Chapter 7

Microbial Growth

Prof. Jung Hoe Kim

Growth

• Increase in cellular constituents that may result in:

– increase in cell number • e.g., when microorganisms reproduce by

budding or binary fission

– increase in cell size • e.g., coenocytic microorganisms have nuclear

divisions that are not accompanied by cell divisions

• Microbiologists usually study population growth rather than growth of individual cells

Prof. Jung Hoe Kim

The Growth Curve • Observed when microorganisms are cultivated in

batch culture

– culture incubated in a closed vessel with a single

batch of medium

• Usually plotted as logarithm of cell number versus

time

• Usually has four distinct phases

no increase

maximal rate of

division

and population

growth

population growth

ceases

decline in

population

size

Lag Phase

• Cell synthesizing new components

–e.g., to replenish spent materials

–e.g., to adapt to new medium or other

conditions

• Varies in length

– in some cases can be very short or

even absent

– factors affecting lag phase length :

inoculum size, activity(age) of cells

Prof. Jung Hoe Kim

Exponential Phase

• Also called log phase

• Rate of growth is constant

• Population is most uniform in terms

of chemical and physical properties

during this phase; balanced growth

Prof. Jung Hoe Kim

cells are dividing and doubling in

number at regular intervals

The data from table for six generations of growth are plotted

directely (red line) and in the logarithmic form (blue line). The

growth curve is exponential as shown by the linearity of the log plot.

Prof. Jung Hoe Kim

Mathematics of growth

Nt = N0 X 2n

ln Nt = ln N0 + n ln 2

= ln N0 + ln 2 / td ∙t

= ln N0 + μ∙t

Nt = N0 ∙ e μt

N0 = initial cell number

Nt = cell number at time t

n = generation number

Where, n = t / td, μ = ln 2 / td

td = doubling time(hr), or

mean generation time :

time required for cell

number in double

μ = mean growth rate constant

(1/hr)

Prof. Jung Hoe Kim

- The generation time can be

determined from a microbial

growth curve.

- The log of the population number

can also be plotted against time

on regular axes.

- The slope corresponds to μ,

td = ln 2 / μ ( μ = ln 2 / td )

( ln Nt = ln N0 + μ∙t )

Generation Time Determination

Prof. Jung Hoe Kim

Generation Times for Selected

Microorganisms

Prof. Jung Hoe Kim

Balanced growth and Unbalanced growth

• During log phase, cells exhibit balanced growth

–cellular constituents manufactured at constant

rates relative to each other

Balanced growth

• Rates of synthesis of cell components vary

relative to each other

• Occurs under a variety of conditions

–change in nutrient levels

•shift-up (poor medium to rich medium)

•shift-down (rich medium to poor medium)

–change in environmental conditions

Unbalanced growth

Prof. Jung Hoe Kim

Effect of nutrient concentration on growth

(a) The effect of changes in limiting nutrient concentration on total

microbial yield. At surfficiently high concentrations, total growth

will plateau.

(b) The effect on growth rate.

(a) (b)

Prof. Jung Hoe Kim

Stationary Phase

• Total number of viable cells

remains constant

–may occur because metabolically

active cells stop growing due to the

limitation of nutrient et al.

–may occur because growth rate is

balanced by death rate

Prof. Jung Hoe Kim

Possible reasons for entry

into stationary phase

• Nutrient limitation

• Limited oxygen availability

• Toxic waste accumulation

• Critical population density reached

Prof. Jung Hoe Kim

Starvation responses

• Morphological changes

–e.g., endospore formation

• Decrease in size, protoplast shrinkage, and nucleoid condensation

• Production of starvation proteins

• Long-term survival

• Increased virulence

Prof. Jung Hoe Kim

Death Phase

• Cells dying, usually at exponential rate

• Death

– irreversible loss of ability to reproduce

• In some cases, death rate slows due to

accumulation of resistant cells

Prof. Jung Hoe Kim

Measurement of

Microbial Growth

• Can measure changes in number

of cells in a population

• Can measure changes in mass of

population

Prof. Jung Hoe Kim

Measurement of Cell Numbers

• Direct total cell counts

– counting chambers

– electronic counters

• Viable cell counts

– plating methods

– membrane filtration methods

Prof. Jung Hoe Kim

Counting chambers

(Hemocytometer)

• Easy, inexpensive, and quick

• Useful for counting both eucaryotes and procaryotes

• Cannot distinguish living from dead cells

(a)

(b)

(c)

(a) Side view of the chamber showing the cover glass and the space

beneath it that holds a bacterial suspension

(b) A top view of the chamber. The grid is located in the center of the slide.

(c) An enlarged view of the grid. The bacteria in several of the central

squares are counted, usually at x400 to x500 magnification. The

average number of bacteria in those squares is used to calculate the

concentration of cells in the original sample.

