Chapter 3 Bio461 (Revised)

Chapter 4
Microbial Growth
4.1 Requirements for growth
4.2 Culture media
4.3 Obtaining pure culture
4.4 Preserving bacterial culture
4.5 Growth of bacterial culture
4.6 Biofilms
2
BIO461:Mic/DNA/UiTM NS_2017
4.1 The requirements for growth
• The requirements for microbial growth can be
divided into two main categories : physical and
chemical.
Temperature
Physical
Osmotic
pressure pH
3
Carbon
Trace
Nitrogen
elements
Chemical
Oxygen Sulfur
Phosphorus
4
Physical requirements : Temperature
III. Heat loving
microbes
 Most microbes grow well at
II. Moderate- the temperatures that
temperature- humans favor.
loving microbes  Each bacterial species grows
at particular minimum,
optimum and maximum
temperatures.
 Min growth temp – lowest
temp at which the species will
grow.
 Optimum growth temp –
temp at which the species will
grows best.
 Max growth temp – highest
temp at which growth is
possible.
I. Cold-loving 5
microbes BIO461:Mic/DNA/UiTM NS_2017
Physical requirements : Temperature
1 3
Psychrophiles 2 Psychrotrophs Mesophiles
 Cold-loving  can grow at 00C.  Moderate-
microbes  Higher optimum temp. temperature-loving
 Organisms that at 20-300C. microbes
capable to grow at  Cannot grow above  Optimum growth
00C. 400C. temperature at 25-
 Group of spoilage 400C.
 Optimum growth
microbes.
temperature at
150C.
 Found in the ocean 4 5 Hyperthermophiles
Thermophiles
depths or in certain  have an optimum
 heat-loving microbes
polar region.  Optimum growth rate of growth temperature of
 Seldom cause 800C or higher.
50-600C.
problem in food  usually live in
preservation. hotsprings associated
with volcanic activity.
6
Physical requirements : pH
Refer to acidity and alkalinity
Most bacteria grow best in a narrow pH near neutrality between pH 4

and 7.5.
Bacteria that are tolerant of acidity are called acidophiles
When bacteria are cultured in the lab, they often produce acids that
eventually interfere with their own growth.
To neutralize the acids and maintain the proper pH, chemical buffers
are included in the growth medium.
Ex. Peptone and acid amino act as buffers and many media also contain
phosphate salts.
BIO461:Mic/DNA/UiTM NS_2017 7
Physical requirements : osmotic pressure
• Microorganisms obtain almost all their nutrients in solution from
the surrounding water.
• This osmotic loss of water causes plasmolysis or shrinkage of the
cell’s cytoplasm.
Obligate halophiles :
Extreme halophiles : organisms from such saline waters
Facultative halophiles :
microorganisms that have adapted and require nearly 30% salt, and do not require high salt
so well to high salt concentrations the inoculating loop used to concentration up to 2%. They can
that they require them for growth. transfer them must be first be tolerate even at 15% salt.
dipped into saturated salt solution.
Chemicals
requirements :
Carbon
As the structural
backbone of
living matters
Needed in all
organic
compounds that
make up living
cell.
Half the dry

weight of a
typical bacterial
cell is carbon.
Chemoheterotrophs
get C from the source
of their energy.
Chemo and
photoautotroph
derived C from
CO2. 9
Chemicals requirements : N,S,P
Nitrogen
 Make up 14% of the dry weight of a bacterial cell.
 N used to form the amino group of the amino acid of proteins.
 Other bacteria use N from ammonium ions (NH4+), which are already in the reduced
form.
 Some important bacteria, photosynthesizing cyanobacteria use gaseous nitrogen (N 2)
directly fro atmosphere, nitrogen fixation.
Sulfur
 Used to synthesize sulfur-containing amino acids and vitamins such as
thiamine and biotin.
 Important natural sources of sulfur include the sulfate ion, hydrogen sulfide
and sulfur containing amino acids.
Phosphorus
 Essential for the synthesis of nucleic acid and phospholipids of cell
membrane.
 Also found in energy bonds of ATP.
 Source of phosphorus BIO461:Mic/DNA/UiTM
is phosphate ions (PO43-).
NS_2017 10
Chemicals requirements : Oxygen
Terms Effects of oxygen on growth

