Production of
Bacteria
Dr. Mayram Hacıoğlu
[email protected]
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Reproduction of bacteria
• Reproduce asexually by binary fission.
• Binary fission involves the division of a single cell,
which results in the formation of two cells that are
genetically identical.
• In binary fission,
the cell elongates,
replicates its chromosome,
separates the newly formed DNA molecules
so there is one chromosome in each half of the cell.
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• Reproduction in bacterial
cell divide equally into two,
following replication
of DNA.
• The cell wall and cytoplasm
also split resulting in the
formation of two daughter
cells.
• Daughter cells are
genetically identical to the
parent cell because they
contain the same number
and type of chromosomes.
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• The generation time is the time taken for the
number of bacteria to double
• The speed of bacteria division depends on the type
of bacteria and the environment of reproduction.
For example; E.coli division time 20 minutes
M.tuberculosis is 14-15 hours.
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• medium: any solid or liquid specially
prepared for bacterial growth
• culture: a liquid or solid medium containing
bacteria which have grown (or are growing)
in or on that medium
• incubation: the process of maintaining a
particular temperature (and/or other
desirable conditions) for bacterial growth
• inoculation: the initial process of adding the
cells to the medium
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Bacterial Growth Curve
• The bacterial growth curve represents the
number of live cells in a bacterial
population over a period of time.
• Suitable liquid medium
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1. Lag phase
No appreciable increase in number
May be an increase in the size of the cells
Adobtion to new environment
2. Log or exponential phase
Increasing number of cells exponentially
Cells are smaller and stained uniformly
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3. Stationary phase
Equal number of dead and viable cells
Show irregular staining
Secondary metabolic product producing
4. Death phase
Lack of nutrients, production of toxic products,
decreased cell number
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Necessary Materials for Nutrition and
Reproduction of Bacteria
1. Water
2. Carbon Source
3. Nitrogen Source
4. Minerals
5. Growth Factor and Vitamins
7. Oxygen
8. Carbon dioxide
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1. Water
• It constitutes 70-90% of bacterial cells. Bacteria can not
reproduce in the absence of water
• Drying is lethal to cells.
2. Carbon Source
• It is necessary for the synthesis of main structures of the
bacterial cell.
• CO2, carbonates and organic sources are used for the
carbons in the structure of polysaccharides, lipids and
proteins.
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3. Nitrogen Source
• It enters the protein structure of the bacteria and is also
found in the structure of the nucleic acids, purine,
primidine and various enzymes.
• Ammonium salts, nitrite, nitrate and amino acids are used
as nitrogen sources.
4. Minerals
• Minerals are found in the structure of microorganisms
and enzymes.
• The most important minerals required for microorganisms
are sulfur, phosphorus, magnesium, calcium, iron and
potassium.
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5. Growth factors and vitamins
• A growth factor is an organic compound which a cell
must contain in order to grow but which it is unable to
synthesize.
• These substances are essential for the organism and are
to be supplied as nutrients.
• Thiamine, nicotinic acid, folic acid and para-
aminobenzoic acid are examples of growth factors
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Effective environmental factors of
bacteria reproduction
1- Temperature 1- Temperature
2- pH * Psychrophiles
3- Osmotic Pressure * Mesophiles
4- Water * Thermophiles and Extreme
Thermophiles
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Psychrophiles
• Optimal growth below 10°C
• Can grow below 0°C (if liquid water is available)
• Found in Artic, Antarctic and ocean environments
• Can be found in refrigerators
• Cause food spoilage
Eg: Vibrio marinus
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Mesophiles
• Grow between 20-40°C
• Many have optimal temperatures of 37°C
• Normal body flora and most pathogens are mesophiles
• Grow rapidly and optimally in the human body
Eg. E. coli
Thermophilic bacteria
• Optimum growth temperature is >45°C
• Occur in compost piles, hot springs and ocean floor
hydrothermal vents
• Streptococcus thermophilus
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2- pH
Bacteria exhibit various tolerances to pH
• Affects the ionic properties of bacterial cell so it affects
the growth of bacteria.
