Transcript Growth in Batch Culture

Measurement of Bacterial Growth
Growth is an orderly increase in the quantity of cellular
constituents. It depends upon the ability of the cell to form new
protoplasm from nutrients available in the environment. In
most bacteria, growth involves increase in cell mass and
number of ribosome, duplication of the bacterial chromosome,
synthesis of new cell wall and plasma membrane, partitioning
of the two chromosomes, septum formation, and cell division.
This asexual process of reproduction is called binary fission.
Growth in Batch Culture
“Growth” is generally used to refer to the acquisition of
biomass leading to cell division, or reproduction
A “Batch culture” is a closed system in broth medium in
which no additional nutrient is added after inoculation of the
broth.
“Generation time” Time taken for a cell population to
double in numbers and thus equivalent to the average length
of the cell cycle
The Bacterial Growth Curve
 Four characteristic phases of the growth cycle are recognized.
1) Lag Phase. Immediately after inoculation of the cells
into fresh medium, the population remains temporarily
unchanged. Although there is no apparent cell division
occurring, the cells may be growing in volume or mass,
synthesizing enzymes, proteins, RNA, etc., and
increasing in metabolic activity.
 The length of the lag phase is apparently dependent on a wide
variety of factors including:
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The size of the inoculum.
Time necessary to recover from physical damage or shock in the
transfer.
Time required for synthesis of essential coenzymes or division
factors.
Time required for synthesis of new (inducible) enzymes that are
necessary to metabolize the substrates present in the medium.
2) Exponential (log) Phase. The exponential phase
of growth is a pattern of balanced growth wherein
all the cells are dividing regularly by binary fission,
and are growing by geometric progression.
The cells divide at a constant rate depending upon the
composition of the growth medium and the conditions of
incubation. The rate of exponential growth of a bacterial
culture is expressed as generation time, also the doubling
time of the bacterial population.
3) Stationary Phase. Exponential growth cannot
be continued forever in a batch culture (e.g. a
closed system such as a test tube or flask).
 Population growth is limited by one of three factors:
1) Exhaustion of available nutrients.
2) Accumulation of inhibitory metabolites or end products
3) Exhaustion of space, in this case called a lack of
"biological space".
4) Death Phase. If incubation continues after the population
reaches stationary phase, a death phase follows, in which the
viable cell population declines. (Note, if counting by
turbidimetric measurements or microscopic counts, the death
phase cannot be observed.). During the death phase, the
number of viable cells decreases geometrically (exponentially),
essentially the reverse of growth during the log phase
Growth in Continuous Culture
A “continuous culture” is an open system in which fresh media
is continuously added to the culture at a constant rate, and old
broth is removed at the same rate.
This method is accomplished in a device called a chemostat.
Typically, the concentration of cells will reach an equilibrium
level that remains constant as long as the nutrient feed is
maintained.
Factors that Influence Growth
1) Temperature.
2) pH.
3) Salt concentration.
4) Oxygen concentration.
Turbidity measurements of
microbial growth
 Optical Density (Counting by Spectrophotometer)
Materials
TSB of Nonpathogenic Escherichia coli
Inoculating loop
Spectrophotometer
Test tube rack
2 tubes of TSB
37C Incubator
Bunson burner
Surface disinfectant
Procedure
1) Wash hands and put on gloves.
2) Assemble equipment and materials and prepare work area.
3) Set the spectrophotometer at 450nm and let it warm up 30
minutes before performing the readings. The meter on the
spectrophotometer should read “0% Transmittance”.
4) Obtain 2 tubes of TSB. Label one C (uninoculated) for control
and one S (inoculated) for sample.
5) Place tube C into the sample holder of the spectrophotometer.
Turn the light control knob until the meter needle is on “100%
Transmittance”. Remove the control tube. The
spectrophotometer is now standardized.
6) Flame and cool the loop.
7) Mix the culture of E. coli and inoculate tube S with 5
loopfuls of the broth.
8) Flame and cool the loop.
9) Start the turbidity readings by placing the S tube into the sample
well and read the OD (absorbance). Record the results in the data
section.
10) Take OD reading over the time period specified in the data
section and return the TSB tube to the 37 C incubator between
each reading.
11) Turn off the spectrophotometer upon completion of the readings.
12) Plot the readings on the graph paper as absorbance vs. time.
13) Clean work area with disinfectant.
14) Remove gloves and wash hands with disinfectant.