Obligate aerobes Only aerobic growth; oxygen required
1
Facultative Both aerobic and anaerobic growth;
2 anaerobes greater growth in presence of oxygen
Obligate anaerobes Only anaerobic growth; ceases in
3 presence of oxygen
Aerotolerant Only anaerobic growth; but continues in
4 anaerobes presence of oxygen
Microaerophiles Only aerobic growth ; oxygen required in
5 low concentration
UNDERSTANDING HOW
ORGANISMS CAN BE HARMED BY
OXYGEN, FURTHER READ
TORTORA PAGE 181
13
Chemicals requirements : Organic growth factor
• Essential organic compounds an organisms is

unable to synthesize.
• They must be directly obtained from the
environment.
– Ex. Vitamins for human
– For bacteria; amino acids, purines, pyrimidines
• Many bacteria synthesize all their own vitamins,
and do not depend on outside sources.
Chemically
defined
media
Enrichment Complex
culture media
Culture
media
Selective Anaerobic
and growth
Differential media and
media methods
Special
culture
media
15
16
Culture media
• Nutrient material prepared
for the growth of
microorganisms in a lab.
• Microbes that are introduced
into a culture medium to
initiate growth are called an
inoculum.
• Microbes that grow and
multiply in or on a culture
medium are referred to as a
culture.
17
• A wide variety of media are available for the
growth of microorganisms in the laboratory.
• Most of the media, which are available from
commercial sources, have premixed components
and require only the addition of water and the
sterilization.
Media are constantly being developed or revised for

the use in the isolation and identification of bacteria
that are of interest of to researchers in such fields as
food, water and clinical microbiology.
18
• For solid medium, agar is added as the solidifying
agent.
• Agar, a complex polysaccharide derived from
marine alga, and has long been used as a
thickener in foods.
Agar media are usually contained in test
tubes and Petri dishes.
• Slant – in test tubes when the agar
medium are allowed to solidify with the
tube held at an angle.
• Deep – agar solidifies in vertical tube
• Petri dishes – a shallow dishes with a lid
that nest over the bottom to prevent
contamination
19
Chemically
Defined Media
• Medium must provide an
energy source to support
microbial growth, as well as
sources of C, N, S and P and
any growth factors that the
organism is unable to
synthesize.
• Chemically defined medium :

when the exact chemical
composition is known.
Complex media
• Usually reserved for laboratory experimental work or for
the growth of autotrophic bacteria.
• Complex media made up of nutrients including extracts
from yeasts, meat or plants or digests of protein from
these and other sources.
• The energy, C, N and S requirements of the growing
microorganisms are provided primarily by protein.
Complex medium in liquid form, it is called NUTRIENT BROTH.
Complex medium in solid form (agar is added), it is called NUTRIENT

AGAR.
24
• For cultivating anaerobic
Anaerobic bacteria, special media called

reducing media must be used.
growth • Example: media containing
media and sodium thioglycolate that will
combine with dissolved O2 and
methods deplete the oxygen in the
culture medium.
Special culture techniques
• Many bacteria have never been successfully grown on
artificial lab media.
• Ex. : Mycobacterium leprae, syphillis spirochete and
viruses, can only reproduce only in a living host cell.
• In clinical lab., special CO2 incubators in which to grow
aerobic bacteria that require concentration of CO2 higher
or lower than that found in the atmosphere.
• Microbes that grow at high CO2
concentration are called CAPNOPHILES.
• Candle jar or commercially available chemical
packets are used to generate CO2 atmosphere in
containers.
26
Selective and Differential Media
Selective media : design to suppress the growth of unwanted
bacteria and encourage the growth of desired microbes.
Ex. : Baired Parker agar – S. aureus etc.
Differential media : make it easier to distinguish colonies of the

