Module 6: Bacterial Growth (Microbial Growth

Requirements)
Growth

• Increase in cellular constituents that may result in:

o increase in cell number
o increase in cell size
• Growth refers to population growth rather than
growth of individual cells

Consequences of Bacterial Growth

• If Streptococcus pyogenes at the back of your throat,

a sore throat
• Growth of microorganisms in refrigerator shortens
the shelf life of the food
• Produce beer, wine, cheese, yogurt and other
products.

Bacterial Division

Chromosomes replication and partitioning and cytokinesis

• Most bacterial chromosomes are circular

• DNA replication proceeds in both directions from the
origin
• Origins move to opposite ends of the cell
• Cell elongates
• Septation – formation of cross walls between
daughter cells and cells separate

Type of bacterial division:

• A hydrophobic alcohol called bactoprenol facilitates
• Binary fission
transport of new glycan units through the cytoplasmic
• Budding
membrane to become part of the growing cell wall
• Certain actinomycetes by coniodiospores
(Figure 6.4).
• Fragmentation
• Transpeptidation bonds the precursors into the
Steps of cell division (binary fission) peptidoglycan fabric (Figure 6.5).

1. Initiation mass reached

2. Initiation of replication
3. Threshold cell length reached
4. Initiation of septum formation

Peptidoglycan Synthesis and Cell Division, P. 139

• New cell wall is synthesized during bacterial growth

by inserting new glycan units into preexisting wall
material
The Mathematics of Growth i. in some cases can be very short or
even absent
Generation (doubling) time
ii. depends on harshness of medium
• time required for the population to double in size 1. is it selective or enrichment medium?
• varies depending on species of microorganism and 2. what is the temperature of medium?
environmental conditions • The general rule of thumb is that microbes
• range is from 10 minutes for some bacteria to several adapt to a shift to improved conditions much
days for some eukaryotic microorganisms more rapidly than they do to a shift to poorer
• This is calculated during log growth phase conditions.
2. Exponential Phase
Calculating No. of Generation and Generation time • Also called log phase or log growth phase
• Rate of growth and division is constant and
log # 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 (𝑒𝑛𝑑) − log 𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 (𝑠𝑡𝑎𝑟𝑡)
# 𝑜𝑓 𝑔𝑒𝑛 = maximal
0.301 (𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡)
• Population is most uniform in terms of
𝑡𝑖𝑚𝑒𝑙𝑎𝑝𝑠𝑒 𝑖𝑛 𝑚𝑖𝑛𝑢𝑡𝑒 chemical and physical properties during this
𝐺𝑒𝑛 𝑡𝑖𝑚𝑒 = = 𝑚𝑖𝑛/𝑔𝑒𝑛
# 𝑜𝑓 𝑔𝑒𝑛 phase
• Bacteria from this stage would be used for
Example:
studies
If 100 cells growing for 5 hours produced 1,720,320 cells: 3. Stationary Phase
• Closed system population growth ceases due
What is the number of generations? to:
o Nutrient limitation
o accumulation of a waste product.
log 1,720,320 − log 100 o Limited oxygen availability
=
0.301 o Critical population density reached
= 41.43 𝑔𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛𝑠 o Bacteria die off and liberate some
What is the generation time? nutrients
• no change in the number of viable cells,
300 active cells stop reproducing or reproductive
=
41.43 rate is balanced by death rate
= 7.24 𝑚𝑖𝑛/𝑔𝑒𝑛
• Can last for long period
The Growth Cycle o microbes in nutrient-poor
environments (like soils and many
• Microorganisms show a characteristic growth pattern (Figure aqueous environments) probably
6.8) when inoculated into a fresh culture medium. spend most of their time in
stationary phase
Phases of Growth Cycle
4. Death Phase
Cell numbers begin to decline due to
• DNA or protein damage or
o perhaps exhaustion of energy reserves
o Accumulation of toxic waste
• Bacteria are dying off opposite to log growth
phase
o do not die all at once
• Two alternative hypotheses
o cells are Viable but Not Culturable
(VBNC)
1. Lag Phase o cells alive, but dormant, capable of new
• Cell synthesizing new components growth when conditions are right
i. e.g., to replenish spent materials • Programmed cell death
ii. e.g., to adapt to new medium or o fraction of the population genetically
other conditions programmed to die (commit suicide)
• Varies in length
Continuous Culture

• The culture is kept in exponential growth phase for as

long as desired by the continuous addition of new
medium and the simultaneous removal of the same
volume of old medium and cells.
• Under these conditions, all cells should be growing
exponentially and samples at various times should be
identical.

