MICROBIAL NUTRITION AND GROWTH

MICROBIAL NUTRITION: Nutrition means nourishment of organisms. It includes the type

of food and the mode of intake of food. Microbes are classified into three types based on their
nutrition. They are autotrophs, heterotrophs and mixotrophs.

AUTOTROPHS: Autotrophs synthesize their own food (auto = self, trophe = nourish) using
inorganic substances. Ex: Green plans, Euglena, Volvox, some bacteria etc. They are self
feeders or lithotrophs, which depend on only inorganic substances and water for their energy.
They are of two types – Photosythetic autotrophs and chemosyntheic autotrophs.

1. Photosynthetic autotrophs: Photosynthetic autotrophs use the energy of sunlight to

synthesize food by the process called photosynthesis and are called photolithotrophs.
They use CO2 for their carbon source. In photosynthesis CO2, mineral salts, water,
sunlight and chlorophyll are used. Ex: Higher plants (maize plant, trees, grass etc),
Euglena, Green algae, Bacteria (Cyanobacteria).

 Bacteria that do not produce oxygen during photosynthesis are known classified as
obligate anaerobes while they produce through a process referred to as anoxygenic
photosynthesis. Ex: The purple bacteria, Green sulfur bacteria, Heliobacteria,
Chloroflexi. While these organisms use light energy to produce their own energy, they
do not use water as the source of protons. Rather, such gases as hydrogen sulfide are used
for reduction. For such organisms as green sulfur bacteria, such pigments as
bacteriochlorophyll (a) and (b) absorb light energy that is then used for photosynthesis
reaction.

2. Chemosynthetic autortophs: Chemosynthetic autortophs are the organisms which

derive energy by oxidizing certain inorganic compounds and are called
chemoautotrophs or chemosynthetic bacteria. Chemoautotrophs are usually bacteria
or archaebacteria, as their metabolisms are usually not efficient enough to support
multicellularity. Ex: Nitrifying bacteria, sulphur bacteria, iron bacteria etc.

The nitrifying bacteria oxidize ammonium salts into nitrites to draw energy to make food from
CO2. NH3 + 3 O2 → 2 HNO2 + 2 H2O + Energy (70K cal)

The sulphur bacteria oxidize hydrogen sulphide to get energy for chemosynthesis.
H2 S + O2 → 2 H2 SO4 + Energy

The iron bacteria oxidize ferrous iron compounds into ferric hydroxides to get energy for
chemosynthesis. Fe2+ + H+ + ½ O2 → Fe3+ + ½ H2O + Energy

The carbon bacteria in the damp coal oxidizes carbon monoxide into carbon dioxide to get
energy for chemosynthesis. CO + ½ O2 → CO2 + Energy (74K cal)
While these organisms live in environments where there is no sunlight, there is sufficient
inorganic material for biosynthesis. Many need to live around deep sea volcanic vents, which
produce enough heat to allow metabolism to occur at a high rate. Essentially, biosynthesis
involves the oxidation of the inorganic material. Here, chemolithotrophs (cells) take in the
electron donor (iron, elemental sulfur and hydrogen sulfide etc) which are then oxidized to
produce energy.
For instance, the oxidation of hydrogen sulfide produces electrons that are transported through
the electron transport chain for oxidative phosphorylation that produces ATP energy. The
chemical energy in the form of ATP is then used in biosynthesis to fix carbon in order to produce
organic compounds.

[Scientists have speculated that life might be able to exist in dark, chemically volatile
environments such as the seas of Jupiter’s moon Titan by using similar metabolisms to those
seen in chemoautotrophs on Earth. No proof of such life has yet been found, but some scientists
believe that the range of metabolic options offered by chemosynthesis drastically expands the
range of places in the universe where we can expect to find life.

It is actually unknown whether photoautotrophs or chemoautotrophs were the first life forms on
Earth. Many favor the idea that the first cells were photosynthetic, since the Sun’s light shines on
the entire surface of the Earth. But some scientists believe that volcanic sites in the deep sea or
on the surface of the Earth could have supplied more concentrated energy and more volatile
chemicals, potentially leading to the creation of the first cells. These cells could then have
evolved photosynthesis as an energy source that would work anywhere on the Earth’s surface
they spread further from their volcanic points of origin.]

