Growth and Culturing of Bacteria

Growth and Cell Division

• In some species, incomplete separation of cells occurs which results in

the formation of special cell arrangements, i.e. tetrads, sarcinae,
sterptococci, etc
• In yeast & a few bacteria cell division occurs by budding, where a
smaller new cell develops from the surface of an existing cell & then
separates from the parent cell.

• Budding vs binary fission;

Although both are asexual forms of reproduction where two
genetically identical cells ‘clones’ are produced, in binary fission the
parent cell is divided into two equally sized new cells, whereas budding
produces a small new cell in addition to the existing parent cell.
 Growth and Cell Division

• Microbial growth is defined as the increase in the number of cells, rather

than in terms of cell size.
• Nevertheless, the ‘mother cell’ usually doubles in size and duplicates its
contents before it divides into two ‘daughter cells’.
• Cell division in bacteria usually occurs by binary fission or sometimes by
budding.
• In binary fission, the cell duplicates its components and a transverse
septum grows in the middle of the cell dividing it into two independent
daughter cells.
• In continuously dividing cells, DNA synthesis is continuous & replicates
the bacterial chromosome shortly before the cell divides.
• The chromosome is attached to the cell membrane which grows &
separates the replicated chromosomes.
Binary Fission Vs Budding (Yeast)
 Phases of Growth

•When bacteria are introduced (inoculated)

into a fresh nutrient medium, they show four
major phases of growth:
1.Lag phase
2.Log (exponential) phase
3.Stationary phase
4.Decline (death) phase

–These phases form the standard bacterial

growth curve.
 Phases of Growth

• Lag phase

–Cells don't increase in number, but are metabolically active.

i.e. the cells are increasing in size, incorporating various
molecules from the medium, synthesizing enzymes & and
producing large quantities of ATP (energy).
–Length of lag phase depends on the characteristics of the
bacterial species and the conditions in the growth media
(both the old medium and the new one).
• Some species adapt to the new medium in 1-2hrs, others take
several days.
 Phases of Growth

• Log (exponential) phase

–Once bacteria are adapted to the new medium, growth (increase

in number) occurs at exponential or logarithmic rate (straight
line if plotted on log y-axis).
–In log phase, organisms divide at their most rapid rate, a regular
genetically determined interval called the generation time.
• Generation time for most bacteria is between 20 min to 20 hrs;
typically less than 1 hr.
–The population of m.o. doubles in each generation time.
– Bacteria are more susceptible to antibiotics.
 Phases of Growth

•Log (exponential) phase

–But bacterial cells don’t all divide precisely
together, rather they show a situation called
‘non-synchronous growth’.
1. Synchronous growth: a hypothetical
situation where all cells divide exactly
together after each generation time → a
stair-step curve.
2. Non-synchronous growth: a natural
situation where each cell divides sometime
during the generation time → smooth
curve.
 Phases of Growth

• Log (exponential) phase

–In a flask or a tube, log phase is limited in time; because as the number of cells
increases nutrients & O2 are used up, waste materials accumulate and living
space is limited. This will reduce the ability of cells to produce ATP & growth
rate decreases.

• In this situation, the log phase levels off & will be followed by a stationary
phase, unless fresh medium is added or the organisms are transferred to
another fresh medium.

• Log bacterial growth can be maintained by using a device called ‘chemostat’

which has a growth chamber where fresh medium is continuously added
(from an attached reservoir) as old medium is withdrawn.
Chemostat
 Phases of Growth

• Stationary phase
–When cell division decreases to a rate equal to that of cell death, the number of
cells remains constant, which appears as horizontal straight line on the
bacterial growth curve.
–In this stage, the medium contains limited amount of nutrients & may contain
toxic quantities of waste materials, O2 is limited to aerobic organisms &
damaging pH changes may occur.

• Decline (death) phase

–Medium is less & less supportive to cell division, so cells lose their ability to
divide & thus die. The no. of live cells decreases at logarithmic rate.
–The duration of this phase is highly variable as the logarithmic phase, both
depend on genetic characteristics of the organism. Some bacteria contain few
bacteria that remain alive after months or years.
 Phases of Growth

•Growth in colonies

–When growing on a solid medium, a cell divides

exponentially forming a small colony containing all the
descendants of the original cell.
–The colony grows rapidly at its edges whereas cells nearer
the centre grow more slowly & begin to die. Thus all phases
of growth occur simultaneously in a colony.
–Each single living bacterial cell will divide to form a colony i.e.
each bacterial cell represents a colony-forming unit (CFU).
 Measuring Bacterial Growth – Enumeration of
Bacteria
• It is measured by estimating the no. of cells that have arisen by
binary fission during a growth phase. Expressed as number of
viable (living) organism per unit volume (i.e. ml).
1.Serial dilution & standard plate count;
• Principle: only living bacterium will divide & form visible colony on
agar plate.
Agar plate: Petri dish containing nutrient medium solidified with agar.
• Serial dilution: series of dilutions e.g. 1/10 → 1/10 → 1/10 etc, then
transfer 0.1ml to agar plate. The transfer is done either by;
a. Pour plate method or
b. Spread plate method
 Measuring Bacterial Growth – Enumeration
of Bacteria
–Pour plate method: add 1ml diluted culture from serial dilutions to
melted nutrient agar, mix, then pour in empty plate → agar cools down
→ solidified → incubated → colonies develop within medium & on
medium surface.
• Disadvantage: damage to colonies exposed to heated agar, smaller
colonies inside agar compared to those on surface.

