Nutrition and cultivation of

bacteria
 Bacteria are usually cultivated and studied under
laboratory conditions. Numerous media (singular,
medium) have been developed for bacterial
cultivation.
 Because the nutritional requirements of bacteria
vary widely, there are great differences in the
chemical composition of the media used in the
laboratory. Bacteria also exhibit wide difference to
the physical conditions favoring their growth. The
successful cultivation of bacteria requires an
awareness of all of these factors.
 Nutritional requirements
for
All forms life
of life, from microorganisms to human beings, have
certain requirements for growth and normal functioning as
follows:
 All organisms require a source of energy. Some rely on
chemical compounds for their energy. Others can utilize radiant
energy (light) as a source of energy.
 All organisms require a source of electrons for their
metabolism. Some organisms can use reduced inorganic
compounds as electron donor while others use organic
compounds.
 All organisms require carbon in some form to synthesize cell
components. Some organism use CO2 as the major source of
carbon while others utilize organic compounds as their carbon
source.
Nutritional requirements
for life….
Other nutritional requirements for life are:

 Nitrogen, oxygen, sulfur, phosphorus

 Metal ions such as K+, Ca++ , Mg++ , Fe++

 Trace elements (low conc. required) Zn++

Cu++ , Mn++ , Mo6+ , Ni++ , B3+ and Co++

 Vitamins

 Water
Microbial Nutrition

 Important for energy and construction

 Nutrients
 substances used in biosynthesis
 energy production

 Environmental factors important in cultivation

Nutrient requirements

 Macronutrients/Macroelements
 Needed in large amounts

 C, O, H, N, S, P – components of
carbohydrates, lipids, proteins, nucleic acids

 K, Ca, Mg, Fe – cations with variety of roles

Nutrient requirements
 Micronutrients/trace elements
 Needed by most cells in small amounts

 Part of enzymes and cofactors

o Aid in catalysis of reactions

o Maintenance of protein structure

 Contaminants are sufficient

 Do not usually limit growth in nature
Requirements for C, H, O
 Carbon needed for backbone of all organic molecules

 Carbon source molecules normally also act as sources

of H and O

 Can serve as energy sources

 CO2 important C source, but lacks H and energy

Nutritional Types of
Bacteria
Bacteria can be divided into many groups on the basis of
nutritional requirements as follows:

 A. According to source of C:

 Autotrophs
Can use CO2 as their sole or principle source of carbon

 Heterotrophs
Use reduced organic molecules as both carbon and
energy sources
Nutritional Types of
Bacteria
 B. According to source of energy:
 Light (phototrophs)

 Oxidizing chemical compounds (chemotrophs)

 C. According to source of electrons:

 Reduced inorganic substances (lithotrophs)

 Organic substances (organotrophs)

Phototrophs:
Bacteria utilizing radiant energy (light) as the source of energy are
known as Phototrophs.

On the basis of the source of electrons required for metabolism,

phototrophic bacteria are further divided into two groups

a) Photolithotrorhs
b) Photoorganotrophs

a)Photolithotrophs
Among the phototrophic bacteria, the species that use inorganic
compound as their source of electrons are known as
Photolithitrophs.
For example, Chromatium okenii uses H2S as its electron donor,
oxidizing it to elemental sulfur.

H2S S + 2e- +2H+

b) Photoorganotrophs

Some phototrophic bacteria use organic compounds such as fatty

acids and alcohols as electron donors and therefore are known as
Photoorganotrophs.

For example, Rhodospirillum rubrum can use succinate as an

electron donor.
Succinate Fumarate + 2e- +2H+
Chemotrophs
Bacteria which rely on chemical compounds for their source of
energy are designated as Chemotrophs.
On the basis of the source of electrons required for metabolism
Chemotrophs bacteria are further divided into two groups
a) Chemolithotrophs
b) Chemoorganotrophs
a) Chemolithotrophs
Among the chemotrophic bacteria that utilize
inorganic compounds as their source of electrons
are known as Chemolithotrophs.

For example,bacteria of the genus Nitrosomonas use

ammonia as their electron source, obtaining energy by
oxidizing ammonia to nitrate.

NH4 + 3/2 O2 + H2O NO2- + 2H3O+

b) Chemoorganotrophs
Many other chemotrophic bacteria use organic compounds,
such as sugars and amino acids as electron donor and are
therefore known as Chemoorganotrophs.