Electronic counter

(Coulter counter)

• Microbial suspension forced through small orifice

• Movement of microbe through orifice impacts electric current that flows through orifice

• Instances of disruption of current are counted

Prof. Jung Hoe Kim

Electronic counter

• Cannot distinguish living from dead

cells

• Quick and easy to use

• Useful for large microorganisms

and blood cells, but not procaryotes

Prof. Jung Hoe Kim

Direct counts on membrane filters

• Cells filtered through special membrane that provides dark background for observing cells

• Cells are stained with fluorescent dyes

• Useful for counting bacteria

• With certain dyes, can distinguish living from dead cells

Prof. Jung Hoe Kim

Membrane Filtration Procedure

Membranes with different pore sizes are used to trap different

microorganisms. Incubation times for membranes also vary

with the medium and microorganism.

Prof. Jung Hoe Kim

Plating methods - Serial dilution of sample for suitable counting on agar plate

- Count number of colonies

- Calculate number of cells in population

Prof. Jung Hoe Kim

Plating methods…

• Simple and sensitive

• Widely used for viable counts of

microorganisms in food, water, and soil

• Population size is expressed as colony

forming units (CFU) on agar plate

Prof. Jung Hoe Kim

Measurement of Cell Mass

• Dry weight

– time consuming and not very sensitive

• Quantity of a particular cell constituent

– e.g., protein, DNA

– useful if amount of substance in each cell

is constant

• Turbidometric measures (light scattering)

– quick, easy, and sensitive

Prof. Jung Hoe Kim

more cells

more light

scattered

less light

detected

Turbidity and Microbial Mass Measurement

Determination of microbial mass by measurement of light

absorption. As the population and turbidity increase, more

light is scattered and the absorbance reading given by the

spectrophotometer increases.

Continuous Culture of

Microorganisms

• Growth in an open system

–continuous feeding of nutrients

–continuous removal of culture medium

• Maintains cells in log phase at a constant biomass concentration for extended periods

• Achieved using a continuous culture system

Prof. Jung Hoe Kim

Chemostat

• Rate of incoming

medium = rate of

removal of

medium from

vessel

• An essential

nutrient is in

limiting

quantities

Schematic diagram of the system. The fresh medium contains a limiting

amount of an essential nutrient. Growth rate is determined by the rate of

flow of medium through the culture vessel.

Prof. Jung Hoe Kim

Importance of continuous

culture methods

• Constant supply of cells in exponential phase growing at a constant known rate

• Study of microbial growth at very low nutrient concentrations, close to those present in natural environment

• Study of interactions of microbes under conditions resembling those in aquatic environments

• Food and industrial microbiology

Prof. Jung Hoe Kim

Environmental Factors Affecting

Cell Growth

• Most organisms grow under fairly

moderate environmental conditions

• Extremophiles(극한미생물)

–grow under harsh conditions that would

kill most other organisms

• Environmental factors :

pH, temperature, oxygen, osmolarity, et al.

Prof. Jung Hoe Kim

Microbial Responses to Environmental Factors

Solutes and Water Activity

• Water activity (aw)

–amount of water available to

organisms = relative humidity of air in

sealed vessel containing substance /

100

– reduced by interaction with solute

molecules (osmotic effect)

higher [solute] lower aw

– important factor specially in solid

culture (food preservation)

Prof. Jung Hoe Kim

Approximate Lower aw limits for

Microbial Growth

Prof. Jung Hoe Kim

Osmotolerant organisms

• Grow over wide ranges of water activity

• Many organisms use compatible solutes to

increase their internal osmotic concentration

• ( Usually a little higher than that of habitat )

– solutes that are compatible with metabolism and

growth at high intracellular conc.

• Some organisms have proteins and

membrane structure that requires high solute

conc. for stability and activity

Prof. Jung Hoe Kim

Effects of NaCl on microbial growth

• Halophiles

(호염균)

– grow optimally

at >0.2 M

• Extreme

halophiles

– require >2 M

Four different patterns of microbial dependence on NaCl concentration

are depicted. The curves are only illustrative and not meant to provide

precise shapes or salt concentrations required for growth.

Prof. Jung Hoe Kim

pH

• Acidophiles

– growth optimum between pH 2 and pH 5.5

• Neutrophiles

– growth optimum between pH 5.5 and pH 7

• Alkalophiles

– growth optimum between pH 8.5 and pH 11.5

Prof. Jung Hoe Kim

pH • Organisms exhibit

distinct cardinal growth

pH

– minimal

– maximal

– optimal

pH

Temperature

• Organisms exhibit

distinct cardinal growth

temperatures

– minimal

– maximal

– optimal

The effect of temperature on growth rate.

Temperature Ranges for Microbial Growth

Microorganisms can be placed in different classes based on their

temperature ranges for growth. They are ranked in order of increasing

growth temperature range as psychrophiles, psychrotrophs,

mesophiles, thermophiles, and hyperthermophiles.

