Microbial Growth

dr. Philip Habib

MicroBiology 2010 Stikes hasanuddin

The Requirements for Growth
 6-1 Classify microbes into five
groups on the basis of preferred
temperature range.
 6-2 Identify how and why the pH of
culture media is controlled.
 6-3 Explain the importance of
osmotic pressure to microbial growth.
Microbial Growth
 Increase in number of cells, not cell size
– Populations
– Colonies
Growth Requirements
 Physical
– Temperature
• Optimum growth temperature- temperature at
which the organism grows its best
• Psychrophiles- cold loving- range from –10 ºC
to < 20 ºC; optimum 15 ºC
• Mesophiles- moderate-temperature-loving-
optimum 25-40 ºC; most common
• Thermophiles- heat-loving- optimum 50-60 ºC
• Psychotrophs- range from 0 ºC to < 40 ºC;
responsible for food spoilage
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Growth Requirements (cont.)
– pH- most bacteria grow best between pH 6.5 and
7.5; fungi prefer more acidic environments,
optimum pH, 5-6
• Acidophiles- acid loving; can live as low as pH
1, e.g. coal mines
– Osmotic pressure
• Halophiles- salt-loving- some can grow in as
much as 30% NaCl

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Fig. 6.1 Typical growth rates…

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 Fig. 6.2 Food
Spoilage
Temperature
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 Fig. 6.3 The
effect of the
amount of
food on its
cooling
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Fig. 6.4 Plasmolysis

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 Why are hyperthermophiles that grow at
temperatures above 100°C seemingly
limited to oceanic depths? 6-1
 Other than controlling acidity, what is an
advantage of using phosphate salts as
buffers in growth media? 6-2
 Why might primitive civilizations have used
food preservation techniques that rely on
osmotic pressure? 6-3
Requirements for Growth
 6-4 Name a use for each of the four
elements (carbon, nitrogen, sulfur, and
phosphorus) needed in large amounts for
microbial growth.
 6-5 Explain how microbes are classified
on the basis of oxygen requirements.
 6-6 Identify ways in which aerobes avoid
damage by toxic forms of oxygen.
Chemical
 Water
 Carbon sources
– Heterotrophs-organics
– Autotrophs- CO2
 Sources of
– Nitrogen- amino acids, nucleic acids
– Sulfur- amino acids, vitamins
– Phosphorous- ATP
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Chemical (cont.)
 Trace elements- cofactors; iron, copper, zinc
 Oxygen- Obligate aerobes
 Anaerobes
– Facultative anaerobes- use oxygen but can grow without it
by fermentation or anaerobic respiration; have catalase and
SOD, e.g. E. coli
– Obligate anaerobes- lack enzymes to neutralize toxic forms
of oxygen
– Aerotolerant anaerobes- tolerates oxygen; SOD
 Microaerophilic aerobes- low oxygen concentration
 Enzymes-
 Organic growth factors- e.g. vitamins, purines
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Enzymes
 Superoxide dismutase- SOD- convert
the superoxide free radical into
molecular oxygen and hydrogen
peroxide
 Catalase- converts hydrogen peroxide
into water and oxygen
 Peroxidase- breaks down hydrogen
peroxide into water

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Toxic Oxygen
 Singlet oxygen: O2 boosted to a
higher-energy state
 Superoxide free radicals: O2–
O2   O2   2H  superoxide dismutase
 H2O2  O2
 Peroxide anion: O22–
 2H2O  O2
2H2O2 catalase
H2O2  2O peroxidase

 2H2O
 Hydroxyl radical (OH•)
Q&A
 Oxygen in the
atmosphere is
essential for
human life. How
can some bacteria
grow in the
absence of
oxygen?
 If bacterial cells were given a sulfur source
containing radioactive sulfur (35S) in their
culture media, in what molecules would the
35S be found in the cells? 6-4

 How would one determine whether a

microbe is a strict anaerobe? 6-5
 Oxygen is so pervasive in the environment
that it would be very difficult for a microbe
to always avoid physical contact with it.
What, therefore, is the most obvious way
for a microbe to avoid damage? 6-6
Biofilms
 6-7 Describe the formation of
biofilms and their potential for causing
infection.
 Biofilms
 Microbial
communities
 Form slime or
hydrogels
– Bacteria attracted
by chemicals via
quorum sensing

Figure 6.5
 Biofilms
 Share
nutrients
 Sheltered from
harmful
factors

Applications of Microbiology, p. 57
Biofilms
 Patients with indwelling catheters
received contaminated heparin
 Bacterial numbers in contaminated
heparin were too low to cause infection
 84–421 days after exposure, patients
developed infections
Biofilms
 Pseudomonas fluorescens was cultured
from the catheters

