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BACTERIA GROWTH & CULTURE
BACTERIA ARE UBIQUITOUS, THEREFORE, THEY CAN BE ISOLATED
FROM ANY ENVIRONMENT ON EARTH.
WE MUST, THEREFORE, APPRECIATE THEIR VARIED HABITATS &
STRATEGIES USED IN ADAPTING TO DIFFERENT HABITATS .
TO BEST STUDY BACTERIA, WE MUST BE ABLE TO GROW THEM IN
CULTURE.
FACTORS AFFECTING THEIR GROWTH MAY BE PHYSICAL &
NUTRITIONAL AND AS SUCH THEY MAY BE CLASSIFIED BASED ON
THESE FACTORS.
BACTERIA GROWTH & CULTURE
PHYSICAL FACTORS:
pH, Temperature, Oxygen, Moisture, Osmotic Pressure; Radiation
pH: (ACIDOPHILES, NEUTROPHILES AND ALKALIPHILES) INDICATE pH
optimum
Acidophiles = OPTIMUM pH < 4
Neutrophiles = OPTIMUM pH 6 - 7
Alkaliophiles = OPTIMUM pH 8 to 9
TEMPERATURE: MOST SPECIES OF BACTERIA MY TOLERATE A 30O
TEMPERATURE RANGE. BASED ON THE TEMP RANGE THEY MAY BE
CLASSIFIESD AS:
Psychrophiles = 15o to 20oC (many grow well at 0oC); None inhabit the
human body but some cause spoilage of refrigerated foods.
Mesophiles = 25o to 40oC (human pathogens included in this group).
Thermophiles = 50o to 60oC (Many found in compost heaps & a few
even tolerate temperatures as high as 110oC).
BACTERIA GROWTH & CULTURE
BACTERIA GROWTH & CULTURE
Oxygen:
Aerobes = Require oxygen to grow
Anaerobes = Do not require oxygen for growth
Obligate aerobes = Must have free O2 for respiration.
Obligate or strict anaerobes = killed by free O2.
Microaerophiles = grow best in presence of small
amounts of free O2.
Facultative anaerobes = carry out aerobic respiration
when O2 is present but shifts to anaerobic respiration in
its absence.
Aerotolerant anaerobes = Survive in the presence of O2
but do not use it in their metabolism.
BACTERIA GROWTH & CULTURE
Note: aerobic growth at top
of broth
BACTERIA GROWTH & CULTURE
Example #2: Growth in agar
BACTERIA GROWTH & CULTURE
WHY IS OXYGEN TOXIC TO SOME BACTERIA?
IT MAY INACTIVATE PROTEINS (e.g., inactivation of sulfhydryl
groups due to oxidation)
PRODUCTION OF TOXIC OXYGEN DERIVATIVES (e.g.,
O2 +e- -----> O2-. (superoxide radical)
O2-. + e- + 2H+ -----> H2O2 (hydrogen peroxide)
H2O2 + e- + H+ -----> H2O + OH. (hydroxyl radical)
Superoxide radical, hydrogen peroxide & hydroxyl radical are extremely toxic.
They are powerful oxidizing agents:
Hydroxyl radical are most toxic With half life of micro-seconds
They react with most organic molecules resulting in their destruction
Obligate aerobes & facultative anaerobes protect themselves because the possess
the enzymes SUPEROXIDE DISMUTASE & CATALASE
2O2-. + 2H+ ---SUPEROXIDE DISMUTASE---------------------> O2 + H2O2
2H2O2 ---CATALASE---------------------> 2H2O + O2
BACTERIA GROWTH & CULTURE
AEROTOLERANT BACTERIAMAY LACK CATALASE BUT ALMOST ALWAYS HAVE
SUPEROXIDE DISMUTASE
ALL STRICT ANAEROBES LACK BOTH ENZYMES & HAVE NO STRATEGY TO
DEAL WITH TOXIC OXYGEN PRODUCTS
BACTERIA GROWTH & CULTURE
SHIFTING BATERIA TO EXTREME TEMPERATURES
(1) RESULT IN DISRUPTION OF MEMBRANE PEARMEABILITY DUE TO
INCREASED FLUIDITY OF THE LIPID BILAYER.
(2) LEADS TO LOSS OF SOLUTES.
(3) HIGH PROTON PERMEABILITY (LOSS OF PROTON CIRCUIT)
ADAPTATION TO EXTREME TEMPERATURES BY THERMOPHILES REQUIRE:
(1) ENZYME SYSTEMS THAT TOLERATE HIGH TEMPERATURES
(2) CHANGES IN THE LIPID COMPOSITION TO MAINTAIN MEMBRANE
FLUIDITY:
- AT LOW TEMPERATURE, SHORT-CHAIN FATTY ACIDS &UN-SATURATED
FATTY ACIDS USED (HELPS MEMBRANE TO REMAIN FLUID DUE TO KINKS
THAT HELP TO PREVENT CLOSE PACKING OF OF LIPID CHAINS).