–The spread plate method: 0.1ml sample is placed on the surface of cool
solidified agar medium. The sample is spread evenly → incubate →
colonies on surface.
–Countable no. of colonies /plate (30-300 CFU).
• It is difficult to count more than 300 colonies on one plate whereas less
than 30 is not statistically representative.
 Measuring Bacterial Growth – Enumeration
of Bacteria
• The colonies are counted by the aid of colony counter (magnifying
lens+ special electrical marker).
• Actual no. of colonies = no. of colonies on plate x dilution factor.
• The concentration of bacterial cells in the original suspension
(culture) is calculated from the number of colonies and is
expressed as CFU/ml.
• To improve accuracy: shake tubes before sampling & make several
plates from each dilution (average).
• Weakness of the process;
Doesn't count the cells that died by the time of plating & does not
include m.o. that cannot grow on the utilized growth medium.
Automated colony counter
 Measuring Bacterial Growth – Enumeration
of Bacteria
2.Direct microscopic count

• A known volume of medium is introduced into specially

calibrated etched glass slide called Petroff-Hausser counting
chamber (hemocytometer).
• Cells are then counted, under the microscope, in specific areas
and their number per unit volume is calculated.
• Disadvantages:
1. Cannot distinguish between living & dead cells.
2. Requires large no. of cells.
3. The bacterial suspension should be homogeneous.
 Measuring Bacterial Growth – Enumeration
of Bacteria
3.Most probable number (MPN) method
• Used in case:
a. The sample contains too few organisms to give reliable measure by plate count
method (e.g. food or water purity studies).
b. If m.o. do not grow on agar.
• Concept: as the dilution factor increases, a point is reached where some tubes
contain only one m.o. while others have none.
• Procedures:
a. A series of dilutions is made (10ml, 1ml, 0.1ml of a sample is added to growth
media), for each volume prepare 5 tubes →incubate → observe for growth.
b. Growth indications: turbidity, production of gas or change in color of indicator
(e.g. acid production).
b. Count the no. of tubes showing growth then check MPN index or table which
shows the count of m.o. in the actual culture at 95% confidence level.
 Measuring Bacterial Growth – Enumeration
of Bacteria
4.Filtration method
–A known volume of fluid (i.e. water or air) is drawn through a filter with
pores smaller than bacteria (e.g. 0.45μm) → filter is placed on solid
medium → incubate → count the no. of cells in each plate → calculate
the number of cells per unit volume (e.g. 100 ml or 1 L).

5.Other methods
–The rate at which metabolic products such as gases and/or acids are
formed by a culture reflects the mass of bacteria present.
 Gas production: can be detected by capturing the gas in small inverted
tubes.
 Acid production: by incorporating pH indicators.
 Turbidity.
 McFarland standards

used as a reference to adjust the turbidity of bacterial suspensions so •

that the number of bacteria will be within a given range to standardize
.microbial testing
Original McFarland standards were made by mixing specified amounts •
of barium chloride and sulfuric acid together. Mixing the two compounds
.forms a barium sulfate precipitate, which causes turbidity in the solution
Now there are McFarland standards prepared from suspensions •
of latex particles, which lengthens the shelf life and stability of the
.suspensions
The standard can be compared visually to a suspension of bacteria in •
sterile saline or nutrient broth. If the bacterial suspension is too turbid, it
can be diluted with more diluent. If the suspension is not turbid enough,
 .more bacteria can be added
 McFarland standards
McFarland standard
0.5 1 2 3 4

Approximate Cell
Density x 10 8 /ml 1.5 3 6 9 12

Absorbance
Specification at 0.08
600nm (ABS) 0.257 0.451 0.582 0.669
to 0.1
 Measuring Bacterial Growth – Enumeration of
Bacteria
– By measurements
 Turbidity can be measured by spectrophotometer or
colorimeter: important to monitor rate of growth
without disturbing the culture.
 No. of cells can be determined by dry weight
measurement.
• To calculate the dry weight of cells, they must be
separated from the medium by some physical means
such as filtration or centrifugation.

• The cells are then dried, and the resulting mass is

weighed.
 Factors Affecting Bacterial Growth –
Introduction
•Microorganisms exist almost everywhere on earth because they:
–Are small in size and easily dispersed.
–Occupy little space.
–Need only small quantities of nutrients.
–Are remarkably diverse in their nutritional requirements.
–Have great capacity for adapting to environmental changes.