Clinical Focus, p 164

 Identify a way in which pathogens find it
advantageous to form biofilms. 6-7
Culture Media
 6-8 Distinguish chemically defined
and complex media.
 6-9 Justify the use of each of the
following: anaerobic techniques, living
host cells, candle jars, selective and
differential media, enrichment medium.
 6-10 Differentiate biosafety levels 1,
2, 3, and 4.
Culture Media
 Culture medium: Nutrients prepared
for microbial growth
 Sterile: No living microbes
 Inoculum: Introduction of microbes into
medium
 Culture: Microbes growing in/on culture
medium
Agar
 Complex polysaccharide
 Used as solidifying agent for culture
media in Petri plates, slants, and deeps
 Generally not metabolized by microbes
 Liquefies at 100°C
 Solidifies at ~40°C
Culture Media
 Types
– Agar- solid or semisolid
• Plates, test tubes (deep or slanted)
– Broth- liquid- NO agar
 Chemically defined media- media whose exact
composition is known
– Useful for “fastidious” organisms
 Complex media- nutrients derived from a
variety of extracts; can vary slightly from one
batch to the next
– Nutrient broth- NB
– Nutrient agar- NA
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Anaerobic Growth Media
 Reducing media- contains ingredients such
as sodium thioglycolate to help deplete the
oxygen from the culture medium
 Anaerobe jar- contains chemicals in a packet
that generate hydrogen and carbon dioxide;
when mixed with water the hydrogen then
reacts with the oxygen present in the air and
forms water, thus removing oxygen from the
jar
 Anaerobe chamber- equipped with air locks
and gases; used when handling a lot of
cultures
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 Fig. 6.5
Anaerobic
jar

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Fig. 6.6 An
anaerobic
chamber

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Special Culture Techniques
 Capnophiles- microorganisms that grow
better at high CO2 concentrations
– Carbon dioxide incubators
– Candle jar

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Fig. 6.7b CO2 generating packet

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 Fig. 6.7a
Candle Jar

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Selective Media- used to suppress the growth
of unwanted bacteria and encourage the growth
of the desired organism
 Bismuth sulfite agar
– Inhibits Gram+ and most G- bacteria
– Used to isolate Salmonella typhi
 Sabouraud glucose agar
– has a pH of 5.6 which inhibits most
bacteria
– used to isolate fungi
 Brilliant Green Agar
– inhibits G+
– Used to isolate Salmonella
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Differential Media
 Medium that makes it easier to
distinguish colonies of the desired
organism
 Blood agar
– used to identify organisms that lyse RBCs
– E.g. Streptococcus pyogenes

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Fig. 6.8 Blood agar, a differential
medium containing RBCs.

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Selective and Differential Media
 Mannitol salt agar (MSA)
– contains 7.5% NaCl- inhibitory
– contains mannitol- carbon source
– contains pH indicator that changes color if acid is
produced from the fermentation of mannitol
– organisms that can tolerate high concentrations of
salt (selective component) and ferment mannitol
(differential component) are likely to be
Staphylococcus aureus

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MacConkey agar
 Contains bile salts and crystal violet
which inhibits G+ bacteria (selective)
 Contains lactose (differential)
– lactose fermenters appear as pink
colonies; e.g. E. coli
– non-lactose fermenters appear as colorless
colonies

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Fig. 6.9 Bacterial colonies on
several different media.

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Enrichment Media
 Provides nutrients and environmental
conditions that favor the growth of a
particular microorganism and not
others; encourages the growth of all
bacteria present in a sample

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Cultures
 Pure cultures
– Colony- arises from a single cell or groups of cells
– Streak Plate method- used to isolate colonies
 Preservation
– Deep-freezing- liquid suspension frozen –50 ºC to –70
ºC; lasts for years
– Lyophilization- freeze-drying- suspension is quickly
frozen and then sublimated- powder like product lasts
for years and the suspension can be hydrated to
“revive” the microorganism
 Growth
– Bacterial division- binary fission
– Generation time- the time required for a cell to divide
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– Logarithmic growth
Fig. 6.10 Streak Plate Method

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Fig. 6.11 Binary Fission in Bacteria

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Fig. 6.12 Cell Division

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Growth Curve
 Lag phase- period of little or no division
 Log phase
– logarithmic increase in the number of
bacteria; exponential
 Stationary phase
– The number of bacteria being produced
equals the number of bacteria dying
 Death or decline phase
– Logarithmic decrease in the number of
bacteria
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Fig. 6.14 Bacterial Growth Curve

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Measurement of Growth
 Plate counts- measures viable cells; CFU-
colony forming units
 Serial dilutions- uses diluted samples
 Pour plates and spread plates-
 Filtration- used for samples with low microbial
numbers; filter concentrates the microorganism
 Most Probable Number (MPN)
 Direct microscopic counts- direct enumeration
of the cells

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Fig. 6.15 Plate counts and serial dilutions

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 Fig. 6.16
Methods for
Plate counts

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Fig. 6.17 Counting bacteria by
filtration.

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Fig. 6.18 MPN Method

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Most Probable Number
 Multiple tube MPN test
 Count positive tubes

Figure 6.19
Fig. 6.19 Direct microscopic counts

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Estimating Bacterial Numbers by
Indirect Methods
 Turbidity
 Metabolic activity
 Dry weight
 Urine culture colony estimates’
– 10,000 or below- contamination
– 10,000-100,000- not conclusive
– > 100,000 - infection

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Fig. 6.20 Turbidity estimation
of bacterial numbers.

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