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bmic chp 2 - Coggle Diagram
bmic chp 2
culture
Mixed culture – a culture that contains more than one species of microbes
Pure culture – a culture that contains a single species of microorganism
subculture: make a culture from a pre-existing culture using inoculum and fresh medium+incubate
culture media
broth
agar
aseptic techniques
specific practices and procedures performed under carefully controlled conditions
so that there is minimal contamination
To protect samples, cultures, sterilized media and equipment from contamination
To protect user from potentially harmful microorganisms
Sterilization techniques
destroy microorganisms in/on them before using
Autoclaving
steam under pressure
thermostable, not sensitive to moisture
Kills vegetative cells and endospores
common settings
121oC, 15 psi, 15 min
115oC, 10 psi, 10 min
Heating in hot air oven
For materials and equipment that are sensitive to moisture
up to 250C for several hours
kills vegetative cells and endospores
Flame sterilization
For non-flammable equipment that are
used over and over again
within a short time frame
Inoculating loop and wire (flame till red hot)
Glass spreader, metal scissors, metal spatula (surface sterilization using alcohol and flame)
Filter sterilization
for fluids and solutions that are
not thermostable
pass fluid/solution through filter membrane
size exclusion: microbial cells are
too large to pass through
pore membrane
common membrane pore sizes
0.45mm – good for removing
bacterial
and larger cells
0.22mm – good for removing large
viruses
Controlled Environment
To provide optimal environmental conditions for microbial growth
e.g Incubators controlling temperature and/level of humidity
Controlled pH
Adjust pH to
optimal pH
to support optimal microbial growth
pH is maintained by using
buffers
(combination of a weak acid and its salt)
Control osmotic pressure
Plasma membrane is a semi-permeable membrane
Osmotic pressure-the pressure exert on the semi-permeable membrane
by the solution
Medium is carefully formulated to be
roughly isotonic(equal solute conc. on both sides of membrane
Controlling Gaseous Atmosphere
Aerobe: an organism that requires O2 for growth (grows in presence of atmospheric air)
Anaerobe: an organism that can only grow in the absence of O2 (cannot grow in presence of atmospheric air)
Obligate
very strict
obligate anaerobes
Environments: anaerobic
glass jar
, anaerobic
chamber
etc. (no O2)
others: capnophiles, microaerophiles-candle jars, CO2 incubators (allow a little bit of O2 in, controls conc. of O2)
CO2 incubators: maintain an optimal environment for cell growth, by providing carbon dioxide control in a humidified atmosphere with constant temperature.
candle jars: Candle jars are used to grow bacteria requiring an increased CO2 concentration (capnophiles). Candle jars increase CO2 concentrations and still leave some O2 for aerobic capnophiles.
facultative
can grow with or without(for facultative anaerobes)
Nutrients
Macronutrients
Required in
fairly large
quantities (g/L)
Elements: C, N, H, O, S, P, Na, K, Ca, Mg, Fe
Micronutrients
Required in
minute
quantities (mg/L or less)
Trace elements: Mo, Mn, V, Co, etc
Growth factors: vitamins
Organic nutrients-carbs, proteins, lipids
Growth factors, organic carbon sources, etc
Inorganic nutrients: mineral salts
All living organisms require carbon and energy
carbon source
heterotrophs
Carbon source from
organic
sources
autotrophs
carbon source from environmental CO2
energy source
phototrophs: energy source from sunlight
chemotrophs: obtain energy from oxidation of
inorganic
chemicals
Classify=energy source+carbon source+troph
chemoheterotrophs: bacteria(most), protozoa, fungi
photoautotrophs: Algae, cyanobacteria
photoheterotrophs :green, purple non-sulfur bacteria
chemoautotrophs: extremophiles(can live in extreme temperatures, pH, salinity levels
Growth factors
Not all microorganisms require growth factors
prototrophs: can synthesize all the growth factors
auxotrophs: require certain growth factors to be included in the growth medium
Histidine auxotrophs – microorganisms that cannot make the amino acid histidine themselves
lack the ability to make histidine, need to get from the culture
often mutants
Culture media
Solid culture(agar)
Microorganism grow as
colonies
on the surface of the agar (each colony arises from a single cell)
Useful for
isolation
to give pure cultures
Easier to
detect contamination
More tedious to prepare
, esp in large quantities
Smaller yields
and more difficult to harvest
Liquid culture(broth)
Growth appears as turbidity
Large quantities
of medium can be
prepared easily
Large quantities of microbial cells can be cultured
Easily harvested and diluted
Difficult to tell whether it’s contaminated, and impossible to salvage if contaminated
types of media
Defined/Synthetic media
known components, exact composition, exactly duplicable
Undefined/Complex media
natural products, exact composition unknown, cannot duplicate exactly and is a batch by batch variation
Natural products
Water-soluble extracts
Eg. beef extract, yeast extract, soil extract
Digests of proteins
Eg. casein hydrolysate (acid
hydrolysed
casein), peptone (peptic digest of animal
proteins
), tryptone (
pancreatic
digest of casein) etc.