desired organism from other colonies growing on the same plate.
Ex. : TCBS agar – Vibrio sp.
• Sometimes selective and differential are combined in

a single medium.
28
29
Enrichment culture
Enrichment culture
The enrichment
was used because It is also a selective
medium usually in
sometimes the medium, but it is
liquid form to
desired bacteria designed to increase
provides nutrients and
present in a small very small numbers of
environmental
number in a sample the desired type of
conditions that favor
and can be missed, organism to
the growth of a
especially if other detectable levels.
particular microbe but
bacteria are present in
not others.
much larger numbers.
30
FURTHER READING ON TORTORA
PAGE 187
31
Obtaining Pure Cultures
• Most bacteriological work requires pure culture of
bacteria.
• The isolation method most commonly used to get pure

culture is streak plate method.
• However, when the microbe to be isolated is present only
in very small numbers , its numbers must be greatly
increased by selective enrichment
32
A B
C D
Preserving Bacterial Cultures
i. Refrigeration : short-term storage
ii. Deep-freezing: process in which a pure culture of microbes is
placed in a suspending liquid (-500c to -950C)
iii. Freeze drying (lyophilization) : a suspension of microbes is
quickly frozen (water remove by a high vacuum) at temp ranging
from : -520c to -720c.
34
Microbial Growth
• Increase in:
Number of cells
(population, NOT cell size!
colonies..)
The Growth of Bacterial Cultures
Bacterial growth
NOT increase in size

an increase in an increase in
of the individual
bacterial number microbial mass
cells.
Bacterial growth rate (µ) : Change in the

number of cell per unit time.
36
• Reproduce by : binary fission & budding
37
Generation time
• Generation : Intervals
between two division
• Generation time : the time
required for a cell to divide to
double, only reproduction by
binary fission which is by far
the most common method.
• It varies depend on :
organisms/environment
conditions such as
temperature
A growth curve for an
exponentially increasing
population,
Plotted logarithmically (dashed
line) and arithmetically
38
(solid line)
Phases of growth
• When bacteria are
inoculated into a liquid
growth medium and the
population is counted at
intervals, it is possible to
plot a bacterial growth
curve.
• 4 basic phase : lag, log,
stationary and death
phases.
39
No of cell Cell begin to Large no of cell could No of deaths eventually
changes very divide and enter arise. exceeds the number of new
little or no cell period of growth Period of equilibrium cell formed.
division @ logarithmic Causes of exponential Also known as
increase. growth to stop : “logarithmic decline phase.
Cellular exhaustion of
reproduction most nutrients/accumulation
active of waste products and
Preferred for harmful changes in pH.
industrial
purposes.
40
Measurement of microbial growth
• The growth of microbial populations can be
measured in a several ways : cell number,
population’s total mass etc.
• Population number = in ml of liquid or g of solid
material.
• Bacterial population usually involve very large
number, counting method are based on direct @
indirect counts of population.
41
Direct
measurement : Plate
counts
• The most frequent method