Adaptations of thermophiles

• Protein structure stabilized by a variety of means

– e.g., more H bonds

– e.g., more proline

– e.g., chaperones

• Histone-like proteins stabilize DNA

• Membrane stabilized by variety of means

– e.g., more saturated, more branched and higher molecular weight lipids

– e.g., ether linkages (archaeal membranes)

Prof. Jung Hoe Kim

Dissolved Oxygen(DO)

Concentration need

oxygen

prefer

oxygen

ignore

oxygen

oxygen is

toxic

< 2 – 10%

oxygen

Obligate

aerobe

Facultative

anaerobe

Strict

anaerobe Microaerobe

100%

0%

Prof. Jung Hoe Kim

Basis of different oxygen

sensitivities

• Oxygen easily reduced to toxic products

(reactive oxygen species, ROS)

– superoxide radical ( ·O2- )

– hydrogen peroxide (H2O2)

– hydroxyl radical (OH-)

• Aerobes produce protective enzymes

– superoxide dismutase (SOD)

– catalase

Prof. Jung Hoe Kim

Reaction of Protective

Enzymes

Prof. Jung Hoe Kim

Each dot represents an individual bacterial colony within the agar or

on its surface. The surface, which is directly exposed to atmospheric

oxygen, will be aerobic. The oxygen content of the medium decreases

with depth until the medium becomes anaerobic toward the bottom of

the tube. The presence and absence of the enzymes superoxide

dismutase (SOD) and catalase for each type are shown.

Oxygen and Bacterial Growth

Prof. Jung Hoe Kim

Pressure

• Barotolerant organisms

–adversely affected by increased

pressure, but not as severely as

nontolerant organisms

• Barophilic organisms

– require or grow more rapidly in the

presence of increased pressure

Prof. Jung Hoe Kim

Radiation damage • Ionizing radiation

– x rays and gamma rays

– mutations death

– disrupts chemical structure

of many molecules, including DNA

• damage may be repaired

by DNA repair mechanisms

• Ultraviolet (UV) radiation

– mutations death

– causes formation of thymine dimers in DNA

– DNA damage can be repaired by two mechanisms

• photoreactivation – dimers split in presence of light

• dark reactivation – dimers excised and replaced in absence

of light

Prof. Jung Hoe Kim

• Visible light

– at high intensities generates singlet oxygen (1O2)

• powerful oxidizing agent

– carotenoid pigments

• protect many light-exposed microorganisms

from photooxidation

Radiation damage…

Prof. Jung Hoe Kim

Growth Limitation by

Environmental Factors

• Leibig’s law of the minimum

– total biomass of organism determined

by nutrient present at lowest

concentration

• Shelford’s law of tolerance

– above or below certain environmental

limits, a microorganism will not grow,

regardless of the nutrient supply

Prof. Jung Hoe Kim

Microbial Growth in Natural Environments

• Oligotrophic environments : microbial environments are

complex, constantly changing, often contain low nutrient

concentrations

• Morphological changes to increase surface area and

ability to absorb nutrients

• Mechanisms to sequester certain nutrients

Microorganisms can change their morphology in response to starvation

and different limiting factors to improve their ability to survive

(a) Caulobacter has relatively

short stalk when nitrogen is

limiting.

(b) The stalks are extremely

long under phosphorus

limited conditions.

50

Biofilms

• Ubiquitous in nature

• Complex, slime enclosed colonies attached to surfaces

• When form on medical devices such as implants often lead to illness

• Can be formed on any conditioned surface

Prof. Jung Hoe Kim

51

Biofilm Formation

• Microbes reversibly attach to conditioned surface and release polysaccharides, proteins, and DNA

• Additional polymers are produced as biofilm matures

• Interactions occur among the attached organisms

• Extracellular matrix and change in attached organisms’ physiology protects them from harmful agents such as UV light and antibiotics

Prof. Jung Hoe Kim

52

Figure 7.26

Prof. Jung Hoe Kim

53

Cell to Cell Communication

• Acylhomoserine lactone (AHL) is an

autoinducer molecule produced by

many gram-negative organisms

• Concentration present allows cells to

access population density

• Process is called quorum sensing

Prof. Jung Hoe Kim

Quorum Sensing and Microbial

Populations

• Quorum(critical

level) sensing

(Autoinduction)

– microbial

communication and

cooperation

– involves secretion

and detection of

chemical signals

(a) generalized structure for acyl homoserine lactone, the best-known quorum

sensing signal (autoinducer)

(b) overview of how quorum sensing functions in many gram-negative bacteria;

R=receptor protein that acts as an inducer; dashed lines indicate that acyl HSL

synthase is not always made in response to autoinducer

Processes sensitive to quorum

sensing: gram-negative bacteria

• Bioluminescence (Vibrio fischeri)

• Synthesis and release of virulence factors (Pseudomonas aeruginosa)

• Conjugation (Agrobacterium tumefaciens)

• Antibiotic production (Erwinia carotovora, Pseudomonas aureofaciens)

• Biofilm production (P. aeruginosa)

Prof. Jung Hoe Kim

Quorum sensing: gram-positive

bacteria

• Mating (Enterococcus faecalis)

• Transformation competence (Streptococcus pneumoniae)

• Sporulation (Bacillus subtilis)

• Production of virulence factors (Staphylococcus aureus)

• Development of aerial mycelia (Streptomyces griseus)

• Antibiotic production (S. griseus)

Prof. Jung Hoe Kim