Transcript SI PPT: Microbioal Growth

Updated: Sept. 8, 2010
Prokaryotic Growth
Supplemental instruction
Fall 2010
For Dr. Wright’s Bio 7/27 Class
Picture from http://www.compostinfo.com/tutorial/microbes.htm
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Pure culture techniques
Pure culture
:Individual organisms are isolated and grown from one single cell.
•_______(free
of microbe)
Sterile
media, instruments, flaming loop or needle
• ________________
Aseptic technique
During handling microbes
•________________
Culture medium
Nutrient for microbes; usually solid
Cell are grow in/on; Colonies from one single cell
Agar
_____
Polysaccharide from algae.
•________________
Media
Nutrient for microbes
Cells are grow in/on
“agar plate”
= petri dish with agar medium
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Streak-method
- Pure culture
- isolated and grown from one single cell.
- Using Aseptic technique
- Sterile (flaming loop) every streak
-Single colony is obtained in 4th .
After streak-method, what?
Stock culture and incubation.
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Bacteria growth
number of cells.
: increase in ______
-How do bacteria grow (reproduce or
multiply)? binary fission
________________________
Generation
time,” or Doubling time
- Time it takes for a population to double in
number. *can be 20minutes or 2hours.
Nt = N0 X 2n
Initial number: 10 cells
Doubling time: 20 minutes
After 4 hours, how many of bacteria?
http://www.bacferm.com.au/silac/micro/files
/page4_3.png
40,960
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Environmental factor:
Temperature, pH, O2, H2O
Microbes live EVERYWHERE!!!
Where they live? Why? How prevent them?
Why do we put food in refrigerator(4oC)?
Why do we cook food?
Why do we have a fever when infected?
Can microbes live in hot spring, artic or North
pole?
Enzymes for growth!!
http://a.abcnews.com/images/Technology/abc_artic5_080509_ssh
http://mayhem-chaos.net/photoblog/images/boiling_hot_spring.jpg .jpg
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http://www.subzerorepairlosan
geles.com/images/ge-cafe-eside-by-side-refrigerator-2.jpg
Environmental factor: Temperature
Psycrophiles
__________ : -5 ~ 15 oC
- in Artic, Antarctic, & lake with glaciers
__________ : -2 ~ 35 oC
Psycrotrophs
- Grow well in low temperature
- Food spoilage
__________ : 15 ~ 45 oC E.Coli
Mesophiles
- Most Human pathogens
- live in Hartnell college soil
__________ : 42 ~ 80 oC
Thermophiles
- hot springs, compot heaps, water heaters,
nuclear power plant cooling towers.
________________ : 68 ~ 108 oC
Hyperthermophiles
-hydrothermal vent deep in the ocean
- Archea
http://www.windenergyplanning.com/wpcontent/uploads/2008/12/didcot_power_station_cooling_tower_zootal
ures.jpg
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Why can some prokaryotes withstand such high
temperatures but most cannot?
Most prokaryotes have enzymes that are
denatured in high temperature.
 With denatured enzymes they can’t grow.
Thermophiles & Hyperthermophiles
-have heat stable enzymes
Many covalent bonds and hydrogen bonds in these
enzymes prevents denaturation of proteins (enzymes).
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Not all microbes cause disease!
Not all microbes spoil food!
____________
Mesophiles
Most human pathogens
 Think the temperature of human body!
Spoilage Microbes
Fungi(yeast, mold), Bacteria
:________________
_______________________
Mesophiles, Psychotrophs in non-refrigerated
foods.
Psycrotrophs,
Psycrophiles in refrigerated foods
______________________
Picture from http://leavingbio.net/FUNGUS/Fungi2.htm
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Archea
-Domain Archea; kingdom Archea
-Heat-stable enzymes
-Most of hyperthermophiles.
can live in hydrothermal vent.
-Live ______________
EVERYWHERE!!
*Sometimes called extremophiles!
http://cvitale.net/ecosystems/pages/ecosystems.html
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http://www.scientificamerican.com/med
ia/inline/what-makes-us-human_1.jpg
We, human, are __________.
Mesophiles
Pathogens for human will be
Mesophiles
__________.
Microbes inside human body
are Mesophiles
__________.
Microbes on the skin are
Mesophiles,
Psychrotrophs
____________
Fever – makes inactive.
inhibit growth.
Refrigerator (4 oC)
-Mesophiles -limits fast-growing.
Psycrotrophs, Psycrophiles
-______________________
:can live or multiply.
