BACTERIAL GROWTH AND

CULTIVATION
Prepared by: Ma.Gladys Bautista
 Bacterial Cell Growth
• In most prokaryotes, growth of an
individual cell continues until the cell
divides into two new cells, a process
called binary fission.
Binary Fission
 Bacterial Cell Growth
• During the growth cycle all cellular
constituents increase in number such that
each daughter cell receives a complete
chromosome and sufficient copies of all
other macromolecules.
 Bacterial Growth
• Growth- is the orderly increase of all
chemical constituents of the cell.
- a process that entails the replication
of all cellular structures, organelles and
protoplasmic components from the
nutrients present in the surrounding
environment.
 Bacterial Cell Growth
• The time required for a complete growth
cycle in bacteria is highly variable and is
dependent on a number of factors, both
nutritional and genetic.
• E. coli- can complete the growth cycle in
about 20 minutes.
 Bacterial Growth
• Generation or doubling time- the average
time required for an organism to double its
number . (E.coli- 20 minutes)

Under favorable conditions almost all

bacteria are able to reproduce very
rapidly.
 Growth Requirements
1. Nutrient Requirements
A. Carbon
Classification:
1. autotrophs(litotrophs)- require only water,
inorganic salts and carbon dioxide for growth

2. heterotrophs (organotrophs)- require an

organic form of carbon for growth
 Nutrient Requirements
B. Nitrogen – nitrogen is a major component
of proteins and nucleic acids of a typical
bacterial cell.

Most microorganisms use NH4 as a sole nitrogen

source and many organisms possess the ability
to produce it from amines.
 Nutrient Requirements
C. Growth Factors- are organic compounds
necessarily needed by a bacterium in order to
grow. These substances are usually provided
in the culture medium.

Examples of Growth Factors

1. B-complex vitamins 4. pyrimidines
2. Amino acids 5. pentoses
3. Purines 6. fatty acids
 Nutrients Requirements
D. Inorganic Salts
Salt in small amounts stimulates the
growth of some organisms. Organisms
requiring high salt concentrations are
called halophilic
 Nutrient Requirements
E. Oxygen
Classification
1. AEROBES – grow in the presence of
atmospheric (free) oxygen
a. obligate aerobes- grow only in the presence of
oxygen ex. Mycobacteria
b. facultative anaerobes- fundamentally aerobes,
but can grow in the absence of oxygen ex.
Staphylococcus, Klebsiella pneumoniae
 Oxygen Requirements
c. Microaerophiles –
grow best at low or reduced oxygen tensions
(lower than atmospheric oxygen =20%
concentration) ex. Borrelia burgdorferi,
Helicobacter pylori, Campylobacter
2. Anaerobes- grow in the absence of
atmospheric oxygen
a. obligate anaerobes – grow only in the
absence of oxygen
 Oxygen Requirements
b. Facultative aerobes- fundamentally
anaerobes, but can grow in the presence
of oxygen
c. aerotolerant anaerobes- do not grow well
but do survive in the presence of oxygen
 Oxygen Toxicity
• The toxicity of oxygen results from its
reduction by enzymes in the bacterial cell
to hydrogen peroxide and by ferrous ion to
the more toxic free radical, superoxide.
• Aerobes and aerotolerant anaerobes, by
the presence of superoxide dismutase
prevents the accumulation of the
superoxide ion
2 O2 + 2 H O2 + H 2 O 2
The hydrogen peroxide formed in the above reaction is rapidly
destroyed by catalase, also present in aerobes and aerotolerant
anaerobes.

2 H 2 O2 2 H2 O + O2
Oxygen relationship of microorganisms
(a) Aerobic (b) Facultative anaerobe (c) Microaerophilic (d)Aerotolerant
(e) Anaerobes
 Other Requirements
• F. Carbon dioxide – some organisms such
as Neisseria, Brucella require for growth a
higher concentration of carbon dioxide (3-
10%) These organisms are called
capnophiles.
• G. Moisture – It serves as a solvent for
food and forms the major portion of
protoplasm
 Physical Requirements
A. Temperature – every bacterium has an
optimal temperature, the temperature at
which the organism grows best.
Classification:
1. Psychrophilic- cold loving-grows at 10-20C
2. Mesophilic- grows at 20-40C (most pathogens grow at
37C)
3. Thermophilic- heat loving- grows at 50-60C
Relationship of Temperature to Growth Rate
 Physical Requirements
B. Hydrogen Ion Concentration- most
pathogenic bacteria have an optimal pH of
7.2 – 7.6
Classification
a. acidophiles- grows at pH 6.5 – 7.0
b. neutrophiles – grows at pH 7.5 – 8.0
c. alkalophiles- grows at pH 8.4 -.9.0
pH Requirements
Classification of bacteria based
on salt requirement
• Halophiles- have a specific requirement for
the sodium ion (3%)
• Mild halophile- 1-6%
• Moderate halophile – 6-15%
Effect of sodium ion concentration on growth of microorganisms of different salt
tolerances or requirements
 The Bacterial Growth Curve
• This characteristic growth curve is obtained by
plotting the logarithm of the number of cells
against the time of growth.