A chemostat A reservoir of sterile

medium is added to the culture
at a constant rate. Spent medium
leaves the culture at the same
rate. Fresh nutrients are
constantly entering the culture
and waste products are
continuously removed.

Environmental Effects on Microbial Growth: Effects of

temperature on Growth

Temperature

• Temperature is a major
environmental factor controlling Environmental Effects on Microbial Growth: Microbial
microbial growth. growth at low or high pH
• The cardinal temperatures
• In water, the hydrogen ion concentration will range
are the minimum, optimum, and
from 1 x 10- 14 M (a pH of 14) to 1 M (a pH of 0). §
maximum temperatures at which
Microbes are found at almost any conceivable pH,
each organism grows
with most common bacteria growing at or near
neutral pH (7).
• The acidity or alkalinity of an environment can greatly
Microbes based on their optimum temperature affect microbial growth. Figure 6.22 shows the pH
scale.
Optimum Habitat
Type
temp. • Some organisms have evolved to grow best at low or
Psychrophiles 15 ⁰c and max Cold environment high pH, but most organisms grow best between pH 6
below 20 ⁰c and 8. The internal pH of a cell must stay relatively
Psychrotolerant 20 ⁰c to 40 ⁰c Function best at cold close to neutral even though the external pH is highly
temp but sensitive to acidic or basic.
warm temp • Organisms that grow best at low pH are called
Psychrotrophs 0 ⁰c to 35 ⁰c acidophiles; those that grow best at high pH are called
Mesophiles 20 ⁰c to 45 ⁰c Midrange; found in alkalophiles.
warm-blooded
animals and Bacteria based on pH Requirements
environment
Thermophiles 45 ⁰c to 80 ⁰c Hot environments Optimum pH Example
Type
such as hot springs level req.
and hydrothermal Acidophile 1.0-5.4 pH Thiobacillus thiooxidans,
vents Bacillus acidocaldarius,
Extreme Above 80 ⁰c Hot environments lactobacillus acidophilus
thermophiles such as hot springs Neutrophiles 5.5-7.9 pH S. aureus, E.coli,
and hydrothermal clostridium sporogenes,
vents P. aeruginosa, S.
pnuemoniae
Alkalophiles 8.0-11 pH
Environmental Effects on Microbial Growth: Osmotic
Pressure

Osmotic Pressure (AW)

• Water availability (water activity) is a measurement

of how much free water is available.
• Pure water has a water activity of 100% and the
activity decreases as solutes are added to the
solution.
• Changes in osmotic concentrations in the
environment may affect microbial cells.
o hypotonic solution (lower osmotic
concentration)
▪ water enters the cell
• Water activity becomes limiting to an organism when
▪ cell swells may burst (plasmoptysis)
the dissolved solute concentration in its environment
o hypertonic (higher osmotic concentration)
increases.
▪ water leaves the cell
▪ membrane shrinks from the cell • To counteract this situation, organisms produce or
wall (plasmolysis) may occur accumulate intracellular compatible solutes (Figure
6.24; Table 6.3) that maintain the cell in positive
Osmotic water balance.