• Most grow in a range between 6.0 and 9.0
• Most medically important bacteria grow at neutral or
slightly alkaline pH (7.2 to 7.6)
• Few bacteria grow below pH 4
• Lactobacilli grow in acidic pH;
Vibrio cholera grow in alkaline pH
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3- Osmotic pressure
• Bacteria are tolerant to osmotic variations because of the
mechanical strength of the cell wall
• High osmotic pressure (hypertonic) removes water
causing plasmolysis
• Low osmotic pressures (hypotonic) cause water to enter
and can cause lysis
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4-Water
• For microorganisms to grow, water must not only be
present, it must also be accessiable
• The determine the availability of an environment,
researchers may calculate the water activity of an
environment (aw)
• Most bacteria require an aw greater than 0.9
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Production of Bacteria
1- In vivo: Production of
bacteria inside the living
cells
2- In vitro: Production of
bacteria outside the living
cells
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1- In vivo
1) Experimental animals: Guinea pig, rat, hamster, rabbit
and monkey.
Some experimental animals are susceptible to some
microorganisms.
Pneumococci grow in the dormouse. The material is injected
into the dormouse. The animal dies 24-36 hours.
Pneumococci are isolated from heart blood as pure culture
2) Eggs with embryo: yellow pouch, amniotic membrane,
allantoic space, embryo.
3) Cell cultures: Hep-2, HeLa, Vero
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2. In-vitro
Media
• Culture media are employed in the isolation and
maintenance of pure cultures of bacteria and are also used
for identification of bacteria according to their
biochemical and physiological properties.
• The media should be selected according to characteristics
of the microorganism
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• Liquid or solid medium containing bacteria which have
grown (or are growing) in or on that medium is
culture.
• A culture is considered a pure culture if only one type
of organism is present and a mixed culture if
populations of different organisms are present.
• A colony is the only way to see microorganisms with
the unaided eye and it should be remembered that
colonies are large groups of microbes, not individuals.
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Preparation and storage of media
1- Rehydrate the powder form of the medium
2- Distribute the medium into tubes or bottles
3- Autoclave to sterilize the media
4- Keep in the incubator at 370C for a sterility control
5- Put in the refrigerator and stored there until it is used.
An autoclave is a pressure device
used to perform sterilization.
Autoclaves sterilize materials by using
pressure, temperature and steam.
120°C, 1 atm pressure, 15 minutes
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Classification of media
According to physical properties
* Solid media
* Liquid media
Buyyon, broth
* Semi-solid media
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• 2.0-3.0% (w / v) agar is added to the liquid media to
obtain a solid medium.
• Jelly-like substance, obtained from red algae
• Agar is an ideal material used for solidification of media.
• Agar melts at 100 °C,
• Solidifies at about 40-42°C (solid at 37 ° C)
• It does not have nutritional value
• No metabolize during the growth of microorganisms
• No change the pH value of the environment.
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Fanny Hesse
• Walther Hesse
• Together they were instrumental in developing agar as a medium for
culturing microorganisms
• Hesse worked in an unpaid capacity to assist her husband through
preparing bacterial growth media, cleaning equipment and
producing illustrations for publications. Hesse suggested that agar
was preferable to gelatin for cultivating bacteria. This led to Koch
using agar to cultivate the bacteria that cause tuberculosis.
• Although Koch, in an 1882 paper on tuberculosis bacilli, mentioned
he used agar instead of gelatin, he did not credit either Hesse, or
mention why he made the switch. Hesse's suggestion never resulted
in financial benefit for the Hesse family.
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1. Solid media
• Used widely for the isolation of pure cultures
• For estimating viable bacterial populations
• Determining colony formation
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2. Liquid media
• Growth, turbity
• Buyyon, broth
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3. Semi-solid media
• Semi-solid media is prepared by adding 0.3-0.5%
agar to the liquid media.
• Craigie is a semi-solid medium containing 0.4% agar
used for the movement examination of bacteria.
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Reproduction characteristics in solid media
• Colony size
• Colony format
• Colony structure
• Colony color
• Colony smell
• Hemolytic activity
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Colony types of bacteria
• S (smooth) colonies
• R (rough) colonies
• M (mucoid) colonies
• L colonies
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S (smooth) colonies R (rough) colonies
Moist and homogeneous Its surface is wrinkled or
colonies with round, flat granular, its edges are
edges, slightly raised, indented, flattened and flat.
smooth surfaces Ex: Mycobacterium
Ex: Staphylococcus aureus tuberculosis
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M (mucoid) colonies • Bacteria that lose their
capsules under inappropriate
• Smooth, sticky, bright conditions form the S type
colonies colony.