The autotrophs are further classified into obligate autotrophs and facultative autotrophs:
Obligate Autotrophs: These self feeders depend only on inorganic substances such
as CO2, minerals and water.
Facultative autotrophs: These self feeders depend on either inorganic substances or organic
substances as per the availability of the nutrient. If inorganic substances are available, they use
inorganic substances and derive their energy. If organic substances are available, they use
organic substances to derive their energy.

HETEROTROPHS: Heterotrophs (Hetero = other, trophe = nourish) obtain their food from
other organisms such as plants and animals. They feed on others. Ex: Animals, some Bacteria,
Fungi etc. They are also called organotrophs. They are further classified into parasites,
saprophytes, saprozoics and symbionts.

Parasites: The organisms which live on other organisms. They feed on the digested food or
blood of other animals. Ex: Entamoeba.

Saprophytes: Saprophytes are plants which absorb dead and decayed organic food materials.
Ex: Fungi, some Bacteria etc.
Saprozoics : Saprozoics are animals, absorbing dead and decayed organic food materials
through their body surface. Ex: Euglena.

Symbionts: Symbionts live in association with another organism where both the partners are
benefited. Ex: Rhizobium and leguminous plants.

MIXOTROPHS: Mixotrophs exhibit both autotropic and heterotrophic pathways

simultaneously. Ex: Euglena.

CULTURE MEDIA: Culture is the term given to microorganisms that are cultivated in the lab
for the purpose of identifying and studying them (Bacteria have to be grown (cultured) for them
to be identified). Medium is the term given to the combination of ingredients that will support
the growth and cultivation of microorganisms by providing all the essential nutrients required for
the growth (i.e. multiplication) in order to cultivate these microorganisms in large number to
study them.

NEED FOR CULTURE MEDIA: It is usually essential to obtain a culture by growing the
organism in an artificial medium. If more than one species or type of organism is present, each
requires to be carefully separated or isolated in pure culture.

Microbiological culture: These are used for growing microorganisms, such as bacteria or yeast.
The most common growth media for microorganisms are nutrient broths and nutrient agar
plates. Specialized media are sometimes required for microorganism and cell culture growth.

COLONY – Macroscopically visible collection of millions of bacteria originating from a single

bacterial cell.

HISTORY: The original media used by Louis Pasteur was urine or meat broth. Liquid
medium shows diffuse growth of the organism. The earliest solid medium is the cooked cut
potato used by Robert Koch. Growth is observed as discrete colonies on solid medium. Gelatin
is not satisfactory as it liquefy at 24oC.

USES OF CULTURE MEDIA:

1. To grow bacteria.
2. To enrich the numbers of bacteria.
3. To select certain bacteria and suppress others.
4. Differentiate among different kinds of bacteria.

BASIC REQUIREMENTS OF CULTURE MEDIA: Nutrients for energy source, carbon

source, nitrogen source, mineral salts like Sulphates, phosphates, chlorides and carbonates of K,
Mg and Ca, a suitable pH of 7.2- 7.4 and growth factors (Arginine for E. coli, Glutathione for
Gonococci, Cholesterol for Mycoplasma,Factor V & X for Haemophilus influenza, Sodium
chloride for Vibrio parahaemolyticus etc.).
AGAR: Fanny Hesse and Walther Hesse for the first time used agar to grow microorganisms.
It is used for preparing solid medium as it melts at 98oC & sets at 42oC. It is obtained from
seaweeds and does not have nutritive value. It will not affected by the growth of the bacteria.
Usually 2% agar is employed in solid medium preparation.

TYPES OF CULTURE MEDIA: I. Based on their consistency:

a) Solid medium
b) Liquid medium
c) Semi solid medium

II. Based on the constituents/ ingredients:

a) Simple medium
b) Natural or Empirical medium
c) Living medium
d) synthetic or defined medium
e) complex medium
f) Minimal medium
g) Special media – Based on their uses.

Special media:
a) Enriched media
b) Enrichment media
c) Selective media
d) Indicator media
e) Differential media
f) Sugar media
g) Transport media
h) Media for biochemical reactions

III. Based on Oxygen requirement:

1. Aerobic media
2. Anaerobic media.

Solid media – Solid media contains 2% agar. It is used to study the colony morphology,
pigmentation, hemolysis etc. Eg: Nutrient agar, Blood agar.

Liquid media – Liquid media does not contain agar. It is used for inoculum preparation, Blood
culture, for the isolation of pathogens from a mixture. Eg: Nutrient broth.