:spoilage occur but slowly.
http://www.subzerorepairlosan
geles.com/images/ge-cafe-eside-by-side-refrigerator-2.jpg
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Environmental factor: Oxygen (O2)
•In the air
•In or beneath the soil of Hartnell College
•In aquatic habitats
•On the skin
•In the human internal organ
-heart, lungs, throat, intestine, stomach…
http://www.geo.arizona.edu/palynology/geos462/pu http://gloucestercitylibrary.files.wordpress.com/2007/11/aquaticlife.jpg
erco1972.jpg
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Environmental factor: Oxygen (O2)
What do you think about Oxygen, O2?
____________
Obligate aerobes
“I love O2. I can’t grow without O2 just like
you.”
http://microbemagic.ucc.ie/ab
out_microbes/good_bad_ugly
.html
-die or not grow in absence of O2
-human=obligate aerobes.
-Aerobic respiration to generate ATP.
____________
Facultative
aerobes
“I like O2, but I can grow without O2.”
-can grow in absence of O2  fermentation or
anaerobic respiration.
-grow better in presence of O2  aerobic respiration.
-E.coli, yeast
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____________
Obligate anaerobes
“I hate O2! I cannot grow with O2.”
-cannot grow in presence of O2
 only fermentation or anaerobic respiration.
____________
Microaerophiles
“I like O2 a little but I’m scared.” “not too much, not too little.”
-Require a small of O2 ; die in high concentration
 fermentation or anaerobic respiration.
____________
Aerotolerants
“I don’t know what that is. I don’t care.”
-Don’t use O2 ; grow with or without O2.
 Neither aerobic or anaerobic respiration.
-strictly fermentative.
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_____________________________
require O2 to grow.
Obligate aerobes, Microaerophiles
_____________________________
Facultative anaerobes, Obligate anaerobes,live in the large intestine or
stomach. aerotolerants
_____________________________
on the skin.
Obligate aerobes, Facultative
anaerobes, aerotolerants
Microaerophiles, Facultative anaerobes,
_____________________________
beneath soil.
aerotolerants
ALL
_____________________________
live in oceans(aquatic habits).
_____________________________
common in aquatic habits
Microaerophiles
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Environmental factor: pH
•Living in our digestive system
- _________
in mouth pH =7
Neutral
- _________
stomach low pH
Acidic
Alkalic
- _________
small intestine high pH
•Acidify food (lower pH)
- inhibit microbial grow
- Pickling food
http://www.paranormalknowledge.com/article
s/wp-content/uploads/2009/01/pickle.jpg
All cells(prokaryotes) maintain internal (neutral/acidic/alkalic) pH.
 How?
-raise environment pH eg) H. pylori(neutrophils) in the stomach.
proton(H+) pump. eg) acidophiles, alkaliphiles
-using _________
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http://health.battlecreekenquirer.com/Test
Factsheet.aspx?id=29
Environmental factor: pH
neutrophils ; pH 5- pH 8
___________
-favor in neutral
-to prevent grow  acidify food
-H. pylori can grow in stomach
produce urease; generates ammonia
to raises pH of immediate
environment
 cause peptic ulcer
___________
acidophiles ; below pH 5.5
-to maintain internal neutral pH
 proton ( in / out )
-Volcanic fissure
http://media.photobucket.com/image/Japan%20volcani
c%20fissure/milljonas/volcanos.jpg
___________
alkalophiles ; above pH 8.5
-to maintain internal neutral pH
 Na+/H+ pump  proton ( in / out )
-Alkaline lakes and soils
http://www.indiana.edu/~sierra2/news_04/day1
2/renee_chemistry.jpg
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Environmental factor: H2O
- interaction b/w H2O and NaCl or Sugars.
- Osmosis; dehydration
- _____________
Plasmolysis
: shrink from the cell wall.
*This is not
prokaryotic cells!
How can bacteria survive high salt
environments? * making itself salty!
Draw water by increasing internal solute
concentration
-solute pump
-synthesize small organic compound
such as amino acid.
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http://farm4.static.flickr.com/3089/2655340763_87ebe676
8b.jpg
http://strawberrygal.wordpress.com/tag/fish/
Environmental factor: H2O
___________
Osmotolerant
- can tolerate up to10% NaCl
- can grow in dry environments (like skin)
___________
Halophiles
-require high level of NaCl to grow.
-Many marine bacteria (3%NaCl)
http://www.niwa.co.nz/__data/assets/imag
e/0009/49239/bacteria3_large.jpg
___________
Extreme halophiles
-require 9% level of NaCl or more to grow.