•However, the type of organisms and their growth rates are

influenced by:
–Physical factors such as pH, temp, O2 concentration, moisture,
hydrostatic pressure, osmotic pressure & radiation.
–Nutritional (biochemical) factors which include the availability
of C, N, S, P, trace elements & vitamins.
 Factors Affecting Bacterial Growth –
Physical Factors
• pH
–Microorganisms usually have optimum pH for their growth.
–According to their tolerance to acidity or alkalinity bacteria are classified as;
1. Acidophiles (pH 0.1-5.4),
2. Neutrophiles (pH 5.4-8),
3. Alkaliphiles (pH 7-11.5).
–Most m.o. (especially pathogens) grow best near neutral pH, i.e.
neutrophiles.
–M.o. usually don't grow well at pH values that are one or more unit above
or below their optimum pH. Because significant changes in pH can lead to
denaturing enzymes & other proteins and interfere with pumping ions at
the cell membrane.
–Organisms that tolerate extreme pH have impervious cell walls that protect
their cell membrane from exposure to extreme pH in the medium and
keeps the inside of the cell as neutral.
 Factors Affecting Bacterial Growth – Physical
Factors

• Acidophiles : Grow best at a pH of 0.1 to 5.4.

• e.g.
– Lactobacillus, which produces lactic acid, but
tolerates only mild acidity.

– Some bacteria that oxidize sulfur to sulfuric acid

(can create and tolerate conditions as low as pH
1.0).
 Factors Affecting Bacterial Growth – Physical
Factors

• Neutrophiles:
Exist from pH 5.4 to 8.0.
Most of the bacteria that cause disease in humans
are neutrophiles.
• Alkaliphiles:
Exist from pH 7.0 to 11.5.
e.g.
Vibrio cholerae (Asiatic cholera) & Alcaligenes
faecalis grow at pH 9.0.
 Agrobacterium grows in alkaline soil of pH 12.0.
 Factors Affecting Bacterial Growth –
Physical Factors
• Temperature
–Most bacterial species grow over 30 °C, but min &
max temp. vary.
–Bacteria are classified to psychrophiles, mesophiles
and thermophiles.
• Within these groups, bacteria are further classified
as:
 Obligate: Means that the organism must have the
specified environmental condition.
Facultative: Means that the organism is able to adjust to
and tolerate the environmental condition, but it can also
live in other conditions.
 Factors Affecting Bacterial Growth – Physical
Factors
Temperature
–Psychrophiles (cold loving): optimal 15-20 °C, some live at 0
°C.
 Obligate psychrophiles: cannot grow over 20 °C, such as
Bacillus globisporus.
 Facultative psychrophiles: optimum below 20 °C but also can
grow above 20 °C, such as Xanthomonas pharmicola.
 Psychrophile usually live in cold water & soil but not in
humans.
 None can live in the human body, except some, such as
Listeria monocytogenes.
 Those are known to cause spoilage of refrigerated foods,
and subsequent disease in humans, sometimes fatal.
 Factors Affecting Bacterial Growth –
Physical Factors
• Temperature

–Mesophiles: optimum 25-40 °C,

• Includes most bacteria & human pathogens.
• Most human pathogens live best at 37 °C.

–Thermophiles (heat loving): optimum 50-60 °C

 Few tolerate temp up to 110 °C in boiling hot springs.
 Obligate thermophiles: can grow only at temp above 37 °C.
 Facultative thermophiles: live at both below & above 37 °C.
 Extreme thermophiles: require very high temp (80°C to 105°C).
–What determines the temp at which m.o. survive is the temp at
which its enzymes function.
 Factors Affecting Bacterial Growth – Physical
Factors
Temperature

• Thermoduric organisms ordinarily live as mesophiles

but can withstand short periods of exposure to high
temperatures.
• Species of bacteria which are thermoduric include
Bacillus, Clostridium and Enterococci.
• Inadequate heating during canning or in
pasteurization
may leave such organisms alive and therefore able to
spoil food.
 Factors Affecting Bacterial Growth –
Physical Factors
• Temperature

–Minimum growth temp: lowest temp at which cells divide.

–Maximum growth temp: highest temp at which cells divide.
–Optimum growth temp: temp at which cells divide most
rapidly (have shortest generation time).
–Enzyme activity usually doubles for every 10 °C rise in temp
until high temp denatures the enzyme.
––Bacteria can survive extremes of cold but not extremes of
heat; because enzymes are denatured by extremes of heat
but not by extremes of cold.
 Factors Affecting Bacterial Growth – Physical
Factors
• Temperature

• Regardless of the type of bacteria, growth gradually

increases from the minimum to the optimum
temperature and decreases very sharply from the
optimum to the maximum temperature.

• Furthermore, the optimum temperature is often very

near the maximum temperature.