Semi solid medium – Semi solid medium contains 0.5% agar. Eg: Motility medium to study
the motility of organisms.
 Figure: Solid medium Liquid medium & Semi-solid medium

Methods of Culturing Bacteria: Bacteria can be cultured in the following methods in the
laboratory: 1. Broth culture 2. Agar plate culture 3. Agar slant culture 4. Agar stab culture
5. Roll tube culture 6. Deep media culture

1. Broth Culture: Cultivation of bacteria in a liquid medium is called broth culture. Broth is a
liquid medium. The broth contains beef extract, peptone, yeast extract, sodium chloride and
water.
Beef extract contains carbohydrates, organic nitrogen compounds, vitamins and salts. Peptone
contains protein. Yeast extract supplies Vitamin B, growth factors, organic nitrogen and carbon
compounds.
Broth culture can be done in a petridish or in a test tube or in a conical flask.
The medium is poured into a test tube or petridish in an aseptic condition. Inoculation
(introducing the bacterium into the medium) is done with a wire-loop after flaming.
The culture tube is incubated at 37°C in an incubator for 24 to 48 hours.
Cloudiness in the broth is an indication of bacterial growth. In broth culture, the bacteria remain
floating.

2. Agar Plate Culture: Cultivation of bacteria in a solid medium in a petri dish is called agar
plate culture. It is also called plate culture. In agar plate culture, agar medium is used. It is a
solid medium. Agar medium is nothing but broth plus agar. Broth is a liquid medium containing
beef extract, peptone, yeast extract, sodium chloride and water. Agar is added as a solidifying
agent.
About 12 to 15 ml of melted agar medium is poured into a petridish. It is allowed to solidify.
Then the bacterium is inoculated and the agar plate is incubated at 37°C for 24 to 48 hours. Agar
plate is used for the cultivation of aerobic bacteria. In plate culture, the bacteria grow in the spot
where they were inoculated.

3. Agar Slant Culture: Cultivation of bacteria on a solid agar medium in a test tube kept in a
slanting position is called slant culture. In slant culture, agar medium is used. It is a solid
medium. It contains broth plus agar.
About 12 to 15 ml of melted agar medium is poured into a sterilized test tube. The mouth of the
tube is plugged with cotton and it is kept in a slanting position. The medium is allowed to
solidify in this position. The medium hardens with a slope. After solidifying, the medium is
inoculated with the bacterium. It is incubated at 37°C for 24 to 48 hours. Agar slant is used for
culturing aerobic bacteria.
4. Agar Stab Culture: Cultivation of bacteria in a solids agar medium in a test tube kept in a
vertical position is called agar stab culture. In a stab culture, agar medium is used. It contains
broth plus agar.
About 12 to 15 ml of melted agar medium is poured into a sterilized test tube. The mouth is
plugged with cotton and it is kept in a vertical position. The medium is allowed to solidify.
The bacterium is inoculated deep into the medium by thrusting the needle right through the
centre of the agar to the bottom of the tube. The tube is incubated at 37°C for 24 to 48 hours.
The agar stab culture is used 1. To study the O2 requirements of bacteria. 2. To study the
motility of bacteria. 3. To preserve culture over a long period.
If the growth has taken place only at the surface of the stab, the bacteria require O, and they are
aerobic. If the growth occurs from the surface to the depth, the bacteria are facultative aerobic. If
the bacteria grow towards the bottom, they do not require O, and they are anaerobic.
To observe motility, semi-solid agar medium is used. If the growth is observed away from
inoculation site, it indicates that the bacteria show motility. Non-motile bacteria will grow at the
centre only.

5. Roll Tube Culture: It is a culture method for anaerobes. The melted agar medium is poured
into a test tube. The medium is boiled for 10 minutes to expel air. The medium is cooled to 45°C.
Then the organisms are inoculated. The tube is then gently rolled by keeping it between the
palms to distribute the organisms. The medium is then quickly cooled by immersing in cold
water.
The tube is incubated at 37°C for 24 to 48 hours. Strict anaerobes will grow at the bottom.
Facultative anaerobes grow at the surface as well as in the depths.

6. Deep Media Culture: It is a culture method for anaerobes. Broth is poured into a tube. Plant
cells or germinating seeds are cultured in the broth to remove (),. The medium is boiled and
cooled to 45°C. The bacteria are inoculated at the depth. Strict anaerobes will grow at the depth.