-Archaea
-salt flats of Utah, Dead sea
http://ianaloni.wordpress.com/2009/03/
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http://www.bluebird-electric.net/save_the_salt.htm
Food preservation using high solutes concentration
 inhibit growth of bacteria. *shrinking bacterial cell.
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Fastidious organisms
-require many many many growth factors.
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CO2
-autotroph : Carbon source from ______________
Organic compounds
-heterotroph : Carbon source from ______________
Photo: Energy sourc from Sunlight
______________
Chemolitho- : Energy sourc from Inorganic
______________
chemicals(H2, NH3…)
Chemoorgano- : Energy sourc from ______________
Organic chemicals
(sugars, amino acids…)
 Heterotroph
Phototroph
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___________
Photoautotrophs
-Energy - sunlight; Cabon – CO2
-make ________
organic compounds
-(primary producers/ consumers/ decomposers)
-Cyanobacteria, Algae
http://www.water.ky.gov/dw/profi/tips/alga
e.htm
___________
Chemolithoautotrophs
(Chemotrophs or Chemolithotrophs)
-Energy – inorganic compounds; Cabon – CO2
(H2, NH3, NO2-….)
-Hot springs, hydrothermal vents
-(primary producers/ consumers/ decomposers)
-Archaea
Primary producers:
Organisms that posses the capacity to produce organic compounds
from inorganic material, with the aid of light or a chemically derived
energy source. Primary producers compose the bottom of various
food chains.
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http://cvitale.net/ecosystems/pages/ecos
ystems.html
___________
Photoheterotrophs
-Energy – sunlight; Cabon – organic compounds;
-purple nonsulfur bacteria
___________
Chemoheterotrophs
-Energy – organic compounds; Cabon – organic compounds;
-(primary producers/ consumers/ decomposers)
We, humans, are _________________
Chemoheterotrophs .
http://www.leanandmighty.com/Shockme.html
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Lithotrophs, Autotrophs, Chemotrophs, Heterotrophs, Phtotrophs
Autotrophs
____________
carbon is obtained from carbon dioxide (CO2).
Heterotrophs carbon is obtained from organic compounds.
____________
____________
an organism whose energy source is inorganicLithotrophs
chemical reactions
Chemotrophs energy is obtained from external chemical
____________
compounds (either inorganic or organic).
____________
energy is obtained from light.
Phtotrophs
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Lithotrophs, Autotrophs, Chemotrophs, Heterotrophs, Phtotrophs
Hydrothermal vent
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Media
•_______________
Complex medium
-most routine laboratory
-nutrient for bacteria *bacteria food
-nutrient broth or agar, TSA, blood agar,
chocolate agar(for fastidious bacteria).
-peptone(protein)
-used for culture including pure culture.
•____________
Defined medium
- amounts of pure chemicals
- specific research experiments.
-To study nutritional requirements of bacteria.
-Glucose-salts or depending on needs for study.
-may add buffer to maintain neutral pH.
*because some bacteria produce acids.
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http://scienceprofonline.googlepages.com/mic
robiologyselectiveanddifferentialmedi
Some media are used to detect or to isolate an
organism in mixed bacterial population.
 selective and differential media
Both media are ( complex / defined ) media.
•________
Selective media
- allow “the organism” to grow.
inhibit the growth of the other organisms.
- MacConkey agar
to isolate Gram-negative bacteria. *inhibit gram+
•_________
Differential media
- to detect “the organism”.
- allow the growth of all organisms.
- Blood agar
hemolysin – lyses RBC.
-MacConkey agar  Both differential and selective
Fermentation sugar(lactose)
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When you have very few bacteria you are interested in……..
An organism we want is present at 1 cell/ml
but, 10,000 fold of other organism.
difficult in pure culture even if selective media are used.
Enrichment cultures
•___________
-helpful in isolating when the organism is
present in very few relatively.
-Broth (liquid) favors the growth of the
target organism.
increase the target population.
 then, isolating by pure culture.
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Measurement of bacterial growth:
Direct
counting * just count!
_____________
-Rapid; Don’t have to wait
-for organisms that can’t grow in medium.
- (Can/Can’t) distinguish live from dead cells.
Hard to count motile organisms.
- Microscopic
Counting chamber with liquid.
To estimate effectively total cells
at least 107 cells/ml must be present.
-Coulter counter (no light)
: Count total cells
-Flow cytometer (light)
:scattering of light by cells
:can count total cells
:can count the target cells after fluorescent dye or tag
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_____________
Viable Cell counts
- living (and growing) bacteria.
- for low number of bacteria.