Certain bacteria can grow as either chemolithotrophs or

chemoorganotrophs.

For example, Pseudomonas pseudoflova can use either

the organic compound glucose or the inorganic compound
H2 as its source of electrons.

C6H12O6 + 6H2O 6CO2 +24H+ + 24e-

H2 2H+ + 2e-
 Autotrophs
All organisms require carbon in some form for
synthesizing cell components. Bacteria utilizing CO2 as their
major source of carbon are termed as Autotrophs.

For example bacteria of the genus Nitrosomonas are able

to oxidize ammonia to nitrite, thereby obtaining sufficient
energy to assimilate the carbon of CO2 into call
components (CO2 fixation).

CO2 + 4e- + 4H+ C6H12 O6 + H2O

Facultative autotrophs
Some bacteria can live either as autotrophs, deriving their
carbon from CO2, or they can live as heterotrophs, deriving
their carbon from organic compounds. These are known as
facultative autotrophs.

For example,P. pseudoflava can live as a heterotroph, using

glucose as a source of carbon; however, if H2 is provided as
the electron sources, then it can use CO2 as its sole carbon
source and can grow as an autotroph.
 Heterotrophs
Bacteria utilizing organic compounds as their source of
carbon are known as Heterotrophs.

Heterotrophic bacteria have been more extensively studied

than autotrophs because all the bacteria causing diseases
to human being, animals and plants are heterotrophs.

Fastidious heterotrophs
Heterotrophs having elaborate requirements for specific
nutrients are designated as fastidious heterotrophs.

As for example Lactobacilli have much nutritional

requirements as E. coli. Hence Lactobacilli are fastidious
heterotrophs
 Q: What are Obligate Parasite?

A: Some bacteria have not yet been successfully

cultivated on an artificial medium, and their nutritional
and physical requirements are not understood. These
are called obligate parasite.

These can be cultivated only in living cell. As for

example, bacterium that causes leprosy, Mycobacterium
leprae, which can be cultivated by infecting mice is an
obligate parasite.
 Bacteriological Media
Bacteria are usually cultivated (cultured) and studied under
laboratory conditions to provide their nutritional
requirements for multiplication and growth media
composed of known chemical compounds are used. These
chemically defined media are known as bacteriological
media.
Materials used as ingredients of media:
Beef extract
• Characteristics:

An aqueous extract of lean beef tissue concentrated to a

paste.
 Nutritional value:

Contains the water soluble substance of animal tissue,

which include carbohydrates, organic nitrogen compounds,
water soluble vitamins and salts.
 Peptone
 Characteristics:

Obtained from digestion of protein materials e.g. Meat, casein and

gelatin; which is accomplished by the treatment of acids or
enzymes.
 Nutritional value:

Principal source of organic nitrogen may also contain some

vitamin and sometimes carbohydrates depending upon the kind of
proteinaceous material digested.
Agar
 Characteristics:

A complex carbohydrate obtained from certain marine algae.

 Function:

Used as solidification agent for media. It forms a gel when the

temperature of the aqueous solution of agar is reduced to 45°C.
Agar is not considered as a source of nutrient to the bacteria.
Yeast extract
 Characteristics:

An aqueous extract of yeast cells, commercially available

as a powder.

 Nutritional value:
A very rich source of the B vitamin; also contains organic
nitrogen and carbon compound.
 Types of media
Bacteriological media are of different types:

 On the basis of physical state:

1) Liquid: Nutrient broth media

2) Solid: Nutrient agar media with 1.5 to 2% agar
which forms firm, transparent gel.
3) Semisolid: prepared with agar at a
concentration of 0.5% or less , have
a soft, custard like consistency.
Composition of Nutrient Broth (liquid) media

Beef extract 3 gm

Peptone 5 gm

Water 1,000 ml

Composition of Nutrient Agar (solid) media

Beef extract 3 gm
Peptone 5 gm
Agar 15 gm
Water 1,000 ml
 On the basis of application:
The microbiologist has developed different types of
media for special purposes such as recognition,
enumeration and isolation of certain types of
bacteria. These are:

1) Selective media
2) Differential media
3) Assay media
4) Media for enumeration
5) Media for characterization
6) Maintenance media
1) Selective media:
This media provides nutrients that enhance the growth and
predominance of a particular type of bacterium and do not
promote (and may even inhibit) other types of organisms
that may be present is known as selective media.