PURE CULTURE: A pure culture consists of a population of only one species of

microorganisms, all derived from a single parent organism.

Isolation and Purification Technique (Isolation of pure culture): Natural environments such
as, soil, water, air and human body contain many different species of microbes. They are mixed
populations. The isolation of one kind of microorganism from a mixture is called isolation and
purification technique or pure culture technique. A pure culture is obtained by any one of the
following methods:
1. Serial dilution technique
2. Streak plate culture
3. Pour plate culture
4. Spread plate technique
5. Enrichment culture
6. Selective medium-culture
7. Differential medium-culture
8. Single cell isolation
1. Serial Dilution Technique: The dilution of sample in successive stages is called serial
dilution. The mixture of microorganism (natural sample or mixed culture) is diluted serially in
test tubes of sterile medium until the last tube contains only a single organism. In this technique,
the dilution factor increases in a regular fashion. Eg. 1/10, 1/100, 1/1000, etc.
Serial dilution technique is used to isolate a single bacterium from a mixture. In this method,
1ml of sample is mixed to 9 ml of sterile water taken in a test tube. This gives a ten fold dilution
and this dilution factor is represented as 1/10 or 10-1.
Now from this 10 fold dilution, 1ml of sample is taken and is added to 9 ml of sterile water taken
in a second test tube. Now the second tube contains a 100 fold dilution and the dilution factor is
represented as 1/100 or 10-2.
Likewise from tube two, 1 ml of sample is taken and is added to 9 ml of sterile water taken in a
third test tube. Now the third tube contains a 1000 fold dilution and the dilution factor is
represented as 1/1000 or 10-3.
Likewise, test tubes 4 and 5 are prepared. Test tube 4 provides 10-4 dilution and test tube 5
provides 10-5 dilution. A 6th tube is prepared as a control containing 10 ml of sterile water only.
Then from each tube, 1 ml of diluted sample is taken and is added to an agar plate (a petridish
containing 10 to 15 ml of melted agar medium). The 6 agar plates are incubated at 30°C for 48
hours.
The agar plate containing 20 to 200 colonies are taken as pure culture of that organism.

2. Streak Plate culture: Streak plate culture is culturing the bacterial sample on an agar plate in
the form of thin irregular long lines. It is a technique of isolating bacteria from a mixed culture.
It is a pure culture method.
In this method, the agar medium is taken in a petridish. The medium is uniformly distributed by
rotating the petridish.
The sample is taken on an inoculation needle. It is streaked on the agar plate. The streaks may be
a continuous streak or quadrant streak or radiant streak or T.streak
The successive streaks thin out the culture. Streaking isolates the individual cells and are
deposited in different regions of the agar medium,
The streaked plates are kept upside down in the incubator for 24-48 hours at 25°C.
The isolated cells will grow into colonies on different regions of the agar medium.

3. Pour Plate Culture: It is an isolation and purification technique. In pour plate method, the
sample is serially diluted in an agar tube and the contents are poured into petridishes and
incubated.
Four tubes A,B,C and D are serially arranged. Agar medium is poured into all tubes. One loopful
of sample is transferred to tube A. It is vigorously shaken.
A looptul of sample is taken from tube A and transferred to tube B. It is vigorously shaken.
Now a loopful is taken from tube B and is transferred to tube C and shaken well. Tube D serves
as control. It is added with agar medium alone.
Now the contents of tubes A,B,C and D are poured into petridishes A,B,C and D.
After solidifying, the petridishes are incubated at 25°C for 24-48 hours.
The plates are observed for colonies.

4. Spread Plate Technique: It is an isolation technique. It is a modification of pour plate

technique. In this method, the mixed culture is serially diluted in sterile distilled water. A small
amount of the diluted mixture is then pour on the surface of an agar plate and it is spread evenly
using a sterile bent glass rod called spreader. The isolated cells grow into colonies.

5. Enrichment culture: It is an isolation technique. In this method, a particular nutrient, which

favours the growth of the desired bacterium, is added to the medium. When the mixed culture is
placed in this enriched medium, the desired bacterium will grow dominantly.
The soil bacterium Nitrobacter oxidizes nitrates to nitrites. An enrichment culture of Nitrobacter
is obtained by adding sodium nitrite in the medium inoculated with soil.