▪ Water can be made unavailable by: Types of solutes:

o Evaporation
o Freezing 1. Amino acid-type and related solutes:
o Bound to solutes • Glycine betaine
▪ Cause dehydration • Ectoine
▪ Water unavailable to enzymes • Dimethylsufoniopropionate
2. Carbohydrate-type solutes:
Microbes based on osmotic… • Sucrose
• Trehalose
▪ Halophiles
o Extreme – 15-30% NaCl 3. Alcohol-type solutes:
o Moderate – 6-15% NaCl • Glycerol
o Mild – 1-6% NaCl • Mannitol
▪ Halotolerant – can endure NaCl; organisms can Oxygen and Microbial Growth
tolerate some reduction in the water activity of their
environment but generally grow best in the absence Oxygen
of the added solute.
• Many microbes and most eukaryotes need oxygen for
▪ Osmophile – lives in high sugar
growth as it is the terminal electron acceptor.
▪ Xerophiles – microbes in dry environments
• Other microbes can live without it and some cannot
▪ Some microorganisms (halophiles) have evolved to
even tolerate its presence.
grow best at reduced water potential, and some
o Obligate aerobes
(extreme halophiles) even require high levels of salts
o Facultative anaerobes
for growth.
o Microaerophiles
o Aerotolerant anaerobes
o Strict anaerobes
• Aerobes requires oxygen to live, whereas anaerobes
do not and may even be killed by oxygen
• Facultative organisms can live with or without oxygen
• Microaerophiles are aerobes that can use oxygen
only when it is present at levels reduced from that in
air.
• Aerotolerant anaerobes can tolerate oxygen and
grow its presence even though they cannot use it.
Oxygen… Magnetospirillum Fresh and microaerophile
magnetotacticum marine
• Strict aerobes and facultative anaerobes are able to water
use oxygen in metabolic processes and generate Camplylobacter Mucosal Microaerophile
more energy per mole of energy source consumed. jejuni surfaces of
• Aerotolerant anaerobes and strict anaerobes do not animals
use oxygen in their metabolism and typically have a and birds
lower energy yield and slower growth rates. Lactobacillus Animals Aerotolerant
acidophilus plants, anaerobe
fermented
foods
Enterobacter Intestines Facultative
aerogenes of warm- anaerobe
blooded
animals,
fresh water
Vibrio fischeri Marine Facultative
water, light anaerobe
organ of
several
marine
species

Toxic forms of oxygen

• A reducing agent such as thioglycolate can e added to
a medium to test an organism’s requirement for • Several toxic forms of oxygen can be formed in the cell,
oxygen. but enzymes are present that can neutralize most of them
(Figure 6.28). Superoxide in particular seems to be a
common toxic oxygen species.

• TA restrict oxygen to the top one-third of the tube. Cell produce toxic form of oxygen during respiration
Aerobes will grow only at the top of the tube (1).
Facultative anaerobes will grow throughout (2 and 3) Enzyme that destroy toxic forms of oxygen
and strict anaerobes will only grow at the bottom of
A. Catalase
the tube (4).
B. Peroxidase
C. Superoxide dismutase
Organism Habitat Oxygen
D. Superoxide dismutase/ catalase in combination
relationship
E. Superoxide reductase
Sulfolobus Hot sulfur Strict aerobe
acidocaldarius spring
Acinetobacter Skin Strict aerobe
calcoaceticus
Bifidobacterium Human Strict anaerobe
bifidum intestines
 The roles trace elements play in metabolism

Element Example of function

Cobalt Part of vitamin B12, which
is used to carry methyl
groups
Zinc Structural role in many
enzymes including DNA
polymerase
Mo Certain reactions
involving nitrogen
assimilation. Found in
nitrate reductase and
nitrogenase.
Cu Catalytic role in some
enzymes that react with
• Special techniques are needed to
oxygen for example
grow aerobic and anaerobic
cytochrome oxidase.
microorganism.
Mn Required by a number of
enzymes in catalytic sites.
Certain photosynthetic
enzymes use manganese
to split water into oxygen
and protons.
Ni Several different enzymes
including some involved in
carbon monoxide
metabolism, urea
Nutrition and culture or microorganisms metabolism and
methanogenesis
Requirements:

• Macronutrients • Some example of growth factors includes

• Micronutrients o Vitamins which are non-protein
• Trace elements components of many enzymes
o Amino acids for protein synthesis
• Temperature
o Nucleic acids for DNA and RNA synthesis
• pH
• Trace elements
• Osmotic pressure (Aw)
o Inorganic elements required in small
• Oxygen
amounts
The Requirements for growth: Chemical Requirements o Usually as enzyme cofactors