The isolation of the gonorrhea-causing organism, Neisseria

gonorrhoeae from a clinical specimen is facilitated by the use
of media containing certain antibiotics; these antibiotics do
not affect N. gonorrhoeae but do inhibit the growth of
contaminating bacteria.
2) Differential media:
Certain reagents or supplements, when
incorporated into culture media, may allow
differentiation of various kinds of bacteria. This type of
media is known as differential media.

For example, if a mixture of bacteria is inoculated on to

blood containing agar medium (blood agar), some of
the bacteria may hemolyse (destroy) the red blood
cells ; others do not. Thus one can distinguish between
hemolytic and non-hemolytic bacteria on the same
medium.
3) Assay media:
Media of prescribed composition used for the assay of
vitamins, amino acids and antibiotics is known as assay
media. Media of special composition are also available for
testing of disinfectant.

4) Media for enumeration of Bacteria:

Specific kind of media used for determining bacterial content
of such material as milk and water.

5) Media for characterization of Bacteria:

A variety of media conveniently used to determine type of
growth produced by bacteria, as well as to their ability to
produce certain chemical changes.
6) Maintenance Media:
A medium, different from that which is optimum for
growth is required for the maintenance of the viability and
physiological characteristics of the culture over time is
known as maintenance media.

For example, glucose in a medium is frequently enhance

growth, but acid harmful to the cell is likely to be
produced in presence of glucose. Therefore, omission of
glucose is preferable in a maintenance media.
 Preparation of media

 Commercially all the ingredients are of the media are

available in powder form.
 Each ingredients or the complete dehydrated medium is
dissolved in the appropriate volume of distilled water.
 The pH of the fluid medium is determined with a pH meter.
 If a solid medium is desired, agar is added and the medium
is boiled to dissolve the agar.
 The medium is dispensed into tubes or flasks.
 The medium is sterilized generally by autoclaving
 Physical conditions required for growth
In addition to knowing the proper nutrients for the
cultivation of bacteria, it is also necessary to know
the physical environment in which the organism will
Grow best.

Bacteria exhibit diverse response to physical condition

such as:
 Temperature
 Gaseous Requirements
 pH
 Miscellaneous
Temperature

Since all processes of growth are depended on chemical

reactions and the rates of these reactions are influenced
by temperature, the pattern of bacterial growth can be
profoundly influenced by this condition.

The temperature that allows for most rapid growth during

a short period of time (12 to 24 hr) is known as the
optimum growth temperature
It can be seen that the maximum temperature at which
growth occurs is usually quite close to the optimum
temperature, whereas the minimum temperature for growth
is usually much lower than the optimum.

On the basis of the temperature required for growth the

microbiologists divide the bacteria into three groups as
follows:
1) Psychrophiles

2) Mesophiles &

3) Thermophiles
1) Psychrophiles:
The bacteria that can grow at 0°C but have an optimum
temperature of 15°C or lower and a maximum temperature
of 20°C is known as Psychrophile.

 Psychrotrophs or Facultative psychrophiles:

The organisms those are able to grow at 0°C but which
grow best at temperature in the range of 20°C to 30°C are
called psychrotroph or facultative psychrophile.
2) Mesophiles:
Mesophiles grow best within a temperature range approximately
25 °C to 40 °C.
For example all bacteria that are pathogenic for human and
warm blooded animals are mesophiles, most growing best at
about body temperature (37°C)

3) Thermophiles:
Thermophiles grow best at temperature above 45°C.

 Facultative thermophiles:
The growth range of many thermophiles extends into mesophilic
range; these species are designated as facultative thermophile.

 True thermophiles/ Obligate thermophiles/ Stenothermophiles:

Thermophiles cannot grow in mesophilic range.
 Gaseous Requirements
The principal gases that affect bacterial growth are
oxygen and carbon dioxide. Bacteria display such a
wide variety of responses to free oxygen that it is
convenient to divide them into four groups as follows:

1) Aerobic bacteria
2) Anaerobic bacteria
3) Facultative anaerobic bacteria
4) Microaerophilic bacteria
1) Aerobic bacteria:
Require oxygen for growth and can grow when incubated
in an air atmosphere (ie. 21% oxygen)

2) Anaerobic bacteria:
Do not use oxygen to obtain energy; moreover oxygen is
toxic for them and cannot grow when incubated in an air
atmosphere.