6. Selective Medium – Culture: It is an isolation technique. A selective medium contains a

chemical, which suppresses or kills unwanted species. But the medium allows the desired
bacterium to grow. For example, crystal violet inhibits gram positive bacteria. When crystal
violet is added to the medium, the medium will select the gram negative bacteria.

7. Differential Medium – Culture: The differential medium gives different appearances to

different species. For example, in eosin-methylene blue agar medium, E.coli will produce
colonies with a brilliant green metallic sheen and Acrobacter aerogenes will produce pink
colonies with dark centres.

8. Single Cell Isolation: It is an isolation technique. In this method individual cells are picked
out using a micromanipulator and a microscope.

Bacterial growth is proliferation of bacterium into two daughter cells, in a process called
binary fission. Providing no event occurs, the resulting daughter cells are genetically
identical to the original cell. Hence, bacterial growth occurs. Both daughter cells from the
division do not necessarily survive. However, if the number surviving exceeds unity on
average, the bacterial population undergoes exponential growth. The measurement of an
exponential bacterial growth curve in batch culture was traditionally a part of the training
of all microbiologists; the basic means requires bacterial enumeration (cell counting) by
direct and individual (microscopic, flow cytometry[1]), direct and bulk (biomass), indirect
and individual (colony counting), or indirect and bulk (most probable number, turbidity,
nutrient uptake) methods. Models reconcile theory with the measurements

Phases

f
Bacterial growth curve\Kinetic Curve

In autecological studies, the growth of bacteria (or other microorganisms, as protozoa,

microalgae or yeasts) in batch culture can be modeled with four different phases: lag phase (A),
log phase or exponential phase (B), stationary phase (C), and death phase (D).[3]

1. During lag phase, bacteria adapt themselves to growth conditions. It is the period where
the individual bacteria are maturing and not yet able to divide. During the lag phase of
the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs.
During the lag phase cells change very little because the cells do not immediately
reproduce in a new medium. This period of little to no cell division is called the lag phase
and can last for 1 hour to several days. During this phase cells are not dormant.[4]
2. The log phase (sometimes called the logarithmic phase or the exponential phase) is a
period characterized by cell doubling.[5] The number of new bacteria appearing per unit
time is proportional to the present population. If growth is not limited, doubling will
continue at a constant rate so both the number of cells and the rate of population increase
doubles with each consecutive time period. For this type of exponential growth, plotting
the natural logarithm of cell number against time produces a straight line. The slope of
this line is the specific growth rate of the organism, which is a measure of the number of
divisions per cell per unit time.[5] The actual rate of this growth (i.e. the slope of the line
in the figure) depends upon the growth conditions, which affect the frequency of cell
division events and the probability of both daughter cells surviving. Under controlled
conditions, cyanobacteria can double their population four times a day and then they can
triple their population.[6] Exponential growth cannot continue indefinitely, however,
because the medium is soon depleted of nutrients and enriched with wastes.
3. The stationary phase is often due to a growth-limiting factor such as the depletion of an
essential nutrient, and/or the formation of an inhibitory product such as an organic acid.
Stationary phase results from a situation in which growth rate and death rate are equal.
The number of new cells created is limited by the growth factor and as a result the rate of
cell growth matches the rate of cell death. The result is a “smooth,” horizontal linear part
of the curve during the stationary phase. Mutations can occur during stationary phase.
Bridges et al. (2001)[7] presented evidence that DNA damage is responsible for many of
the mutations arising in the genomes of stationary phase or starving bacteria.
Endogenously generated reactive oxygen species appear to be a major source of such
damages.[7]
4. At death phase (decline phase), bacteria die. This could be caused by lack of nutrients,
environmental temperature above or below the tolerance band for the species, or other
injurious conditions.

Measurement of Bacterial Growth: Growth in bacteria and other unicellular organisms is

measured by measuring either increases in mass or increases in numbers in relation to time.