Major Phases of the Growth Curve

a. Lag Phase (phase of rejuvenescence or phase
of physiologic youth) – the period of adaptation
of the organism to their new environment
characterized by little or no multiplication.
 Phases of Bacterial Growth Curve
B. Exponential Phase (logarithmic phase)
The period at which the cells are in a state
of balanced growth characterized by
maximal rates of cell division and mass
increase. It is during this phase that the
generation time is constant.
 Phases of Bacterial Growth Curve
C. Stationary Phase (Phase of Equilibrium
or Plateu Phase)
The period at which the rate of cell
production equals the rate of cell death.
Growth ceases due to:
1. accumulation of waste products
2. exhaustion of nutrients
3. change in pH and other factors
 Phases of Bacterial Growth Curve
D. Death Phase (Phase of Decline)
The period at which complete cessation of
multiplication occurs such that the death
rate in the medium increases rapidly.
Growth Cycle
 Measurement Of Growth
1. Total Cell Count – counts viable and non-
viable bacteria
A. Direct Microscopic Count
- samples dried on slides
- samples in liquid
2. Direct plate count – by growing dilutions of broth
cultures on agar plates, or by the use of
calibrated loop, cells are reported as the
number of colony forming unit per milliliter
3. Turbidimetric measurements of cell number
 Measurement of Bacterial Growth
B. Determination of cell number
1. stained slide method
2. counting chamber
3. coulter counter
4. calibrated wire loop
5. pipet dilution method
6. pour plate method
Direct Microscopic Counting Procedure
 Limitations of Direct Microscopic Counting

1. Dead cells are not distinguish from living

cells.
2. Small cells are difficult to see
3. Precision is difficult to achieve.
4. A phase contrast microscope is required
when sample is not stained.
5. Not suitable for cell suspensions of low
density.
 Viable Count
• Viable – one that is able to divide and form
offspring
Colony count/Plate count- done by
determining the number of cells in the
sample capable of forming colonies on a
suitable agar medium.
1. Spread plate method
2. Pour-plate method
3. Dilutions
 Turbidimetric measurements of cell
number

Turbidity of the cell suspension is measured

with:
1. Photometer
2. Spectrophotometer
both measures scattered light
the more cells present, the more light
scattered and hence the more turbid the
suspension
 Bacterial Cultivation
• Cultivation- is the process of growing
microorganisms in culture by taking bacteria
from the infection site and growing them in the
artificial environment of the laboratory.

