Bacterial Growth

Definition
A complex process involving numerous
anabolic (synthesis of cell constituent and
metabolites) and catabolic (breakdown of
cell constituents and metabolites)
reactions resulting in cell division.
- Under ideal conditions a cell can divide in
as little as 10 mins., however, some may
take as long as 100 years in some
subsurface terrestrial environment.
- Such slow growth is the result of a
combination of factors including the fact
that most subsurface environments are
both nutrient poor and heterogenous. This
inhibits a metabollic state that is efficient
enough to allow exponential growth.
 APPROACHES TO MICROBIAL
GROWTH:
Two approaches to the study of microbial growth
using pure cultures.
(1) batch culture
• is a population of microorganisms grown in a
closed system.
• Resources are fixed and added from the onset.
• limited amounts of the resources needed for
survival.
(2) continuous culture
• A steady influx of growth medium and substrate
such that the amount of available substrate
always remains the same.
 Bacterial Growth in the natural environment
- Level of complexity is tremendous due to factors such
as:
1. Variation in soil surfaces

2. Microenvironment that have altered physical and

chemical properties.

3. A limited nutrient status

4. A consortia (group) of different microorganisms

competing for the same limited nutrient supply.
 Requirements for growth
(1) Physical
- pH
- temperature
- osmostic pressure

(2) Chemicals
- Carbon
- nitrogen
- sulphur
- phosphorus
- trace elements
- oxygen
- organic growth factors
 How is temperature tolerance used to classify
microbes?
1. psychrophiles- cold loving microbes, 0-150C max. 25
Eg. Arthrobacter sp. And Psychrobacter sp.
2. psychrotrophs- 0-300C max. 40, can slowly degrade food (cause
mold, slime etc) ie. sac fungi
3. Mesophiles- moderate temperature, 25-400C (spoilage and
disease causing microbes) ie. Staphylococcus aureus, Salmonella
4. Thermophiles- heat loving microbes- 50-600C min. 45
5. Hyperthermophiles- 800C , lives in hot springs associated with
volcanic activity and therefore sulphur is important in their
metabolic activity ie. Genus Pyrodictum, thermococcus litoralis
Note : they all grow at a minimum, optimum and maximum
temperature.
Above the optimum temperature growth drops because enzymes
systems within the cells are inactivated.
- micro-organisms becomes dormant and freezing temperatures
 pH
- Most bacteria grow best between pH of 6.5-7.5
- Yeast and molds grows best between 5-6
- Few grow at an acidic pH

- Acidiophiles ie chemoautotrophic bacteria in coal mines oxideizes

suphur to form sulphuric acid. Grow between pH of 1-4

- Alkalinity also inactivate growth but rarely use to preserve food

- When the bacteria are cultured in the lab they often produce acids
that eventually interfere with their own growth. To neutralize the
acid and maintain proper pH, chemical buffers are included in the
growth medium. Ie. Peptone , amino acids, phosphate salts (non
toxic)
 Osmotic pressure
- Microbial cells are made up of 80-90% water

- In hypertonic conditions water passes out through the plasma

membrane

- Result: plasmolysis, shrinking

WHAT HAPPENS IN A HYPOTONIC AND A ISOTONIC CONDITION?
- Importance: growth inhibited as the plasma membrane pulls away
from the cell wall. Thus addition of salt or another solute to increase
osmostic pressure can be used to preserve food.

- Halophiles require between 2-30% salt solution for growth. Those

that require 2% are called facultative halophiles and those that
require 30% are called obligate halophiles.
- Cell may burst in a hypotonic solution
- Agar not more than 1.5% salt
 Chemical requirements
1. carbon: ½ the dry weight of the cell, derived from carbon dioxide

2. Nitrogen- 14% dry weight of cell, derived from ammonium salts,

nitrate ions or nitrogen gas. Use in synthesizing proteins such as
DNA, enzymes etc

3. Sulphur and Phosphorus-4% dry weight of the cell

- Sulphur is used to synthesize sulphur containing amino acids and
vitamins such as thiamine and biotin
- phosphorus- synthesis of nucleic acid and phospholipids of cell
membrane; source – phosphate ions

4. Trace Elements
Iron, copper, zinc : use to make essential enzymes function
 Oxygen
- Microbes use it to produce maximum energy from nutrients. They
are called obligate aerobes ie. Genus Clostridium that causes
tetanus and botulism.

- Facultative anaerobes use oxygen when present but are capable of

continued growth when absent by using fermentation.
Note: microbes may also substitute an electron acceptor such as nitrate
ion for oxygen.

- Aerobes, facultative anaerobes and aerotolerant anaerobes must

have the enzyme superoxide dismutase to neutralize them and
either catalase or peroxidase to break down toxic bi-products.
 Organic Growth Factors
- Some bacteria lack the enzymes necessary to synthesize the
necessary vitamins.
- Other growth factors required by bacteria are amino acids, purines
and pyrimidines.
 Types of Media
1. Chemically defined- the extract chemical composition is known.
2. Complex media- one in which the extract chemical composition
varies from batch to batch. Ie. Nutrient agar
3. Reducing media- oxygen starving media, is employed for growing
obligate anaerobes. Reducing medium particularly contains
chemicals (reducing agents) that deplete molecular oxygen.
• Selective media- Differential growth suppression: Selective medium
is designed to suppress the growth of some microorganisms while
allowing the growth of others (i.e., they select for certain microbes).
• Solid medium is employed with selective medium so that individual
colonies may be isolated.
• Examples of selective media include: mannitol salts agar (selects
against non-skin flora)
• MacConkey agar (selects against gram-positives)
• eosin-methylene blue agar (selects against gram-positives)
• phenylehyl alcohol agar (selects against gram-negatives)
5. Differential- allow the growth of more than one microorganism of
interest but with morphologically distinguishable colonies.
• Examples of differential media include:
– mannitol salts agar (mannitol fermentation = yellow)
– blood agar (various kinds of hemolysis)
– MacConkey agar (lactose fermentation = yellow)
– eosin-methylene blue agar (various kinds of differentiation)