• These growth properties are due to changes in enzyme

activity.
 Factors Affecting Bacterial Growth – Physical
Factors
Temperature
• Temperature is important not only in providing
conditions for microbial growth, but also in
preventing such growth by:

1. Refrigeration of food, at 4oC, to prevent the growth

of most bacteria.
2. Freezing; being stored at temperatures of -20oC if
they are to be kept for long periods of time.
 Factors Affecting Bacterial Growth –
Physical Factors
• Oxygen

• In general, bacteria can be divided into aerobes (require O2 to

grow) and anaerobes (don’t require O2 to grow).
• Obligate aerobes: must have free O2 for aerobic respiration (e.g.
Mycobacterium tuberculosis).
• Microaerophiles: grow best in presence of little O2, typically 2–
10% O2 (e.g. Helicobacter pylori). Some are also capnophiles which
grow best in presence of high conc. of CO2 (e.g. Campylobacter
spp.). They grow below the surface of the medium in a culture
tube at the level where oxygen availability matches their needs.
• Obligate anaerobes: produce energy by anaerobic respiration or
fermentation and they are killed by free O2 (e.g. Clostridium spp.
such as C. botulinum and C. tetani).
 Factors Affecting Bacterial Growth – Physical
Factors
Oxygen
• Facultative anaerobes: ordinarily carry on aerobic metabolism
when O2 is present but shift to anaerobic metabolism when O2 is
absent (e.g. E. coli and Staphylococcus spp.).
• They have the most complex enzyme systems???
• They have two sets of enzymes that enables them to use oxygen as
an electron acceptor and another molecule as an electron
acceptor when oxygen is not available.

• In contrast, other groups have one set which is limited to either

aerobic or anaerobic respiration.

• Aerotolerant anaerobes: can survive in presence of O2 but do not

use it in their metabolism (e.g. Propionibacterium acnes).
 Factors Affecting Bacterial Growth –
Physical Factors
• Oxygen

• In presence of free oxygen, a toxic form of oxygen called superoxide

(O2-) can be produced in living cells by some oxidative enzymes.
• O2- is a highly reactive oxygen species (ROS) that can damage the
cells causing their death.
• Superoxide can be metabolized into molecular oxygen and hydrogen
peroxide (H2O2) by an enzyme called superoxide dismutase (SOD).
• Although it is weaker than O2-, hydrogen peroxide is still considered
as a ROS and may cause cellular damage.
• Hydrogen peroxide can also be metabolized into molecular oxygen
and water by an enzyme called catalase (Cat).
• 2H2O2 (aq) → 2H2O (l) + O2 (g)
 Factors Affecting Bacterial Growth – Physical
Factors

• Oxygen

 Obligate aerobes & most facultative anaerobes have

both SOD and Cat.
 Obligate anaerobes have neither SOD nor Cat.
 Aerotolerant & some facultative anaerobes have SOD
but not Cat.
 Factors Affecting Bacterial Growth – Physical
Factors
• Oxygen

• For aerobes, oxygen is often the environmental factor that

limits growth rate.

• There are various methods employed to maintain a high O

concentration in cultures, by bubbling air through a culture.

• This is especially important in such commercial processes as the

production of antibiotics and in sewage treatment using
bacteria.
 Classification of microbes according to oxygen requirements

Obligate Facultative Obligate Aerotolerent Micro-

aerobes anaerobes anaerobes anaerobes aerophiles
Factors Affecting Bacterial Growth – Physical
Factors
• Moisture
–Presence of enough available water is important for the growth of
bacteria.
–Most vegetative bacteria can live only few hours without moisture
while spores can exist in dormant state in dry environment.
–Halophilic bacteria can tolerate lower water levels.

• Radiation
–Gamma rays & UV usually cause mutational changes in DNA & kill the
m.o.
–Some m.o. have pigments that protect them from radiation while
others have enzymes that repair certain DNA damage.
 Factors Affecting Bacterial Growth – Physical
Factors
• Radiation

• The bacterium, Deinococcus radiodurans can survive 10,000 Grays (Gy)

of radiation.
• The Gy is a unit of measurement for absorbed dose of radiation.
• 5 Gy will kill a human, and 1,000 Gy will sterilize a culture of E. coli.
• Bacteria that can withstand high levels of radiation may be valuable for
use in cleaning up contaminated sites (nuclear energy waste).
• Deinococcus radiodurans is an extremophilic bacterium, one of the
most radiation-resistant organisms known. It can survive cold,
dehydration, vacuum, and acid, and is therefore known as
a polyextremophile and has been listed as the world's toughest
bacterium in The Guinness Book Of World Records.
 Factors Affecting Bacterial Growth – Physical
Factors
• Hydrostatic pressure

–Pressure exerted by standing water in proportion to its depth (10 m increase in

depth doubles atmospheric pressure).
–Barophiles (or piezophiles): bacteria that live at high pressure & die at
atmospheric pressure. Have a high proportion of fatty acids in their
cytoplasmic membrane, which allows membranes to remain functional and
keep from gelling at high pressures.
• Their membranes and enzymes do not simply tolerate pressure but require
pressure to function properly.
• Pressure is needed to maintain their enzymes & membrane in the proper 3D-
configuration. Without it, the enzymes lose their shape and denature, and the
organisms die.
• E.g. Salinicola salarius.
Factors Affecting Bacterial Growth – Physical
Factors
• Osmotic pressure

–Hyperosmotic (hypertonic) environment usually causes the cells to shrink

(plasmolysis or crenation).
–Hypoosmotic environment causes the cells to gain water & become highly turgid
(distended).
• but usually protected from bursting by cell wall.
• Most bacterial cells can tolerate a fairly wide range of concentrations of
dissolved substances.
• Their cell membranes contain transport systems that regulate the movement of
dissolved substances across the membrane.
–High osmotic pressure (high salt conc.) → water loss → can inhibit growth or kill
the bacterial cells.
that’s why high concentrations of salt or sugar can be used to preserve food.
 Factors Affecting Bacterial Growth – Physical
Factors
• Osmotic pressure