I.
Record the absorbance value for each sample
reading starting at 0 time.
II. Plot the absorbance values vs. time on graph paper.
Label the x and y axis. Each of the readings will
represent the growth of the E. coli in the culture.
III.Label each part of the
growth curve: Lag phase,
Exponential growth phase,
and the Stationery phase.
Microbial Control Agents
Many microorganisms are beneficial and necessary for human
well-being.
However, microbial activities may have undesirable
consequences, such as food spoilage and disease.
It is essential to be able to kill microorganisms or inhibit their
growth to minimize their destructive effects.
Physical, chemical, and mechanical methods to destroy or
reduce undesirable microbes in a given area
Radiation
Radiation is example for Physical Methods of
Microbial Control.
Types of Radiation:1) Ionizing radiation.
2) Nonionizing radiation.
1) Ionizing radiation: deep penetrating power that
has sufficient energy to cause electrons to leave
their orbit, breaks DNA.
 E.g. Gamma rays, X-rays, Cathode rays.
 Used to sterilize medical supplies and food products.
2)Nonionizing radiation: have little penetrating
power and must be directly exposed.
 UV light creates thymine dimers, which interfere
with replication.
Terminology of Microbial Control
Sterilization: Killing or removing all forms of microbial life
(including endospores) in a material or an object.
Disinfection: are chemicals used on nonliving surfaces to
inhibit the growth of microorganisms.
Antiseptic: are chemicals used on living tissues to inhibit the
growth of microorganisms.
Chemotherapeutic agents (antibiotics): are chemicals
used to destroy or inhibit the growth of microorganisms in living
tissues.
Antimicrobial Chemotherapy
 Antimicrobial Chemotherapy : is the use of chemicals to
inhibit or kill microorganisms in or on the host.
 Based on their origin, there are 2 general classes of antimicrobial
chemotherapeutic agents:
 Antibiotics: substances produced as metabolic products of one
microorganism which inhibit or kill other microorganisms.
 Antimicrobial Chemotherapeutic Chemicals: chemicals
synthesized in the laboratory which can be used therapeutically on
microorganisms.
Antimicrobial Agents
 Antimicrobial agents are :
 Cidal in action: they kill microorganisms (e.g., penicillins,
cephalosporins, streptomycin, neomycin).
 Static in action : they inhibit microbial growth long enough for
the body's own defenses to remove the organisms (e.g.,
tetracyclines, erythromycin, sulfonamides).
 Selective Toxicity: This means that the agent used must inhibit
or kill the microorganism without seriously harming the host.
 Antimicrobial agents also vary in their spectrum:
 Broad spectrum : Drugs which are effective
against a variety of both gram-positive and gramnegative bacteria : (e.g., tetracycline,
streptomycin, cephalosporins, ampicillin,
sulfonamides).
 Narrow spectrum : Drugs which are effective against just
gram-positive bacteria, just gram negative bacteria, or only a few
species are termed (e.g., penicillin G, erythromycin,
clindamycin, gentamicin).
 If a choice is available, a narrow spectrum is preferable since it
will cause less destruction to the body's normal flora. In fact,
indiscriminate use of broad spectrum antibiotics can lead to super
infection by opportunistic microorganisms.
Antimicrobial Drugs
 Antibiotic Resistance: bacteria gain ability to grow; no longer
sensitive to drug
 Antiretroviral: act specifically against viruses
 Combination of drugs:
 Synergism: action of two antibiotics greater.
 Antagonism: action of drug is reduced; less effective.
 Minimum Inhibitory Concentration (MIC):The lowest
concentration of chemotherapeutic agent capable of preventing
growth of the test organism.
 Minimum Bactericidal Concentration (MBC): MBC is the
lowest concentration of the chemotherapeutic agent that results in
no growth (turbidity) of the subcultures.
Antimicrobial Activity
Five Modes of Antimicrobial Activity:
1) Injury to Plasma Membrane (polymixin B).
2) Inhibition of Cell Wall Synthesis (penicillins, bacitracin).
3) Inhibition of Protein Synthesis (translation).
4) Inhibition of Nucleic Acid replication & transcription.
5) Inhibition of essential metabolites.
Haw To Choose Antimicrobial Drugs?
Narrow spectrum OR broad spectrum.
Old age OR child.
Male OR female.
Pregnant OR lactating women.
In patient OR out patient.
Type of microorganisms.
Site of infection.
END OF LECTURE