BRANCHED CHAIN FATTY ACIDS ARE SOMETIMES REPLACE THE
UNSATURATED FATTY ACIDS.
- AT HIGH TEMPERATURES SATURATED FATTY ACIDS USED IN MEMBRANE.
BACTERIA GROWTH & CULTURE
BACTERIA GROWTH & CULTURE
pH HOMEOSTASIS (MAINTAINING CYTOPLASMIC pH (pHi ) DESPITE
FLUCTUATIONS IN IN THE EXTERNAL pH).
- THE CYTOPLASMIC MEMBRANE ACTS AS FIRST BARRIER SINCE IT
IS RELATIVELY IMPERVIUS TO IONS.
- ACIDOPHILES & NEUTROPHILES USE PROTON PUMPS &
POTASSIUM TRANSPORT SYSTEMS TO MAINTAIN pHi IN NEUTRAL
RANGE.
- ALKALOPHILES Na+ CIRCUITS ARE OF MAJOR IMPORTANCE.
Neutrophiles &
acidophiles:
exchange K+ for
H+ using antiport
transport system
Alkalophiles: exchange internal
Na+
Ions for external H+
BACTERIA GROWTH & CULTURE
BACTERIA GROWTH IN CULTURE
TWO TYPES OF CULTURES
(1) BATCH CULTURE OR CLOSED SYSTEM
- SINGLE BATCH OF GROWTH MEDIA IN CLOSED VESSEL
- NO FRESH MEDIA ADDED
- DURING INCUBATION, NUTRIENT CONCENTRTION
DECLINES AS WASTE CONCENTRATION INCREASED
(2) CONTINUOUS CULTURE OR OPEN SYSTEM
- CONTINUAL PROVISION OF NUTRIENTS
- CONTINUAL REMOVAL OF WASTE
- EXPONENTAL GROWTH MAINTAINED FOR EXTENDED
PERIOD
- MAINTAINS CONSTANT BIOMAS CONCENTRATION (# OF
CELLS)
- TWO MAJOR TYPES OF SYSTEMS:
(A) CHEMOSTAT
(B) TURBIDOSTAT
BACTERIA REPLICATION IS BY BINARY FISSION
GROWTH CAN BE PLOTTED AS THE LOGARITHYM OF CELL # VERSUS
INCUBATION TIME
BACTERIA GROWTH & CULTURE
BACTERIA GROWTH & CULTURE
WHEN BACTERIA ARE INTRODUCED INTO FRESH CULTURE MEDIUM
THE GROWTH CURVE FOR A BATCH CULTURE HAS FOUR PHASES:
(1) LAG PHASE
(2) LOG OR EXPONENTIAL PHASE
(3) STATIONARY PHASE
(4) DEATH PHASE
LAG (“A RETOOLING PERIOD”):
MAY BE DUE TO SWITCHING FROM ONE MEDIUM TO ANOTHER, REQUIRING
NEW ENZYME SYNTHESIS; CELLS MAY REQUIRE THE SYNTHESIS OF NEW
FACTOR, RIBOSOMES OR ATP
NO INCREASE IN CELL # OCCURS
PHASE VARIES IN LENGTH DEPENDING ON THE CONDITIONS FROM WHICH
THE INNOCULUM IS TAKEN (OLD VS NEW CULTURE; SWITCHING FROM
MEDIUM WITH A DIFFERENT COMPOSITION e.g., GLUCOSE AS CARBON &
ENERGY SOURCE TO LACTOSE AS CARBON & ENERGY SOURCE)
BACTERIA GROWTH & CULTURE
EXPONENTIAL PHASE:
RATE OF GROWTH AND DIVISION IS MAXIMUM
RATE OF GROWTH IS CONSTANT, i.e., DOUBLING OCCURS AT REGULAR
INTERVALS
CELLS ARE MOST UNIFORM IN CHEMICAL & PHYSIOLOGICAL PROPERTIES
STATIONARY PHASE:
POPULATION GROWTH CEASES (DEATH RATE = REPLICATION RATE)
OCCURS IN BACTERIA CULTURES AT CONCENTRATIONS ABOUT 109 CELLS/ml
FACTORS THAT TRIGGER THIS PHASE INCLUDE
- ESSENTIAL NUTRIENT DEPLETION,
- OXYGEN (AEROBES)DEPLETION,
- TOXIC WASTE ACCUMULATION (e.g., ORGANIC ACIDS DUE TO
FERMENTATION* [VERY SIGNIFICANT IN ANAEROBIC CULTURES]
DEATH PHASE:
DEATH RATE GREATER THAN REPLICATION RATE
CELL NUMBER MAY NOT APPEAR TTO DECREASE EXPONENTIALLY DUE TO
LACK OF LYSIS. VIABILITY COUNT IS NECESSARY TO CONFIRM DEATH

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