• Halophiles: Salt-loving organisms which require moderate to high

concentration of salt (sodium chloride) to grow well.
• Their membrane transport systems actively transport sodium ions
out of the cells and concentrate potassium ions inside them.
• Halophiles require sodium chloride (salt) for growth, in contrast to
halotolerant organisms, which do not require salt but can grow
under saline conditions.
• Halophiles are typically found in the ocean, where the salt
concentration (3.5%) is optimum for their growth (e.g.
Halobacterium).
 Factors Affecting Bacterial Growth –
Nutritional Factors
Nutrients needed by m.o. include carbon, nitrogen, sulphur, phosphorous, certain-
.elements & vitamins
Few m.o. are fastidious, i.e. have special nutritional needs that can be difficult to meet in-
.the lab
Some fastidious pathogens grow well in the human body but are difficult to grow on
.nutrient medium

Carbon sources•
:Carbon containing compounds are used by bacteria as–
.Energy source .1
.Building blocks to synthesize cell components .2
.Heterotrophs: obtain C & energy from organic compounds e.g. glucose–
Autotrophs: obtain C from CO2. Derive energy by oxidation of inorganic compounds, or–
.from light (photoautotroph)
.Photoautotrophs: convert CO2 to glucose & other organic molecules (photosynthesis) –
 Factors Affecting Bacterial Growth –
Nutritional Factors
Nitrogen sources•

.All organisms need N2 to synthesize enzymes, other proteins & nucleic acids–
Some m.o. obtain N2 from inorganic sources and few m.o. even obtain–
.energy by metabolizing inorganic N-containing compounds
Many m.o. reduce nitrate (NO3-) to amino (-NH2) and then use it to make–
.amino acids
Some m.o can synthesize all 20 amino acids found in proteins while others–
.must have few amino acids provided in the medium
Purines (adenine and guanine) & pyrimidines (cytosine, thymine and uracil)–
.are used to build DNA, RNA
The processes by which proteins and nucleic acids are synthesized are –
.directly related to the genetic information contained in the cell
 Factors Affecting Bacterial Growth –
Nutritional Factors
Sulfur and Phosphorous (S & P) •

.They are important cell components–

Sulfur is obtained from inorganic sulfate salts & from S- –
.containing amino acids (cysteine and methionine)
Sulfur is used for the synthesis of coenzymes, proteins & –
.other cell components
Phosphorous is mainly obtained from inorganic –
.phosphate ions (PO43-)
.Used to synthesize ATP, phospholipids & nucleic acids –
 Factors Affecting Bacterial Growth –
Nutritional Factors
Trace elements•

Microorganisms need a variety of trace elements; (minute–

.amount) of minerals such as copper, iron, zinc and cobalt
.Trace elements usually serve as cofactors in enzymatic reactions–
.Cobalt is required by m.o. that synthesize vitamin B12–
.Na, Cl are required by all m.o–
.K, Zn, Mg & Mn are used to activate certain enzymes–
Ca is required by G+ve bacteria for the synthesis of cell walls &–
.by spore-forming bacteria for spore synthesis
 Factors Affecting Bacterial Growth –
Nutritional Factors
Vitamins•

Are organic substances that an organism require in small–

.amounts & that are used as coenzymes
.e.g. vitamin B12, folic acid (vitamin B9) and vitamin K
.Many m.o. make their own vitamins, others need it in nutrients–
Human pathogens often require variety of vitamins; thus grow–
well in host but in the lab they require complex medium that
.contains all nutrients they obtain from host
.Microbes in intestines make some vitamins (B & K)–
 Factors Affecting Bacterial Growth –
Nutritional Factors
Nutritional complexity•

.It is the no. of nutrients an organism must obtain to grow–

Nutritional complexity is determined by the kind & no. of organism’s–
.enzymes
Absence of certain enzyme means that the organism cannot –
synthesize specific substance & this substance must be obtained from
nutrients; i.e. nutritional complexity reflects deficiency in biosynthetic
.enzymes
.So m.o. with many enzymes → need simple nutrients –
While m.o with fewer enzymes → need complex nutritional –
.requirements
 Factors Affecting Bacterial Growth –
Nutritional Factors
Locations of enzymes•

Most m.o. have endoenzymes: i.e. used within cells for metabolism & transportation of –
.molecules
.Many bacteria & fungi have exoenzymes: i.e. released through the cell membrane –
:Exoenzymes are divided into–
Extracellular enzymes: produced by G+ve rods & act in the medium around the .1
.organism
.Periplasmic enzymes: produced by G-ve & act in periplasmic space .2
Most exoenzymes are hydrolases (they add water as they split molecules); e.g.–
.carbohydrases, amylases, proteases, lactases, lipases, sucrases
Although microbes cannot move large molecules across membranes, in nature they –
use large molecules from other organisms by digesting those molecules with
.exoenzymes before absorbing them
 Factors Affecting Bacterial Growth –
Nutritional Factors
Adaptation to limited nutrients•