provide amino acids, organic carbon source, nitrogen and sulfur
Others
blood, serum
provides complex growth factors for fastidious microorganisms
purpose of media
1) General media
Support the growth of a
wide variety
of microorganisms
except the fastidious species
(nutritionally demanding; a wide variety of growth factors need to be supplied)
Eg. nutrient broth/agar, tryptone soya broth/agar
contains natural products
2) Minimal media
Chemically defined, contains C source, inorganic minerals and water
Contain
minimum nutrients
to support the growth of a
specific
species/strain of microorganism
Useful for screening of
auxotrophic mutants
and to
investigate the minimal nutritional requirements of the study organism
and define them.
3) Selective media
Contain specific
inhibitory
substances against specific microorganisms
Suppress growth of undesired microorganisms
, favour growth of particular microorganisms
Allow
rapid isolation
as well as
preliminary identification
of desired microorganisms
eg. potato extract agar
The high carbohydrate and low nitrogen content
limits growth of most bacteria
.
Lactic
acid
lowers the pH to ~3.5,
allowing only acidophiles to grow.
4) Differential media
Give visual differentiation by appearance (colour) to distinguish colonies of one microorganism from another
Usually involve
nutrient utilization
pH indicators usuualy involved when there is acid/alkaline production
eg. sheep blood agar
a-hemolysis – partial lysis of blood cells greenish halo around colonies
b-hemolysis-complete lysis of blood cells, clearing around the colonies
g-hemolysis-no lysis of blood cells, agar appears unchanged
hemolysis: destruction of red blood cells
Different bacteria lyse blood cells differently
5) Enrichment media
6) Specialized media
used for purposes other than isolating/growing/differentiating microorganisms
Transport, reducing, identification, assay, carbohydrate fermentation, enumeration, enriched
Both MacConkey and Mannitol salt agar are selective and differential agars
Mannitol salt agar
This medium is used for the differentiation of
pathogenic staphylococci from other staphylococci
in clinical samples.
The 7.5% salt inhibits most bacteria growth;
only salt-tolerant bacteria can grow.
Pathogenic staphylococci (usually S. aureus) ferments mannitol.
Acids produced lowers the pH, and in the presence of phenol red, changes the colour of the surrounding agar yellow.
Mannitol-sugar alcohol, and high salt concentration
Test to see if the Bacteria can ferment mannitol or not
Colonies that can ferment mannitol appears yellow, cannot ferment will remain pinkish
MacConkey agar
This medium is used for the
detection of coliforms
from environmental and food samples.
Bile salts and crystal violet
largely inhibit Gram-positive bacteria(no growth).
Fermentation of lactose
produces lactic acids,
lowering the pH, which in the presence of neutral red, changes
the colour of the colonies and/or the surrounding agar to red/pink.
This differentiates the coliforms (lactose-fermenting) from non-coliforms (non-lactose fermenting).
Gram+/- 2. Lactose+/-
no growth, gram +ve, got growth, gram -ve
lactose +ve, pink, Lactose -ve, tan(pH indicators)
Other parts and functions of media
Glucose: carbon and energy source
H2O is a solvent
NaCl maintains osmotic balance
Media for viruses-living cell media
examples
1) Bacterial
cells
cultures
2) Embryonated eggs
3) Living animals/plants
4) Tissue/Cell cultures (plant or animal)
viruses are obligate intracellular parasites, need living host cell to replicate