- need special media (selective & differential)
- Dilute sample
Plate counts
Membrane filtration MPN (Most Probable
Number)
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1)____________
Plate counts
- only used if more than 100
organisms/ml
- dilute sample with distilled water or
0.85 % NaCl to prevent lysis .
- colony from one single cell.
- First, dilute sample
 pour plate or spread plate
 incubation  count colony as a
single cell (=colony-forming unit)
 Calculation
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2)____________
Membrane filtration
- Relatively low # of organisms in a
sample; dilute env.
eg) natural water
-sterile membrane filter
 plate
incubation  count colony as a
single cell (=colony-forming unit)
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3)____________
Most Probable Number (MPN)
- Statiscal estimation
theory of probability
- used commonly in coliforms.
- Dilution 10-1, 10-2, 10-3
 incubation
 note gas production
 compare MPN table.
estimate cell concentration.
Coliforms – lactose-fermenting, Gram-negative rods
reside in the intestine.
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Turbidity (cloudiness)
_______
- spectrophotometer
- can measure (#of cells / cell mass) *commonly cell mass
can estimate # of cells after done to
determine correlation b/w cell concentration
and tubidity.
-relatively assay
- Cells grown in liquid medium.
Turbid culture indicate bacteria
are present.
HOWEVER, a clear solution =
bacteria absence????? NO!
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Growth can be measured either by _______
_______
turbidity or by counting
to determine growth curve.
C
D
E
B
A
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A: Lag phase
B: Log phase
C:Stationary
D:Death phase
E: Prolonged decline
___________
Growth curve
-As a population in a closed system grow, a pattern of stage.
 in closed system.
-Grow pattern in broth culture.
http://ultrapixelshots.blogspot.c
om/2009_04_01_archive.html
1) Lag phase
- “ready to multiply”
- rich nutrient
- synthesize macromolecules.
enzymes, ribosomes, nucleic acids
- generate ATP.
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2) Log phase “Happy bacteria”
- exponential Phase
- Active multiplication
- Binary fission
- Susceptible to antibiotics
- Important medically
metabolites
- Primary
___________________
required for growth
Commercial flavors by gene manipulation
Late log phage (resistant) “Ready to be transformer”
- transition to stationary phase
- depletion of nutrients; build up of waste
- initiate the process of sporulation(to be endospores)
- Change cell walls, cytoplasmic membranes
-______________________
Secondary metabolites
Synthesizing different enzymes & proteins  Antibiotics!!!!
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3) Stationary Phase (more resistant)“Bacteria gangs”
- Dying  release nutrient for growth
of other cells.  Multiplying
- secondary metabolites
- Growth rate = the death rate
4) Death phase
- “Bacteria death”
- Exponential death; faster death; slower multiply
- Growth rate << the death rate
5) Prolonged Decline “What a bacteria!”
- Dynamic changes
- Use nutrient source from dead cells.
- “Survival of the fittest”
- Growth rate < the death rate
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Growth pattern in a solid medium
-Colony growth
-The center of colony
High density of cells  rapidly deplete O2
and nutrients.
-The edge of colony
grow rapidly  little competition and can
use more O2 and nutrients
Log phase
Stationary phase
Death phase
Picture from http://scienceblogs.com/digitalbio/2009/01/how_well_do_you_know_your_bact.php
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Protective structures
glycocalyx
endospores
biofilm
Biofilm
-polysaccharides-encased community
-attach to surface.; protect communities of microbes
-Interactions each other
dental plaque, scum in toilet, coats kitchen drains
-Resistant to antibiotics
65% of human bacterial infections
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Biofilms
-a layer of slim with open channel for
nutrient and waste.
- protects communities of microbes in
nature (not in the lab.)
-Unrelated cells can attach and grow.
-Communication with chemicals
-So Bad…
Resistant to antibiotics, disinfectant
Tooth decay (dental plaque)
in Pipes, drains, and cooling water
towers; damage equipment.
-May be good…
Bioremediation- degrade harmful chemical.
Wastewater treatment
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http://www.cs.montana.edu/ross/personal/intro-biofilmss1_files/r2003_PSTO_BFIN3STEPS.jpg
Biofilm (cont’d)
-But, beneficial to human
______________
Bioremediation
:natural process of cleaning up organic
contaminants through the use of microbes.
degrade harmful chemicals
Waterwater treatment
Biofilm
Youtube.com
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Mixed Microbial Communities
- Interaction, cooperation
- The activities of one species
benefit another
1) Creating anaerobic condition
Aerobes consume O2
 anaerobic  allows the growth of
other organism that cannot multiply in the
presence of O2
2) Wastes of one species
 can be nutrient for others
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