:Microorganisms adapt to nutrient limitation by–

Synthesizing increased amounts of enzymes (upregulation).1
.for uptake & metabolism of the limited nutrients
Synthesizing enzymes to use different nutrients e.g. if low.2
.glucose → synthesize enzymes to use lactose (if present)
Adjust rate of growth & metabolism to fit the available.3
nutrients i.e. slow down & no energy is wasted on
.synthesizing products that are not used
 Sporulation

Sporulation or Sporogensis is the formation of endospores which usually•

.occurs in Bacillus, Clostridium and a few other G+ve genera
Endospores are formed inside the bacterial cell & generally produced in•
stationary phase in response to environmental, metabolic & cell signals
.(endospores are considered as insurance from extinction)
.While fungal spores are produced in large no. & are a form of reproduction–
When nutrients (C, N) are limited → resistant endospores form inside•
mother cells (very few exceptions, endospores form even when nutrients are
.available)
Although endospores are metabolically inactive, they can survive long•
periods of drought, resist killing by extreme temp, radiation and toxic
.chemicals
 Sporulation

They cannot divide; a parent cell produces only •

.one endospore
i.e. sporulation is protective or survival mechanism–
.not means of reproduction
• Endospore size may be the same as, smaller than,
or larger than the size of the vegetative cell.
• Depending on the species, endospore might be
located terminally, sub-terminally, or centrally
inside the vegetative cell.
 Sporulation

Endospore formation•

.DNA replicates & forms long compact axial nucleoid–

The 2 chromosomes formed by replication separate & move to different –
.locations in the cell
.Endospore form either in the middle or at one end –
.DNA where endospore is forming directs the process –
RNA & proteins gather around DNA to make the core (living part of–
.endospore)
The core has dipicolinic acid & Ca2+ ions (responsible for heat resistance by –
.stabilizing protein structures)
Endospore septum then grows around the core: double cell membrane but–
.lacking a cell wall
 Sporulation

Endospore formation•

Both layers of this membrane release peptidoglycan into the–

.space between them
This forms a laminated layer called cortex which protects the –
.core from osmotic pressure like that resulting from drying
Spore coat of keratin-like protein is then layered around the–
.cortex (to protect against chemicals)
In some endospores, an exosporium (a lipid-protein–
.membrane) is formed outside the coat (unknown function)
.Under lab. conditions, sporulation takes about 7 hours–
The vegetative and sporulation cycles
 Sporulation

If favorable conditions return, germination occurs; i.e. the spore•

.returns to its vegetative state & loses its resistance

Germination stages•
Germination is initiated by activation step which requires some traumatic–
.agent (e.g. low pH, heat) to damage the coat, otherwise germination is slow
:Germination’ then occurs‘–
.Water penetrates the damaged coat.1
.Cortex (peptidoglycan) is broken down.2
The living cell (inside the core) takes water & loses its resistance to heat &.3
.staining
Outgrowth finally occurs; proteins & RNA are synthesized, then DNA synthesis –
.occurs, the cell becomes vegetative & undergoes binary fission
 Other Sporelike Bacterial Structures

1. Cysts:
• Certain bacteria, such as Azotobacter, form resistant
cysts, or spherical, thick-walled cells, that resemble
endospores.
• Like endospores, cysts are metabolically inactive
and resist drying. Unlike endospores, they lack
dipicolinic acid and have only limited resistance to high
temperatures.
• Cysts germinate into single cells and therefore are not
a means of reproduction.
 Other Sporelike Bacterial Structures

2. Conidia:
• Some filamentous bacteria, such as Micromonospora and
Streptomyces, form asexually reproduced conidia, or chains
of aerial spores with thick outer walls.
• These spores are temporarily dormant but are not especially
resistant to heat or drying.
• When the spores, which are produced in large numbers, are
dispersed to a suitable environment, they form new
filaments.
• Unlike endospores, these spores contribute to reproduction
of the species.
 Culturing Bacteria - Introduction

To study bacteria, it is important to obtain pure culture (i.e. a •

.culture that contains only a single species of organisms)
Pure culture is necessary to study nutritional needs, growth •
characteristics, pathogenicity and antimicrobial susceptibility of
.individual spp
.Pure cultures are usually obtained using ‘solid’ growth media •
Agar is an ideal solidifying agent for microbiological media, •
?why
It doesn’t melt below 95°C, and after melting it solidifies at –
.~40°C (hysteresis)
.Inert substance: only very few organisms can digest it –
 Culturing Bacteria – Methods of Obtaining
Pure Cultures
The streak plate method•

:Procedures–
.Pick bacteria on sterile wire loop.1
.Move the wire along the agar surface depositing streaks of bacteria on surface.2
.Loop is flamed.3
.Pick bacteria from the bacteria deposited on agar & streak new regions on agar .4
…Flame & repeat.5
Individual organisms are deposited in the region streaked last (i.e. after –
.incubation, isolated colonies usually appear on agar surface in that region)
isolated colonies, that represent an individual m.o., can then be picked up and –
.transferred to fresh medium for further studying
The streak plate method
Culturing Bacteria – Methods of Obtaining Pure
Cultures
The pour plate method•

