-hemolysis
• β-hemolysis
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e.g., SYTA
Strep. salivarius forms a capsule
(or glycocalyx) when grown on sucrose, but
not when grown on glucose
see the General Media module
on the CD-ROM
Experiment 1 – Introduction to the
microbial world
• read experiment in the lab manual
• inoculating selective plates and indicator
plates from various sources -- keep track!
• air, soil, water
• skin, throat, tongue
• make microscopic observations, do Gram stains
• ask yourself why certain plates are used in each part
• e.g., cycloheximide, GYC, SYTA,…
Experiment 1 – Introduction to the
microbial world
“Air” plates… exposed to air for ten minutes
• some plates contain cycloheximide;
some do not.
• why? What purpose does this serve?
Experiment 3A – Bacteria in soil: isolation
from enrichment cultures
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enrichment with
“unusual” C-sources
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the start of a multi-expt. project
(3A, 3B, 3C)
• isolating and characterizing a
Pseudomonad
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inoculate the first enrichment
culture
• read pp. 45-46 carefully; read
again in a month
Experiment 2 – Morphology
microscopic observations
• prepared slides
• bacterial types, flagella, capsules, spores, etc.
• wet mounts
• Bacillus megaterium, Pseudomonas aeruginosa,
colonies from your SYTA and blood agar plates
• become proficient at using the microscope!
see the Techniques module -- “The Microscope”
(first 11 sections) on the CD-ROM