Download FE-206 Food Microbiology1 Spring 2016

FE-206 Food Microbiology1
Spring 2016
LECTURE3
Microbial Growth Characteristics in
Foods
Outline
I.Introduction
II.General principles of Microbial Growth andSurvival
A)Reproduction
Yeast,Mold, bacteria
B)Growth Curve
C)First order Kinetics
1)Growth Kinetics
2)Death Kinetics
D)Importance of Being Small Size
E)Microbial Growth Characteristics in Foods
III.Chemical Changes Caused by micrrorganisms in Foods
Reproduction
• An increase in the number (or mass)of
vegetative cellsbacteria,yeast and molds is
used to reflect growth for microorganisms.
– Bacteria-binary fisson
– Molds-sexually and asexually
– Yeast-Budding
– Viruses-by lytic and lysogenic cycles
General principles of Microbial Growth
and Survival
• Microbial Growth (cell multiplication),except
viruses in raw and processed foods are
important with respect to foodborne
disaeases, food spoilage and food
bioprocessing.
The factors inflencing the microbial growth are
helpful designing methods either control(against
spoilage,heath hazard) or stimulate their
growth(in biprocessing,fermentation)
Viral Reproduction
• Steps of Lytic Cycle
1.Attachment
2.Entry
3.Replication
4.Assembly
5.Lysis/Release (lyses the cell)
How do viruses replicate?
2 methods of replication:
1. Lytic Cycle – the virus enters the cell, replicates
itself hundreds of times, and then bursts out of the
cell, destroying it.
2. Lysogenic Cycle – the virus DNA integrates with
the host DNA and the host’s cell helps create more
virus DNA. An environmental change may cause the
virus to enter the Lytic Cycle.
In the lytic cycle, the
virus reproduces
itself using the host
cell's chemical
machinery. The red
spiral lines in the
drawing indicate the
virus's genetic
material. The orange
portion is the outer
shell that protects it.
In the lysogenic
cycle, the virus
reproduces by first
injecting its
genetic material,
indicated by the
red line, into the
host cell's genetic
instructions.
What is Bacterial Growth?
• Bacterial Growth - an increase in bacterial
numbers
- does not refer to an increase
in size of the individual cells
How to determine Microbial
Numbers?
 directly – through counting
 indirectly – through measuring their metabolic
activity
Bacterial Division
Binary Fission - most common method of
reproduction, asexual reproduction, splitting of
parent cell into two daughter cells
Budding - another form of bacterial division, also
asexual reproduction, it forms from outgrowths
(buds) of mature organisms, it is a form of mitotic
cell division, when the bud reaches the size of the
parent cell, it separates
Binary Fission
Filamentous bacteria (some actinomycetes) reproduce
by producing chains of conidiospores carried
externally at the tips of the filaments. Other
filamentous bacteria simply fragment and the
fragments initiate the growth of new cells.
Generation Time
• In binary fission, one cell’s division produces two
cells, two cells’ divisions produces four cells and so
on. When the arithmetic number of cells in each
generation is expressed as a power of 2 (2x), where x
is the exponent that tells the number of doubling
(generations) that have occurred.
Generation time is the time required for a cell to divide
(and its population to double). The generation time
among organisms vary according to environmental
conditions such as temperature or pH level. Most
bacteria have a generation time of 1 – 3 hours while
other species can require up to 24 hours per
generation.
Phases of Growth
Lag phase:
• No increase in cell number
• Period of adaptation of cells to a new environment
• No change in number, but an increase in mass
• Multiple lag phases may sometimes be observed more than one carbon source
(Diauxic growth)….why?
• Length of the lag phase – characteristics of microbial
species and in part by the media conditions
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Cont.…
Log Phase:
• Growth rate is higher
• Increase in cell mass and cell number with time exponentially
• This phase results in straight line… why?
• Hence, it is also known as Exponential phase.
• Period of balanced growth, in which all the components of a
cell grow at the same rate
• Composition of biomass remains constant
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Cont.…
• The exponential growth rate is the first order reaction
• The rate of biomass is correlated with the specific growth
rate(µ) and the biomass concentration or cell number, X
• A measure of the rapidity of growth has dimension T-1
dX/dt = µ.X
Integration of the eq. between the limits X0 at the time t=0 and
X at sometime t gives:
ln (X/X0) = µt (or) X=X0 e µt
Taking the neutral log,
ln X = ln X0 + µt
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Stationary Phase
• Period of equilibrium
• Metabolic activity of surviving cells slows
down which stabilizes the population
• Cause of discontinuity of exponential growth is
not always clear
• May play a role: exhaustion of nutrients,
accumulation of waste products and harmful
changes in pH
• Chemostat – continuous culture used in
industrial fermentation
Death Phase
• Also known as Logarithmic Decline Phase
• Continues until a small fraction of the
population is diminished
• Some population dies out completely
• Others retain surviving cells indefinitely while
others only retain for a few days
Cryptic Growth
• After a long tim,some cells may still
remainviable due toreduced competitionand
the population.This survival is called cryptc
growth.
Quantifying Cell Concentration
Why ?
– To determine the kinetics & stoichiometry of microbial
growth
– Basically we’re trying to figure out how much stuff we can
make given the starting amounts (yields)
How ?