used for measuring bacteria
population.
• Advantages : only count the
viable cells.
• Disadvantages : it takes
sometimes more than 24h for
visible colony to form.
• Plate counts are often reported
as colony-forming units (CFU).
• Plate count unit : CFU/ml atau
CFU/g.
42
Serial dilutions
https://www.youtube.com/watch?v=MCrNjHcfcpY
https://www.youtube.com/watch?v=fGTVFk2a2Gg 43
(a) Pour plates (b) Spread plates
Disadvantages :
Advantages :
• Not useful for
• Colonies will be
heat sensitive
on surface and
organisms
not exposed to
• Colonies appear melted agar.
under agar
surface
45
46
47
Direct measurement : Filtration
• When the quantity of bacteria is very small.
• Ex. : Fecal bacteria in lake, ocean or water.
• A large sample (100ml or more) is filtered to retain
bacteria.
• Filter is transfer into petri dish and incubate. After that
count the colonies.
49
Direct measurement : Most Probable Number (MPN)
•Method determining the number of bacteria in a
sample.
•Most useful when microbes being counted will not
grow.
50
51
• Multiple tubes MPN test (5 tubes & 3 tubes)
- Count the positive tubes (turbid tubes) only.
52
Direct measurement : Direct Microscopic
• The measured volume of a bacterial suspension is
placed within a defined area on a microscope slide.
• Ex. : used to count no. of bacteria in milk.
• A special design slide called a Petroff-Hauser cell
counter is used in direct microscopic count.
Advantages : Disadvantages :
• No incubation time is • Motile bacteria are difficult
required to count by this method,
• An electronic counter dead cells are likely to
known as Coulter counters count as live ones.
which automatically count • High concentration of cell is
the number of cells in a required to be counted.
measured volume of liquid.
53
54
55
Indirect measurement : Turbidity
• Practical way of monitoring bacterial growth.
• Instrument used : spectrophotometer @ colorimeter
– Beam of light transmitted through a bacterial suspension to
alight sensitive detector.
– This change of light will register on the instrument’s scale as
the percentage of transmission.
– Also printed on the instrument’s scale is a logarithmic
expression called the absorbance (sometimes called optical
density, or OD which calculated as Abs = 2-log of %
transmittance).
– More than a million cells per mililiter must be present for first
traces of turbidity to be visible.
56
• Therefore, turbidity is not a useful measure of contamination
of liquid by relatively small numbers of bacteria.
57
Indirect measurement : Metabolic activity
• By assuming that the amount of a certain metabolic product such
as acid or CO2 is in direct proportion to the number of bacteria
present.
Indirect measurement : Dry weight

• Usually used to measure the filamentous organisms such as molds.
• By removing the fungus from its growth medium, filter to remove
extraneous material and dried in a desiccator.
• It is then weight.
• Can also be used for bacteria.
58
Biofilms
• Microorganisms typically live in communities called biofilms.
• After the development of confocal microscopy, we start noticing
the presence of biofilms in nature.
• Biofilms reside in a matrix made up primarily of polysaccharides,
but also containing DNA and proteins called slime.
• Can be considered as hydrogel, a complex polymer containing
many times its dry weight in water.
• Cell to cell chemical communication or QUORUM SENSING, allows
bacteria to coordinate their activity and group together into
communities that provide benefits not unlike those of
multicellular organisms.
• Biofilms are always attached to a surface, such as rock in a pond, a
human tooth or a mucus membrane.
• Within the biofilms community, the bacteria are able to share nutrients and are
sheltered from harmful factors in the environment, such as dessication,
antibiotic and the body’s immune system.
• The microorganisms in biofilms can work cooperatively to carry out complex
task.
• Biofilms are an important factor in human health.
• One approach to preventing biofilms formation is to incorporatre antimicrobial
into surfaces on which biofilms might form.
Exercise :
(Tortora Ref Try to answer all questions.
Book. Page
200-201)

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Chapter 4

Most bacteria grow best in a narrow pH near neutrality between pH 4

Bacteria that are tolerant of acidity are called acidophiles

Half the dry

Terms Effects of oxygen on growth

• Essential organic compounds an organisms is

Media are constantly being developed or revised for

• Chemically defined medium :

Complex medium in liquid form, it is called NUTRIENT BROTH.

Complex medium in solid form (agar is added), it is called NUTRIENT

Anaerobic bacteria, special media called

Differential media : make it easier to distinguish colonies of the

• Sometimes selective and differential are combined in

• The isolation method most commonly used to get pure

NOT increase in size

Bacterial growth rate (µ) : Change in the

• The most frequent method

Indirect measurement : Dry weight

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