Makes use of serial dilutions so that the final dilution contains–

.about 1000 organism
1ml of this dilution is then placed in 9ml of melted agar medium–
.(at 45°C) & the medium is quickly poured into a sterile plate
The resulting plate will contain small no. of bacteria some of–
.which will form isolated colonies on the agar
Since some m.o. are embedded in agar medium, this method is –
useful for growing microaerophiles that cannot tolerate exposure
.to atmospheric levels of oxygen
The pour plate method
 Culturing Bacteria - Culture Media

Growing bacteria in the lab requires knowledge of•

their nutritional needs & the ability to provide these
.substances in a medium

Although many bacteria can be grown in the lab•

nowadays, some m.o., such as those causing syphilis &
leprosy, still cannot be cultured in lab media but rather
.need cultures containing living human or animal cells
 Culturing Bacteria - Culture Media

Requirements of growth media

Energy source.1
Protein source.2
Buffer system.3
Defined salt concentration.4
 Culturing Bacteria - Culture Media

Types of media•

Lab medium is a synthetic medium prepared from materials of precise or –

.reasonably well-defined composition
Defined synthetic medium: synthetic medium that contains specific kind &–
.amount of chemical substances
Complex medium (chemically non-defined medium): contains reasonably –
.familiar materials but varies slightly in chemical composition from batch to batch
Complex media may contain peptone, blood or extracts from beef, yeasts,
.soybean, etc
Peptone: a product of enzymatic digestion of proteins (from meat or fish) that
.provides small peptides that m.o. can use
.Both liquid nutrient broth & solid agar medium are used to culture bacteria –
 Culturing Bacteria - Culture Media

:Commonly used media•

Most routine lab culture media contain peptone, such media can be–
:enriched by
Yeast extract: contains a number of vitamins, coenzymes and.1
.nucleosides
Casein hydrolysate: made from milk protein and contains many.2
.amino acids
Blood (or serum): contains many nutrients needed by fastidious.3
.pathogens
Blood agar (usually sheep’s blood) is also used to identify m.o. that
.cause hemolysis ( β-hemolytic streptococcus)
 Culturing Bacteria - Culture Media

:Selective, differential and enrichment media•

:These media are very important in diagnostic medicine

Selective medium: it encourages the growth of some m.o. but suppresses–
.the growth of others
e.g. an antibiotic can be added to the growth medium so as only m.o. that
.are resistant to this antibiotic can grow
Differential medium (indicator media): has an indicator constituent that–
causes an observable change (color change or pH change) in the medium
.when a biochemical reaction, that is characteristic to a certain m.o., occurs
This will allow to distinguish a certain type of m.o. (colony) from others
.growing on the same plate
.E.g. blood agar can be used to distinguish hemolytic bacteria
 Culturing Bacteria - Culture Media

:Selective, differential and enrichment media•

Some media can be selective and differential at the same time,–
:examples

:MacConkey Agar
It has crystal violet & bile salts which inhibit the growth of G+ve –
.bacteria but allows the growth of G-ve ones → selective
It also has sugar lactose and pH indicator that turns colonies of –
lactose-fermenters (Lac+) into red colonies & the non-lactose-
.fermenters (Lac-) into colorless colonies → differential
E.g. it can be used to differentiate between E. coli (Lac+) and
Salmonella (Lac-)
 Culturing Bacteria - Culture Media

:Sulfite Polymyxin Sulfadiazine (SPS) Agar

.Used for the detection of Clostridium botulinum –

The two antibiotics inhibit the growth of most m.o. –

.other than Clostridium spp. → selective

Sulfite is reduced by Clostridium botulinum to sulfide –

.which is a black iron sulfide precipitate → differential
 Culturing Bacteria - Culture Media

SPS agar plate showing black Clostridium MacConkey agar with Lac+ colonies (left) and
colonies Lac- colonies (right)
 Culturing Bacteria - Culture Media

Mannitol Salt Agar (MSA)

the differential ingredient in MSA is the sugar mannitol. Organisms
capable of using mannitol as a food source will produce acidic by
products of fermentation that will lower the pH of the media. The
acidity of the media will cause the pH indicator, phenol red, to turn
yellow. Staphylococcus aureus is capable of fermenting mannitol
.while Staphylococcus epidermidis is not (differential)
The high concentration of salt (7.5%) selects for members of the
genus Staphylococcus, since they can tolerate high saline levels.
Organisms from other genera may grow, but they typically grow
.very weakly (selective)
 Culturing Bacteria - Culture Media

:Selective, differential and enrichment media•

Enrichment medium: it contains special nutrients that allow the–

growth of particular m.o. that might not otherwise be present in
.sufficient numbers to allow it to be isolated and identified
e.g. Salmonella typhi may be in very small no. in fecal samples, so it is
cultured on a medium containing the trace element selenium which
.supports the growth of this m.o
.Unlike selective medium, it doesn’t suppress the growth of other m.o –
Blood agar and chocolate (heat-treated blood) agar are also –
considered as enrichment media and are frequently used to grow
.fastidious m.o
 Culturing Bacteria – Controlling Oxygen
Content of Media
:Obligate aerobes•
Usually obtain O2 from nutrient broth or on the surface of solidified–
.agar, but some may need more
So O2 can bubbled through medium (with filters to prevent–
.contamination)