• The method used is classified as either:
1.) Direct
or
2.) Indirect
• Direct method is usually not feasible due to the presence
of suspended solids or interfering compounds in the
medium.
Quantifying Cell Concentration
Indirect Methods
• Petroff-Hausser slide or a hemocytometer
• Plate Counts from agar plates
• Ring-mounted microscope slide
(miniture culture dish)
• Commercial particle counters
Cell Number Density - Hemocytometer
• A Petroff-Hausser slide or a hemocytometer is often used for
cell counting.
• A calibrated grid is placed
over the culture chamber
and cells per grid square
are counted using a microscope.
– To be statistically reliable at least
20 grid squares must be counted.
• Suitable for non-aggregated cultures.
• Stains can be used to distinguish
between live and dead cells.
Cell Number Density – Plate Counts
• Plates with growth medium and agar gel are used for counting viable
(capable of reproduction) cells.
• Samples are diluted, spread on agar and incubated.
• Colony-forming units (CFU’s)
CFU/mL for liquid
CFU/g for solids
• More suitable for bacteria and yeast
compared to mold.
• Viable count may vary depending on the composition of the growth
medium and culture conditions chosen.
• A large number of colonies must be counted in order to obtain a
statistically reliable value.
Cell Number Density - Ring Mounted Slides
• Agar-gel medium is placed in a small
ring mounted on a microscope slide
• Cells are spread on this miniature
culture dish.
• Cells are incubated and then examined
under a microscope.
• Much quicker than plate count with
the same limitation.
Cell Number Density – Particle Counters
• Relatively high electrical resistance of cells
One electrode is placed in a tube with an orifice,
a vacuum is applied to this tube causing the
electrolyte solution (which contains the cells) to
be sucked through the orifice
Vacuum
• Uses 2 electrodes and an electrolyte solution
Electrical
Voltage
• Electrical potential is applied across the
electrodes
• As cells pass through the orifice, electrical
resistance increases and causes pulses in
electrical voltage
Orifice
• # of pulses = # of particles
• Height of the pulse = a measure of cell size
cells
Electrodes
Determining Cell Mass Concentration
• Direct vs. Indirect Methods
1.) Direct
a) Dry Weight
b) Packed Cell Volume
c) Optical Density
2.) Indirect
a) Measurements of substrate consumption
b) Measurements of product formation
Determining Cell Mass Concentration
1.) Direct
Dry Weight: most commonly used, only used for cells grown in
solids-free medium, process may involve centrifuging, filtering,
washing & drying
Ex.) Sometimes cellulose, molasses or corn steep are present in
which case dry weight would measure these as well and
therefore be inaccurate
Packed Cell Volume: used for rough but rapid estimates of
fermentation broth, process involves centrifuging under standard
condition and measuring volume.
Optical Density: based on light absorption of suspended cells, uses
a spectrometer, fast, inexpensive and simple.
Determining Cell Mass Concentration
1.) Indirect
Measurement of Substrate Consumption or Product Formation
Useful for molds and other fermentation processes
o Intracellular components of cells that change with time during the growth cycle:
-DNA, RNA & Protein (kits available)
-ATP concentration (luciferase activity)
o Nutrients used for production but not in product formation
- Nitrate, phosphate, sulfate
-Utilization of carbon or oxygen uptake rates
o Products produced that are growth associated
-Production of ethanol, lactic acid
o Changes in Physiochemical Properties
- pH changes
-viscosity of broth
DIRECT MEASUREMENT OF
MICROBIAL GROWTH
Plate Counts
• Measures the number of viable cells
• It takes about 24 hours or more for visible colonies
to form
• Reported as colony-forming units (CFU)
• Only a limited number of colonies must be
developed in the plate because when too many
colonies are present, some cells are overcrowded
and do not develop.
• The original inoculum is diluted several times in a
process called serial dilution to ensure that colony
counts will be within 25 – 250 colonies.
• Serial Dilutions
Example:
A milk sample has 10,000 bacteria per milliliter. If 1 ml
of this sample were plated out, there would
theoretically be 10,000 colonies formed in the Petri
plate of the medium. Obviously, this would not
produce a countable plate. If 1 ml of this sample were
transferred to a tube containing 9 ml of sterile water,
each milliliter of fluid in this tube would now contain
1000 bacteria. If 1 ml of this sample were inoculated
into a Petri plate, there would still be too many
potential colonies to count on a plate. Therefore,
another serial dilution could be made.
One milliliter containing 1000 bacteria would be
transferred to a second tube of 9 ml of water.
Each milliliter of this tube would now contain
only 100 bacteria, and if 1 ml of the contents
of this tube were plated out, potentially 100
colonies would be formed– an easily
countable number.
Plate counts and serial dilutions
• Pour Plates and Spread Plates
• A plate count is done by either the pour
plate method or the spread plate method
Methods of preparing
plates for plate counts
• Pour Plates: Disadvantages
• Some relatively heat-sensitive microorganisms
may be damaged by the melted agar and will
then be unable to form colonies
• When certain differential media are used, the
distinctive appearance of the colony on the
surface is essential for diagnostic purposes.
Colonies that form beneath the surface of a
pour plate are not satisfactory for such tests.
• To avoid these problems, the spread plate
method is used instead