CHE 315: Microbiology

Microbial Nutrition and Growth

• Growth requirements and classification
• Physical parameters that effect growth and
classification based on growth patterns
• Chemical parameters that effect growth and
classification based on growth patterns
• Population growth -- growth curve
• Population growth -- Methods

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Environmental Effects on Bacterial Growth

• Temperature

• pH

• Osmotic pressure

• Oxygen classes

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 Temperature and Microbial Growth
• Cardinal temperatures
– minimum
– optimum
– maximum
• Temperature is a major
environmental factor
controlling microbial
growth.

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 Temperature
• Minimum Temperature: Temperature below which
growth ceases, or lowest temperature at which
microbes will grow.
• Optimum Temperature: Temperature at which its
growth rate is the fastest.
• Maximum Temperature: Temperature above which
growth ceases, or highest temperature at which
microbes will grow.

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Classification of Microorganisms by Temperature
Requirements

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 Temperature Classes of Organisms
• Mesophiles ( 20 – 45C)
– Midrange temperature optima
– Found in warm-blooded animals and in terrestrial and
aquatic environments in temperate and tropical latitudes
• Psychrophiles ( 0-20C)
– Cold temperature optima
– Most extreme representatives inhabit permanently cold
environments
• Thermophiles ( 50- 80C)
– Growth temperature optima between 45ºC and 80ºC
• Hyperthermophiles
– Optima greater than 80°C
– These organisms inhabit hot environments including boiling
hot springs, as well as undersea hydrothermal vents that can
have temperatures in excess of 100ºC

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 pH and Microbial Growth
• The (measure of [H+]) acidity or alkalinity of an environment
can greatly affect microbial growth. Internal pH regulated
by BUFFERS and near neutral adjusted with ion pumps
• Most organisms grow best between pH 6 and 8, but some
organisms have evolved to grow best at low or high pH. The
internal pH of a cell must stay relatively close to neutral even
though the external pH is highly acidic or basic.
– Acidophiles (optimum in pH range 1-4) : organisms that
grow best at low pH( Helicobacter pylori, Thiobacillus
thiooxidans, lactic acid bacteria – 4-7 )
– Alkaliphiles (optimum in pH range 8.5-11): organismsa
that grow best at high pH ( Vibrio cholera)
– Most of pathogenic
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bacteria are neutrophiles 7
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 Osmotic Effects on Microbial Growth
• Osmotic pressure depends on the surrounding solute concentration and
water availability
• Water availability is generally expressed in physical terms such as water
activity (aw)
• Water activity is the ratio of the vapor pressure of the air in equilibrium
with a substance or solution to the vapor pressure of pure water ( aw
1.00).
aw= P solu
P water

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Environmental factors and growth
1. Osmotic Effect and water activity
organisms which thrive in high solute – osmophiles
organisms which tolerate high solute – osmotolerant
organisms which thrive in high salt – halophiles
organisms which tolerate high salt – halotolerant
organisms which thrive in high pressure – barophiles
organisms which tolerate high pressure – barotolerant

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 Microbial Nutrition

The hundreds of chemical compounds present inside

a living cell are formed from nutrients.

• Macronutrients : elements required in fairly large

amounts
• Micronutrients : metals and organic compounds
needed in very small amounts

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 Main Macronutrients
Carbon:
(C, 50% of dry weight) and nitrogen (N, 12% of dry
weight)
• Virtually all chemical substances in microorganisms
contain carbon in some form, whether they are
proteins, carbohydrates, or lipids. 50% of a bacterium's
dry weight is carbon. Carbon can be obtained from
organic materials in the environment, or it may be
derived from carbon dioxide.
• Autotrophs are able to build all of their cellular organic
molecules from carbon dioxide

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 Classification of organisms based on sources
of C and energy used