:Microaerophiles•
A broth tube or agar plate can be incubated in an airtight jar in which–
.a candle is lit before the jar is sealed
Burning candle uses O2 & adds CO2, when the candle extinguishes →–
.suitable conditions (old technique)
 Culturing Bacteria – Controlling Oxygen
Content of Media
:Obligate anaerobes•

.All molecular O2 must be removed–

Addition of oxygen-binding agents like thioglycolate, cysteine (amino acid)–
.or sodium sulfide, prevent O2 from exerting its toxic effects on anaerobes
If the culture is in plates, special jars are used where special bags–
.containing a chemical substance are placed to remove O2 & generate CO2
Stab cultures: a culture of anaerobic bacteria can be made quickly by–
stabbing a straight wire coated with m.o. into a tube of agar-solidified
.medium
Using anaerobic cabinet/ chamber (atmosphere control units designed to –
be used when working with oxygen sensitive materials, product
.containment needs, and/or general isolation control)
 Culturing Bacteria – Maintaining Cultures
:Stock culture•

.A pure culture prepared and stored for future usage–

It is not used in the experiments by itself but rather it can be used to prepare–
.subcultures (by inoculating fresh medium)
:Preparation
add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw.1
.top tube and gently mix
Freeze the glycerol stock tube at -80°C. The stock is now stable for years, as.2
.long as it is kept at -80°C. Subsequent freeze and thaw cycles reduce shelf life
To recover bacteria from your glycerol stock, open the tube and use a sterile.3
loop, to scrape some of the frozen bacteria off of the top. Do not let the
.glycerol stock unthaw
 Culturing Bacteria – Maintaining Cultures

Because organisms in stock cultures go through –

growth phases, deplete nutrients, and accumulate
wastes just as those in any culture do. As the
culture ages, the organisms may acquire odd
.shapes or other altered characteristics

Bead stocks: porous beads, which are intended as –

carriers to support the viability of microorganisms
.during storage in the freezer
Bead stocks for bacterial preservation
 Culturing Bacteria – Maintaining Cultures

:Aseptic techniques •
Taking precautions & measures to prevent contamination of and from the bacterial–
.cultures

:Preserved culture•
.A culture in which organisms are maintained in dormant state–
.To avoid the risk of contamination and to reduce the mutation rate in stock cultures –
e.g. lyophilization (freeze-drying): freezing with vacuum to dry the culture, placed in vials–
.which can be stored at room temp for long periods

:Reference culture (microbial standards)•

A preserved culture that maintains the m.o. with the characteristics as originally defined–
:(these m.o. are usually well characterized), examples
ATCC: American Type Culture Collection (USA)–
NCTC: National Collection of Type Cultures (UK)–
 Culturing Bacteria – Biochemical profiling –
metabolic profiling
:Biochemical tests determine ability to •
Ferment/oxidise sugars.1
Digest a substrate – gelatin.2
Produce a product (H2S) or reduce a target (nitrate).3
Use a substrate as a sole carbon source (citrate).3
Deaminate amino acids.4
Culturing Bacteria – Methods of Performing
Multiple Diagnostic Tests
Many kits use culture systems that contain a large no. of differential & •
.selective media to identify different m.o
.e.g. Analytical Profile Index (API) and Enterotube Multi-test System –
Advantages: use small amount of media, occupy little space in an –
incubator, efficient & reliable means of identifying infectious organisms
:API kit•
.Plastic tray with 20 micro-tubes containing different dehydrated media –
The micro-tubes are rehydrated & inoculated with bacterial suspension –
from an isolated colony → incubate → write the no. → check the list for
.identification
.Other tests depend on immunological properties of the m.o •
 ®The Enterotube System

• The Enterotube System® is used to identify enteric

pathogens, or organisms that cause intestinal diseases
such as typhoid and paratyphoid fevers, shigellosis,
gastroenteritis, and some kinds of food poisoning.
• The causative organisms are all Gram-negative rods
indistinguishable from one another without
biochemical tests.
• The Enterotube System® consists of a tube with
compartments, each of which contains one or more
different media, and a sterile inoculating rod.
 ®The Enterotube System

• Each compartment is inoculated when the tip

of the rod is touched to a colony and the rod is
drawn through the tube.
• After the tube has been incubated for 24
hours at 37oC, the results of 15 biochemical
tests can be obtained by observing:
whether gas was produced and )1(
.the color of the medium in each compartment )2(
  Viable but non-culturable (VBNC) bacteria
Refers to bacteria that are in a state of very low metabolic activity •
and do not divide, but are alive and have the ability to become
.culturable once resuscitated
Bacteria in a VBNC state cannot grow on standard growth •
media. Bacteria can enter the VBNC state as a response to stress,
due to adverse nutrient, temperature, osmotic, oxygen,
.and light conditions
The cells that are in the VBNC state are morphologically smaller, •
and demonstrate reduced nutrient transport, rate of respiration,
.and synthesis of macromolecules
Sometimes, VBNC bacteria can remain in that state for over a •
.year