• The encapsulated bacteria • However, under appropriate
are form sticky mucoid conditions are again form M
colonies. colonies.
• Ex: Klebsiella pneumoniae
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L colonies
• They are very small,
colonies that look like nail
elongated into media
• Colonies of the L form
bacteria
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Reproduction characteristics in fluid media
1. Ring or pellicle formation on the surface
2. Flocculent (small parts)
3. Turbidity
4. Sediment
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Hemolysis of Bacteria
• Hemolysis is the breakdown of red blood cells.
• A substance that causes hemolysis is a hemolysin.
• Blood agar is used to induce the hemolysis.
• This is particularly useful in classifying streptococcal
species
Blood agar plates; contain mammalian
blood (usually sheep or horse), typically
at a concentration of 5-10%.
Enriched, differential media used to
isolate fastidious organisms and detect
hemolytic activity
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Brown (1919) introduced three terms alpha, beta and
gamma to indicate three types of streptococci based on
haemolytic reactions observed on blood agar plates.
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Alpha-hemolysis (α-hemolysis)
• α-hemolysis, is a partial, “green” hemolysis associated
with reduction of red cell hemoglobin.
• Caused by hydrogen peroxide produced by the bacteria,
oxidizing hemoglobin to green methemoglobin.
• Examples: Streptococcus pneumoniae and a group of
oral streptococci (Streptococcus viridans or viridans
streptococci)
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Beta-hemolysis (β-hemolysis)
• β-hemolysis, is associated with complete lysis
of red cells surrounding the colony.
• Transparent color change around colony
Example: Streptococcus pyogenes
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Gamma-hemolysis
• Non-haemolytic
• Colonies show neither typical alpha nor beta haemolysis.
• The streptococci included in this group are usually not
pathogenic.
• Examples: Enterococcus faecalis (formerly called
“Group D Strep”)
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Types of medium (Basis of purpose)
1. General purpose media/ Basic media
Basic or simple media
Enriched medium
2. Special medium (Added growth factors)
Addition of extra nutrients in the form of blood, serum,
egg yolk etc, to medium makes them enriched media
Selective media
Differential/indicator media
Enhancing media
Anaerobic media
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1- General purpose media
• Basically simple media that supports most non-fastidious
bacteria.
• Peptone,salt, water, nutrient broth and nutrient agar are
considered as basal medium.
• These media are generally used for the primary isolation
of microorganisms.
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• Basic medium: The media are prepared from substances
such as broth, pepton, salt.
The solid medium is obtained by adding agar to buyyon
• Enriched medium: Medium obtained by the addition of
nutrients like blood, egg to the basic medium, Ex.
Blood agar
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2- Special medium (Added growth factors)
Selective medium: Inhibits or prevent the growth of
certain types or species of bacteria and/or promote the
growth of desired species,
• Ex: Desoxycholate citrate agar
Deoxycholate Citrate Agar: Selective and differential
medium for recovery of Salmonella and Shigella from
clinical, water and dairy products.
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• Differential medium: It allows distinguishing betweent
different types of bacteria based on some observable trait
in their pattern of growth on the medium,
Ex: Eosin Methylene Blue Agar (EMB)
EMB: Slightly inhibits the growth of Gram + bacteria and
provides a color indicator distinguishing between organisms
that ferment lactose and those that do not
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• Enhancing medium: (Broth) Inhibits or prevent the
growth of certain types or species of bacteria and/or
promote the growth of desired species,
Ex: Selenite buyyon
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Main Substances Used in Preparation of Media
Water
• Distilled/double distilled water in the preparation of pure
synthetic media.
Agar
• Agar agar (agar for short) is a sea algae that is more
abundant in the Indian and Japanese seas.
• It is a long chain polysaccharide structure made of d-
galactopyranose units.
• Agar cannot be used by the microorganisms
Sodium chloride
•It is putting into the media about 0.5% to adjust the osmotic
pressure 50
Peptone
• Peptone is a medium containing polypeptide, dipeptide
and amino acid.
• Obtained by hydrolyzing proteins such as meat, casein,
soybean meal with enzymes such as pepsin, trypsin and
papain.
• Peptone, easily soluble in water and not coagulated when
heated
• It is used as a nitrogen source by bacteria.