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Nitrogen:
• is used for the synthesis of proteins, amino acids,
DNA, and RNA. Bacteria that obtain nitrogen directly
from the atmosphere are called nitrogen-fixing
bacteria. They include species of Rhizobium and
Azotobacter, both found in the soil.
• Most Bacteria can use Ammonia -NH3 and many can
also use NO3-
• Nitrogen fixers can utilize atmospheric nitrogen (N2)

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 Classification of organisms based on O2 utilization
• Utilization of O2 during metabolism yields toxic
by-products including O2-, singlet oxygen (1O2)
and/or H2O2.
• Toxic O2 products can be converted to harmless
substances if the organism has catalase (or
peroxidase) and superoxide dismutase (SOD)
• SOD converts O2- into H2O2 and O2
• Catalase breaks down H2O2 into H2O and O2
• Any organism that can live in or requires O2 has
SOD and catalase (peroxidase)

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 Classification of organisms based on O2 utilization

• Obligate (strict) aerobes require O2 in order to grow

• Obligate (strict) anaerobes cannot survive in O2
• Facultative anaerobes grow better in O2
• Aerotolerant organisms don’t care about O2
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Microaerophiles require low
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 Toxic Forms of Oxygen and Detoxifying Enzymes

Hydrogen
peroxide

Superoxide

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Environmental factors and growth
4. Oxygen
anaerobes lack superoxide dismutase and/or catalase
anaerobes need high -, something to remove O2
chemical: thioglycollate; pyrogallol + NaOH
H2 generator + catalyst
physical: removal/replacement

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 Special Culture Techniques
Candle Jar

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Special
Culture
Techniques
Gas Pack
Jar Is Used
for
Anaerobic
Growth
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 Other Macronutrients
• Phosphate (P), sulfur (S), potassium (K), magnesium
(Mg), calcium (Ca), sodium (Na), iron (Fe)
• Iron plays a major role in cellular respiration, being a
key component of cytochromes and iron-sulfur
proteins involved in electron transport.
• Siderophores : Iron-binding agents that cells produce
to obtain iron from various insoluble minerals.

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 Microbial growth

• Microbes grow via binary fission, resulting in exponential

increases in numbers
• The number of cell arising from a single cell is 2n after n
generations
• Generation time is the time it takes for a single cell to grow
and divide
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Binary Fission

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Rapid Growth of Bacterial Population

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 Growth curve

• During lag phase, cells are recovering from a period of no growth and
are making macromolecules in preparation for growth i.e.
• no apparent cell division occurring but the cells are growing in volume
or mass, synthesizing enzymes, proteins, RNA e.t.c and increasing in
metabolic activity. The duration of lag phase depends on;
• Inoculum size
• Recovery period of cells from physical damage or shock during
transfer to a fresh medium
• Time required for synthesis of new inducible enzymes necessary to
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metabolize substrates present in a medium
log phase;
• cultures are growing maximally
• All cells are dividing at a constant rate and the population is
growing by geometric progression
• Composition of medium and the conditions of incubation
determine the rate of cell division
• Rate of exponential growth of a bacterial culture is expressed as
generation time or doubling time of the bacterial population
• Generation time G is defined as the time (t) per generation (n-
number of generations). Hence, G = t/n
The Stationary phase;
• occurs when nutrients are depleted and wastes accumulate
(Growth rate = death rate)
• Exhaustion of available nutrients
• Accumulation of inhibitory metabolites or end products
• Exhaustion of space (biological space)

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In this phase, the rate of cell division = rate of cell death
and the bacteria population remains constant. Some
bacteria produce secondary metabolites i.e metabolites
produced after the active stage of growth (antibiotics
and pigments). In spore-forming bacteria, the
sporulation process is induced during the stationary
phase.
Death phase/decline phase:-
If the incubation continues after the stationary phase,
death phase follows. Viable cell population declines.
Turbidimetric measurements or microscopic counts
cannot detect death phase in culture since living and
dead cells are counted. In death phase, number of
viable cells decreases geometrically or exponentially. It
is the reverse of growth during the log phase.