Nitrogen Nutritional classification of microbes

• In amino acids, proteins Energy source:

• Most bacteria decompose proteins
Phototrophic – utilizes light as a source of energy
• Some bacteria use NH4+ or NO3-
• A few bacteria use N2 inn nitrogen fixation Chemotrophs – obtain energy by oxidation of either
inorganic or organic compounds
Sulfur
Carbon source:
• In amino acids, thiamin, biotin
• Most bacteria decompose protein Autotrophs – obtain their carbon from carbon dioxide
• Some bacteria use SO4 2- or H2S
Heterotrophs – rely on pre-made organic compounds for
Phosphorus carbon

• In DNA, RNA, ATP, and membranes

• PO4 3- is a source of phosphorus
 Electron source: • Movement of microbe through orifice impacts
electric current that flows through orifice.
Organotrophs – obtain their electrons from organic
• Instances of disruption of current are counted = result
compounds
in count of individual cells
Lithotrophs – obtain electrons from inorganic compound
Limitations
Measuring microbial growth
• Without special staining techniques dead cells cannot
Parameters used as a measure of growth of a population be distinguished from live cells
of bacteria. They include: • Small cells are difficult to see under the microscope,
and some cells are inevitably missed.
• Change in cell number. • Precision is difficult to achieve
• Change in the turbidity or light scattering of the • A phase-contrast microscope is required if the sample
culture. is not stained.
• Change in the amount of a cell component • Cell suspension of low density have few if any bacteria
Measurement of microbial growth in the microscope field unless a sample is first
concentrated and resuspended in a small volume
• Microscopic counts: • Motile cell must be immobilized before counting
o Counting chambers • Debris in the sample may be mistaken for microbial
o Electronic counters – flow cytometry cells.
o On membrane filters
• Viable counting methods: Viable counting: Alive or dead?
o Spread and pour plate techniques
o Membrane filter technique
o Turbidity for most probable number (MPN)
• Measurement of cell mass
o Dry weight analysis
o Measurement of cell component
o Turbidity

Direct counting: Counting

chambers

• Easy, inexpensive, and

quick
• Useful for counting both
eukaryotes and
prokaryotes
• Cannot distinguish living
from dead cells

Direct counts on membrane filters

• Cell filtered through special membrane that provides

dark background for observing cells
• Cells are stained with fluorescent dye
• Useful for counting bacteria
• With certain dyes, can distinguish living from dead
cells

Direct count: Flow cytometry

• Microbial suspension forced through small orifice

with a laser light beam
Viable counting methods • Plate counts can be highly unreliable when used to
assess total cell numbers of natural samples such as
• Spread and pour plate techniques
soil and water. Direct microscopic counts of natural
o Diluted sample of bacteria is spread over
samples typically reveal far more organisms than are
solid agar surface or mixed with agar and
recoverable on plates of any given culture medium.
poured into petri dish
o After incubation the number of organisms is
• This is referred to as "the great plate count anomaly,"
determined by counting the number of
and it occurs because direct microscopic methods
colonies multiplied by the dilution factor
count dead cells whereas viable methods do not, and
• Results expressed as colony forming units (CFU)
different organisms in even a very small sample may
have vastly different requirements for resources and
conditions in laboratory culture.

Measurements of cell mass

• Dry weight
o Time consuming and not very sensitive
• Quantity of a particular cell constituent
o E.g., protein, DNA, ATP or chlorophyll
o Useful if amount of substance in each cell is
constant
• Turbidometric measure (light scattering)
o Quick, easy and sensitive

• Turbidity measurements are an indirect but very

rapid and useful method of measuring microbial
growth (Figure 6.12). However, to relate a direct cell
count to a turbidity value, a standard curve must first
Viable counting methods be established.