 Nonstringent or tolerant anaerobes:

These anaerobes can tolerate low level of oxygen.

 Stringent or Strict anaerobes:

These anaerobes cannot tolerate even low levels and
even may die upon brief exposure to air.
3) Facultative anaerobic bacteria:
Do not require oxygen for growth, although they may use
it for energy production if it is available.
They are not inhibited by oxygen and usually grow as
well under air oxygen as they do in the absence of
oxygen.

4) Microaerophilic bacteria:
Require low levels of oxygen for growth but
cannot tolerate the level of oxygen present in an
air atmosphere.
 pH (Acidity or Alkalinity):
 Organisms sensitive to changes in acidity because H+ and
OH- interfere with H bonding in proteins and nucleic acids
 Most bacteria and protozoa grow best in a narrow range

around neutral pH (6.5-7.5) – these organisms are called

neutrophiles.
 Other bacteria and fungi are acidophiles – grow best in

acidic habitats
 Acidic waste products can help preserve foods by

preventing further microbial growth

 Alkalinophiles live in alkaline soils and water up to pH 11.5
GROWTH
The most common means of bacterial reproduction is binary fission; one cell
divides producing two cells. Thus if we start with a single bacterium, the increase
in population by geometric progression:
20 21 22 23 24 2n
Where n = the no of generations. Each succeeding generation, assuming no cell
death,doubles the population. The total population N at the end of a given time
period would be expressed as.

N = 1 × 2n
However, under practical conditions the number of bacteria N0 inoculated at
time zero is not zero but more likely several thousands, so the formula now
becomes
N = N 0 × 2n
logN = logN0 + n log2
n = ( logN - logN0)/ log2
If we now substitute the value of log2, which is 0.301,in the above equation, we
can simplify the equation to
n = ( logN - logN0)/ 0.301
n = 3.3 ( logN - logN0)
Bacterial Growth Curve

 All microorganisms undergo similar growth

patterns.
 Each growth Curve has 4 Phases
 1 Lag Phase
 occurs immediately after inoculation
 cells do not grow; cells per volume do not increase
Microbial Growth Curve

 2.) Growth Phase

 Exponential Phase
 Log Phase
 During this phase the microbe is growing at the
maximum rate possible.
 Cells per volume increases dramatically
 Most research is performed on cells during log
phase
Microbial Growth Curve

 3.) Stationary Phase

 Growth levels off.
 Cells per volume does not increase or decrease
 Growth Rate = Death Rate
 Due to
 Depletion of Nutrients

 Increase in Waste Products

Microbial Growth Curve

Stationary
# cells / ml

Log Death

Lag

Time
Microbial Growth Curve

 4.) Death Phase

 Death Rate exceeds Growth Rate
 Cells per volume decreases
 Due to:
 Very low concentrations of Nutrients

 Very high concentrations of Waste Products

 Advantage
 Direct microscopic counts can be made rapidly and simply
with a minimum of equipment;
 the morphology of the bacteria can be observed as they are
counted.
 Very dense suspensions can be counted if they are diluted
properly
.
Disadvantage
 Suspensions having low number of bacteria, e.g. at the
beginning of a growth curve cannot be counted accurately.
 We can’t distinguish between the viable and nonviable
bacteria.
 Advantage
 The plate-count method is routinely and with satisfactory results for the
estimation of bacterial population in milk, water, foods, and many other
materials.
 It is easy to perform
 It has the advantage of sensitivity, since very small numbers of
organisms can be counted. Theoretically, if a specimen contains as few
as one bacterium per milliliter, one colony should develop upon the
plating of 1ml.
Disadvantage
 One limitation of the plate-count technique is that the only bacteria that
will be counted are those, which can grow on the medium used and
under the conditions of incubation provided. This can be an important
consideration if a mixture of bacteria is to be counted.
 The development of one colony from one cell can occur when the
bacterial suspension is homogeneous and no aggregates of cells are
present; however, if the cells have a tendency to aggregate, e.g., cocci
in clusters (staphylococci), the resulting counts will be lower than the
number of individual cells. For this reason the “counts” are often reported
as colony-forming units per ml rather than number of bacteria per
milliliter