• The Culture Medium

A culture medium is any material containing
the necessary nutritional and environmental
requirements for bacterial growth
 Bacterial Cultivation
• Classification of Culture Media
A. According to physical state or consistency
1. liquid- without any trace of agar or solidifying
substances (e.g. nutrient broth, thioglycollate)
2. semi-solid- contains 0.5-1.5% of agar
(e.g.SIM)
3. solid- contains 2-3% of agar (e.g. BAP, CAP, TSI)
agarose-most commonly used solidifying agent
4. biphasic- contains both a liquid and a solid phase
(e.g. certain blood culture methods)
• Bacterial growth in a liquid media is
indicated by a change in appearance of
the broth or the liquid media from clear to
turbid.
• Bacterial colony- visible cluster of bacteria
growing on the surface or of within a solid
medium.
Bacterial cultures derived from a single
colony or clone are considered “pure”
 Bacterial Cultivation
B. According to composition
1. synthetic- exact chemical composition of the
ingredients is known (commercially prepared
culture media)
2. non-syntethic- precise composition of some or
all of the nutritive substances used is not known
( meat extract broth)
3. tissue culture media- contains living tissue
(embryonated egg)
 Bacterial Cultivation
C. According to how the medium is
dispensed
1. plated- distributed in sterile petri dishes
2. tubed- distributed in sterile test tubes
types:
a. slant c. butt/slant
b. butt
 Bacterial Cultivation
D. According to use:
1. Simple or supportive- contains nutrients
that allow most non-fastidious organisms
to grow at their natural rates without
providing a growth advantage to any
particular organisms; utilized for routine
cultivation of microorganisms
(e.g. nutrient agar, brain heart infusion
broth)
 Bacterial Cultivation
2. Enrichment- design to enhance the
growth of particular organisms (usually
pathogens) and suppress the growth of
normal flora present in the specimen
( selenite, GN and tetrathionate broth)
3. Differential – distinguishes organisms
growing together by their differences in
cultural characteristics (MacConkey agar)
 Bacterial Cultivation
4. Selective- contains one or more agents
inhibitory to all organism except the
organism being sought except
( bismuth sulfite agar, phenylethyl alcohol
and Salmaonella Shigella agar)
 Bacterial Cultivation
• Some of the inhibitory agents added to
media:
a. Gentian violet-inhibit gram (+) cocci
b. Sodium deoxychocolate and other bile
salts– inhibit gram(+) cocci
c. Basic fuchsin- inhibit gram (+)cocci
d. Alcohol- prevent swarming of Proteus
e. Chloral hydrate- prevent swarming of
Proteus
 Bacterial Cultivation
f. Potassium tellurite- inhibit growth of gram (-)
organism
g. Sodium azide- inhibit growth of gram (-)
organisms
< Many media used in diagnostic bacteriology
provide more than one function. For example
MacConkey agar is both differential and
selective. Sheep blood agar is both supportive
and differential.
 Bacterial Cultivation
5. Selective and Differential- some media
are both selective and differential like the
media used in the primary isolation of
enteric gram (-) organisms
(McConkey agar, XLD and Eosin methylene
blue agar)
 Bacterial Cultivation
6. Special- specially prepared to support
growth of specific microorganisms
Examples:
a. Middlebrook 7H-10 agar
b. Fletcher medium- Leptospira
c. Bordet-Gengou agar- Bordetella pertusis
d. Thayer-Martin- Neisseria
f. McBride- Listeria monocytogenes
 Most Commonly Used Artificial
Media for Routine Bacteriology
• Brain-Heart Infusion Broth- often used as
major component of media developed for
culturing patient’s blood. Key ingredients
include animal tissue, peptone and
dextrose.
 Most Commonly Used Artificial
Media for Routine Bacteriology
• Sheep Blood Agar- contains a protein
source (tryptones), soybean protein digest,
sodium chloride, agar and 5% sheep blood
 Most Commonly Used Artificial
Media for Routine Bacteriology
• Chocolate Agar- essentially the same as blood
agar except that during preparation the red
blood cells are lysed when added to molten agar
base.
The lysis releases intracellular nutrients
such as hemoglobin, hemin (“X” factor) and the
coenzyme nicotinamide adenine dinucleotide
(NAD or “V” factor) into the agar for utilization of
fastidious bacteria.
 Most Commonly Used Artificial
Media for Routine Bacteriology
• Columbia CNA with blood- contains three
peptones and 5% defribinated sheep
blood. CAN refers to the antibiotics colistin
(C) and nalidixic acid (NA) which are
added to the medium which suppress the
growth of most gram negative organisms
 Most Commonly Used Artificial
Media for Routine Bacteriology
• Phenylethyl alcohol (PEA) agar-
essentially sheep blood that is
supplemented with phenylethyl alcohol to
inhibit the growth of gram negative
bacteria
 Most Commonly Used Artificial
Media for Routine Bacteriology
• Hektoen Enteric (HE) agar- contains bile
salt and dyes (bromthymol blue and acid
fuchsin) to selectively slow the growth of
most non-pathogenic gram-negative bacilli
found in the GI tract and allow Salmonella
and Shigella spp. to grow
• MacConkey agar- commonly used for the
cultivation of gram negative bacilli differentiate
lactose from non lactose fermenters
contains crystal violet- inhibit gram (+) and fungi
pH indicator-neutral red; bacterial fermentation of
lactose results in acid production which
decrease medium pH and causes the neutral
red indicator to give bacterial colonies a pink to
red color.
 Most Commonly Used Artificial
Media for Routine Bacteriology
• Thioglycollate broth- enrichment broth,
contains casein, yeast and beef extracts
and vitamins, indicator resazurin, dextrose
and hemin.
• This agar supplement and the presence
of thioglycolic acid, which acts as reducing
agent to create an anaerobic environment
deeper in the tube, allows anaerobic
bacteria to grow.
• Xylose Lysine and Desoxychoxolate agar-
selective and differential for Shigella spp. and
Salmonella spp. The salt sodium
desoxychocolate inhibits many gram positive
organism.
phenol red indicator- media will turn pink to red if
xylose, lactose and sucrose are fermented.
Shigella do not ferment these carbohydrate so
their colonies remain colorless. Colonies of
Salmonella are also colorless on XLD.
• Because of the decarboxylation of lysine,
which results in a pH increase that causes
the pH indicator to turn red. These
colonies exhibit a black center that results
from Salmonella spp. Producing H2S.
• Several of the non-pathogens ferment one
or more of the sugars and produce yellow
colonies.
 Preparation of Artificial Media

1. Weighing
2. Dissolving- add the exact amount of solvent to
the ingredients and then dissolved by heating
3. Titration- adjustment to the right pH (pH 7.2)
4. Sterilization- autoclace 121C for 15 mins
5. Cool to approximately 50C
6. Distribute to individual petri dish