6. Enrichment culture is used to increase the relative concentration of

certain microorganisms in the culture prior to plating on solid
selective medium.
• Unlike selective media, enrichment culture is typically used as broth
medium.
 Preserving cultures
• Preserving cultures is important for:
– scientific reasons
– identification
– vaccine production
– industrial use
– etc.
• Methods of preserving cultures include:
– refrigeration
– stabs
– slants
– lyophilization
– freezing
• Stabbing- Cultures are stabbed deeply into agar using a inoculating
needle. The stabs are incubated until visible cultures form, then
sealed and stored at room or lower temperature.
• Slant method- Cultures may be streaked onto the surface of the
solid medium in a slant tube.
• The slants are incubated until visible culture formation then sealed
and stored at room or lower temperature.
• Lyophilization Freeze drying:
– Lyophilization is the freeze-drying of cultures.
– Cultures are first frozen and then dried under high vacuum.
– To revive cultures they are rehydrated by broth.
• Freezing Freezing with protection:
– Broth cultures are mixed with various ingredients eg. glycerol to
limit damage upon freezing and then frozen to temperatures
ranging from -50�C to -95�C.
– To revive cultures they are thawed, pelletted, and resuspended
into broth.
 GROWTH IN PURE CULTURE IN A FLASK:
• cells are placed in a flask in which the nutrient supply
and environmental conditions are controlled.

• If the liquid medium supplies all nutrients required for

growth and environmental parameters are optimal, the
increase in numbers or bacterial mass can be measured
as a function of time to obtain a growth curve.
 LAG phase
- period before growth begins
• Cells are adapting to a new source of nutrients.
CAUSES:
1. Required for induction of specific mRNA and protein synthesis to
meet new cultural requirements.
2. Low densities of microorganisms that results in dilution of
exoenzymes and nutrients that leak from the growing cell.

- The length of this period varies but can be controlled by:

1. Type of medium (nutrient constituent ie. Trypticase soy broth –
TSB vs. Mineral salt with glucose –MS)
2. Initial inoculums size (104 vs 106)
 When can a lag phase be observed?
1. If the inoculum is taken from an old culture and
inoculated into the same medium.
Why? because the cells are usually depleted of various
essential constituents and time is required for their
resynthesis.
2. When the inoculum consists of cells that have been
damaged, but not killed, by treatment with heat,
radiation, or toxic chemicals.
Why? because of the time required for the cells to repair
the damage.
3. When a population is transferred from a rich culure
medium to a poorer one.
Why? For growth to occur in a particular culture medium
the cells must have the complete complement of
enzymes for synthesis of metabolites not present in that
medium. Therefore, lag time is required for synthesis of
the new enzymes.
 EXPONENTIAL PHASE
- All cells are growing and dividing and the resultant
progeny cells are also growing and dividing.

- Cell growth during the exponential phase can be

described using the following equation:

dX/dt=µX
X- the number of mass of cells (mass/volume)
T- time
µ- the specific growth rate constant (1/time)

- The time it takes for cell division to occur is called the generation
time or doubling time.

(* explain calculation 1 &2 pg 46-47)

 Other characteristics of the exponential phase
• Reserve or storage products may be accumulating in all cells.

• Only a small fraction of the total number of cells dies during this
phase.

• The rate of exponential growth varies greatly since it is influenced

by environmental conditions such as temperature, composition of
the culture medium and the genetic characteristics of the
organism.
Ie. In general, prokaryotes grow faster than eukaryotes, and small
eukaryotes grow faster than large ones.

• - if exponential growth starts with a low number of cells then the

number of cells produced will initially be small but as the number
of cells increases, then the number of new cells will begin to
increase explosively.
 STATIONARY PHASE
• No net growth
• Cells still grow and divide but is balance with
death.
# of cells growing= # of cells dying
• Cells are smaller and rounder than cells in the
exponential phase
• Mathematical model
dX/dt=0

• Nutrient sources are depleted and toxic wastes

accumulate.
• - Cells alter their physiology to slow metabolism,
use up storage materials, and slow the cell
division process until the division rate equals the
death rate.
When does the stationary phase occur?
1. Carbon and energy sources or essential
nutrients become completely used up.
- Growth still occurs cause dying cells lyse
and becomes nutrients.
(ENDOGENEOUS METABOLISM)

2. Waste products build up to a point where

they begin to inhibit cell growth or are
toxic to cells.
- Occur in culture with high cell density.
 DEATH PHASE
• Net loss of cultural cells
• Cells may still be metabolize and divide
but more is loss than made.
• The number of cells that die as a result
of irreversible damage to cellular,
metabolic, genetic, or structural
components increases.
• In some cases, death is accompanied
by actual cell lysis.