Serum
• It is obtained by separating the blood taken from sheep,
horse, rabbit or human under sterile conditions. It is used
by adding 5-10% of the medium.
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Blood
• Obtained from various animals and is added to the media.
the blood should be sterile and prevented from coagulation.
• Defibrined blood is added to the medium at a rate of 5-10%.
Used for the construction of blood agar and chocolate agar.
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Culture Methods of Bacteria (aerop and anaerobe)
• Bacterial culture: It is obtained by cultivation of
bacteria in suitable temperature by cultivation in solid
or liquid medium.
• Bacteria produce homogeneous turbidity, flocculent,
ring, sediment in liquid media
• In a solid media, bacteria grow by forming a layer or
colony.
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Bacterial culture types
• Stab cultures: Bacteria is introduced via an inoculation
needle or a pipette tip being stabbed into the center of the
agar. Bacteria grow in the punctured area.
In this way, motility and gas formation of bacteria can
easily examined
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• Pour plate method, a small amount of inoculum from a broth
is added by pipette to the centre of a Petri dish. Cooled, but still
molten (45-50°C), agar medium in a test tube or bottle is then
poured into the Petri dish.
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Mixed culture: A mixed culture is one that contains more than
one type of organism
Pure culture: Culture containing a single species of
organism.
Pure cultures of microorganisms can be obtained by spreading
microorganisms out and permitting the individual cells to form
masses of growth called colonies.
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With a pure culture;
* Reproduction and colony characteristics of bacteria
* Staining and morphological characteristics
* Biochemical properties
* Antigenic structures
* Antibiotic susceptibility tests
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Methods for obtaining pure culture
1. Streak plate technique
• Streak plate technique is used for the isolation of pure
culture (mostly bacteria), from mixed population.
• Usually by the third or fourth quadrant only a few
organisms are transferred
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An inoculation loop, is a simple tool used mainly by
microbiologists to get the inoculum from a culture of
microorganisms.
The loop is used in the cultivation of microbes on plates
by transferring inoculum for streaking.
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2- Pour plate method
• Pour plate method, a small amount of inoculum from a
sample is added by pipette to the centre of a Petri dish.
• Cooled, but still molten (45-50°C), agar medium in a test
tube or bottle is then poured into the Petri dish.
- Petri dishes are incubated in the incubator for 24-48
hours.
- The colonies formed on the surface and examined one by
one.
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Cultivation of the material after a special process
To obtain spore bacteria
• The material is heated for 5 minutes at 850C or for 30
minutes at 650C.
To obtain tuberculosis bacteria
• Treatment of 30 minutes with 4% NaOH to obtain
At this stage, non-resistant bacteria die.
Tuberculosis bacteria are grown in special media.
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Oxygen Needs of bacteria
• Aerobic: They need free oxygen in the air for their
growth.
• Anaerobic: These microorganisms grow in an oxygen-
free environment.
• Facultative anaerobe: They can grow in both aerob
and anaerobic conditions.
• Microaerofil: These bacteria grow in atmospheres with
reduced oxygen by 1 to 2%
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Aerobic Bacteria
• Main Principle: Provide Oxygen
• Atmospheric condition is generally adequate for
culture of aerobes or facultative anaerobes
Anaerobic Bacteria
• Main Principle: Reduce the O2
• Can not grow in the presence of oxygen, oxygen is
toxic for them. They use other substances as terminal
electron acceptor.
• Their metabolism frequently is a fermentative type in
which they reduce available organic compounds to
various end products such as organic acids and
alcohols. 65
• Cannot carry out oxidative phosphorylation
• Lack such enzymes as catalase, peroxidase and
superoxide dismutase that can convert various
molecules to produce oxygen among other
molecules
• Can only get their energy from glycolysis
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Oxygen is present in the air so special methods are
needed to culture anaerobic microorganisms.
1- Special Anaerobic Culture Media
During preparation, the culture medium is boiled for
several minutes to drive off most of the dissolved oxygen
2- Anaerobic Chambers
This refers to a plastic anaerobic glove box that
contains an atmosphere of H2, CO2, and N2
3- Anaerobic Jar
Anaerobic jar is a jar with a gas tight seal within which
tubes, plates, or other containers to be incubated are
placed along with H2 and CO2 generating system (GasPak
system) .
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