• Membrane filter technique (used in our lab during

water testing)
o Bacteria from aquatic samples are trapped
on membranes
o Membrane placed on culture media
o Colonies grow on membrane
o Colony count determines # of bacteria in
sample
• If microbe cannot be cultured on plate media
• Dilutions are and added suitable media
• Turbidity determined to yield the most probable
number (MPN)
Module 7: Isolation and cultivation of microorganisms o E.g. eosin
methylene blue agar
Pure culture (Axenic culture) (EMBA)
• A culture which contains a single species of
microorganism
• A population of cells arising from a single cell

Cultivation

• Increasing the population of microorganisms by

providing their nutritional and physical requirements

Nutrients

• Extracellular substances which provide the cell with

materials for building protoplasm and for energy
generation

Culture medium

• Any nutrient material for growth and cultivation of

• Selective media – allows growth of
microorganisms in the laboratory
specific type of microorganism
Use of culture media only
o With selective agents (ex.
• For growth and maintenance of microbial cultures Salts, dye, antibiotics
• To favor the production of particular compounds etc.)
• To study microbial action on some constituent of the o E.g. bacillus cereus agar
medium (BCA)
• Enrichment
Types of culture media
media – used to
According to physical state… increase the number
of microorganisms
• Liquid (broth) – no solidifying agent with unusual
• Semi-solid – 0.1-0.5% solidifying agent physiological
• Solid – 1.5-2.0% solidifying agent characteristics
o With special
Solidifying agent: agar or gelatin
nutrients (ex. blood)
According to chemical composition… o E.g. blood agar

• Synthetic – all components are chemically defined

• Complex – not all components are chemically defined
o E.g. potato infusion, beef extract, yeast
extract

According to principal function, purpose or application: • Assay media – of prescribed composition used for
assay of vitamins, amino acids and antibiotics
o Used to determine qualitative/quantitative
• General purpose – can
production of such compound by an
support most or almost all types of
organism
species
o E.g. nutrient agar (NA)

• Differential media – distinguishes one type of

bacteria from another; with special reagents lie pH
indicators or dyes
Isolation techniques 4. Single-cell isolation technique
a. Uses a micropipette or a
1. Plating
microprobe to physically
a. Colony – a microscopically visible (surface or
pick a single cell and
subsurface) growth or cluster of microorganisms on
transfer it on an agar
a solid medium
medium

5. Membrane filter
technique
a. For samples with
low population
b. Uses a sterile
membrane filter having a
pore size that retains
microorganism

Steps in preparing pure cultures

2. Enrichment culture
a. Isolation of specific types • Isolation
of microorganisms by a • Transfer
combination of nutrient • Verify the purity
and physical conditions • Make stock cultures
b. Used for the isolation of Culture preservation
unusual physiological
types of microorganisms Objective
which are present in
A. To retain the viability of the stock culture for a long
small numbers and which
period of time while maintaining its purity and trait of
grow slowly.
being “true-to-type”
3. Serial Dilution
a. Used if the desired microorganism is present at a Culture preservation methods
higher level than any other microorganism
b. Outcome is 10-fold reduction of cells/cfus in every 1. Periodic transfer to fresh media
transfer a. Consideration
i. Time interval of transfers
ii. Proper medium
iii. Proper storage temperature
2. Overlaying cultures with mineral oil
a. Aim: limit the availability of O2
b. Advantages: simple; enables one to remove some
growth under the oil and inoculate it in a fresh
medium and still preserve the initial culture
c. Disadvantages: viability of microorganisms varies
with species
3. Freeze-drying (lyophilization)
 a. Temperature = -70 C
b. Employs: rapid drying in frozen state; dry ice in
alcohol
c. Advantages: long-term survival, less opportunity
for changes in the characteristics of culture
d. Smallness of storage containers

4. freezing with liquid nitrogen

a. temperature = -196 c

b. considerations: cryoprotective agent (glycerol),

liquid-nitrogen refs

5. drying

a. drying temperature = 45 c

b. limitation: for spore- and cyst- formers

Banking microbes

Culture collections

• Organization which maintain authentic pure cultures

of microorganisms
• Provide ‘type’ strains to microbiologist throughout
the world
• Example:
o American type culture collection (Maryland)
o National collection of type culture (London)
o Japanese type culture collection (Japan)
o Philippine national collection of
microorganisms (biotech-UPLB)