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 Calculation of Generation Time
If we start with one cell, when it divides, there are 2 cells in
the first generation, 4 cells in the second generation, 8 cells in
the third generation, and so on. The generation time is the
time interval required for the cells (or population) to divide.
G (generation time) = (time, in minutes or hours)/n(number
of generations)
G = t/n
t = time interval in hours or minutes
B = number of bacteria at the beginning of a time interval
b = number of bacteria at the end of the time interval
n = number of generations (number of times the cell
population doubles during the time interval)
b = B x 2n (This equation is an expression of growth by binary
fission)
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Example:
What is the generation time of a bacterial population
that increases from 10,000 cells to 10,000,000 cells in
four hours of growth?
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 Methods used to measure microbial growth
Growth;-- the formation of new protoplasm from
available nutrients involves the increase in cell mass
and the number. The time interval for a bacterial cell to
divide into 2 new cells or for a population of bacterial
cells to double is called generation time. Generation
time for bacteria growing under natural conditions
ranges from a few minutes to several days. In bacteria
and other unicellular organisms, growth can be
measured in terms of 2 parameters;
a) Changes in cell mass
b) Changes in cell numbers

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Measurement of cell mass
This may be direct or indirect.
• Direct determination of cell dry weight, wet weight or volume of
cells is done after centrifugation. This is a physical method. The
dry weight of the bacterial mass is directly proportional to their
number.
• Direct determination of a chemical constituent of the cells e.g.
total N, total protein or total DNA content. This is a chemical
method. Total N can be determined by the micro-Kjeldahl
method.
• Indirect measurement of a chemical activity e.g. the rate of
respiration using optical oxygen sensors, e.t.c.
• Turbidity measurements involve the use of an instrument such as
a spectrophotometer to determine the amount of light scattered
by cell suspensions. Cells such as bacteria scatter light in
proportion to their numbers. Turbidity of a suspension of cells is
directly related to the cell mass/cell number.
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 Methods used to measure microbial growth
• Count colonies on plate or filter (counts live cells)
Indirect viable cell counts/plate counts methods. This
involves inoculating a known volume of culture on a
suitable growth medium in plates. Each viable cell
grows into a colony. Colonies are then counted to
determine the number of colony forming units (CFUs)
and hence the viable number of bacteria/unit volume
can be calculated.
• Microscopic counts
• Flow cytometry (FACS)
• Turbitity

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 Viable counts

• Each colony on plate or filter arises from single live cell

• Only counting live cells
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Direct Count
Pour Plate

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 Microscopic counts

Direct microscopic counts are possible using special slides known as

counting chambers e.g. Petroff-Hausser counting chambers. The
disadvantage is that both dead and living cells are counted. Only
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populations can be counted.
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 Turbitity

• Cells act like large particles

that scatter visible light
• A spectrophotometer sends a
beam of visible light through
a culture and measures how
much light is scattered
• Scales read in either
absorbance or %
transmission
• Measures both live and dead
cells

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 Inoculation
• Sample is placed on sterile medium providing
microbes with the appropriate nutrients to
sustain growth.

• Selection of the proper medium and sterility of

all tools and media is important.

• Some microbes may require a live organism or

living tissue as the inoculation medium.
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 Incubation
• An incubator can be used to adjust the proper
growth conditions of a sample.

• Need to adjust for optimum temperature and

gas content.

• Incubation produces a culture – the visible

growth of the microbe on or in the media
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 Isolation
• The end result of inoculation and incubation is
isolation.

• On solid media we may see separate colonies, and

in broth growth may be indicated by turbidity.

• Sub-culturing for further isolation may be

required.
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 Inspection
• Macroscopically observe cultures to note color,
texture, size of colonies, etc.

• Microscopically observe stained slides of the

culture to assess cell shape, size, and motility.

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 Identification
• Utilize biochemical tests to differentiate the
microbe from similar species and to determine
metabolic activities specific to the microbe.

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