1. Measurement of Cell Mass: It is an indirect method of measurement of bacterial growth.

The cell mass can be measured in the following ways:
a. Dry Weight Method: In this method, a known volume of culture is removed from the
medium, washed effectively, filtered, dried and weighed accurately. Such determinations are
time consuming and relatively insensitive.
b. Cell Volume Method: This method involves placing a standard volume, say 10ml, of the
liquid culture in a kind of calibrated centrifuge tube called a Hopkin's tube. It is centrifuged at a
standard speed. The pellet is measured in volume. From a knowledge of the average volume of
the individual cell, the estimation of numbers is possible.
c. Chemical Method: Chemical estimates of cell masses are made by measuring the amount of
some chemical component, say protein, DNA, RNA, free amino acid, phosphorus, etc. that are
always present in fairly constant amounts in living cells. Such methods are hardly applicable to
bacteria but are much used in measuring heavy growth of filamentous microorganisms.
d. Turbidimetric Method: This is a widely used technique. In this, we measure turbidity or
scattering of light in the liquid culture due to evenly dispersed cells suspended in it. This
technique is based on the fact that small particles scatter light proportionally, to their
concentration. When a beam of light is passed through a suspension of bacteria, the reduction in
the amount of light transmitted is measured to represent the bacterial mass. Such measurements
are usually made in the photoelectric colorimeter or spectrophotometer.

These instruments are convenient for estimating cell concentration. When calibrated against
bacterial suspension of known concentration, they provide an accurate and rapid way to estimate
the dry weight of bacteria per unit volume of culture.

More sensitive instrument for measuring scattering is called nephelometer. It has a light sensing
device kept at right angles to the incident beam of light and hence directly measures the scattered
light.

2. Measurement of Cell Number: Counting the bacterial cells using a microscope is direct
count. Total count is made directly with a microscope. This is a direct method and not precise as
culture methods. However, it is less time consuming and easy to perform.

Generally specimens that contain large numbers of bacteria (more than 10+ cells per ml) are
diluted from 1:10 to 1:10$ or more depending on sample and counting method, to make the
number more manageable and simplify the counting. The following methods are used to measure
the cell number.
a. Using Counting Chamber: A number of counting chambers are available for counting the
number of cells under the microscope. They are Petroff-Hausser slide, Haemocytometer or
special counting chamber.
In this method, a measured volume of a sample is spread over a measured area of the slide and
the cells therein are counted under a microscope. The counted number is multiplied by an
appropriate factor to calculate the actual number of cells in the whole volume of culture/sample.

The counting chambers available have depressions of the known depth and volume, marked off
into squared areas. The organisms in an area are counted (say 50 or 100 squares) and the total
number of bacteria in the sample can be calculated from the proportion of the total volume, This
method is applicable to count upto 1 x 107 cells/ml of suspension.
b. Spread Method or Smear Count: In this method, a known volume of sample is smeared
over an exact area on a slide (say 1 square centimetre), dried, fixed and stained with methylene
blue or with any other dye. The cells are counted in a known portion of the total area. Knowing
the diameter of the microscopic field from previous measurements (by means of stage
micrometer), one can calculate the number of organism per ml of culture.

c. Membrane Filter Count: In this method, a measured sample of fluid may be passed through
sterile, porous membrane filters and the microorganisms on the filter are then counted directly.
The organisms must not be too numerous and must be uniformly distributed. They are stained in
situ on the membrane and then counted in calibrated fields. Before counting, the filter is made
transparent by saturating it with immersion oil. This gives a total count.

d. Electronic Colony Counter: This method gives an accurate counting of thousands of cells,
alive or dead (total count) in a few seconds. This instrument works on the principle of electronic
gating or electronic eye. Basically, it depends on interruptions of an electronic beam that
traverses a space between two closely adjacent electrodes. Each particle, as it passes between the
electrodes, causes an interference with the electron beam due to different conductivities of cells
and fluid. The interruptions are taken up by instruments and recorded electronically.

The petriplate in which bacteria to be counted is placed on the machine and each distinct colony
that is separated from its neighbours is counted as if it were a single cell. This operation is
performed automatically and records the total plate count on the display panel.

e. Plate or Colony Count: The enumeration of unicellular organisms can also be made by plate
count, because single viable cells separated from one another in space give rise to separate
colonies. The theoretical assumption in this is that one bacterial cell or clump of cells gives rise
to one colony. By counting the number of macroscopically visible colonies that develop on the
agar plate one can know the bacterial count. The colony count is widely used for determining
appropriate number of microorganisms in milk, water and many other materials.

4. Dilution Methods: This method involves making a serial dilution with the given sample of
bacterial suspension. In a typical procedure, Iml of the sample diluted to ten-fold, four-fold or
two-fold, are placed in tubes of broth. After incubation of the tubes of broth, if presence of
growth in the tube that received 1:1,000 dilution (1:10-3), but no growth in the tube receiving the
1:10,000 (1:10-4) dilution, then there were between 1000 and 10,000 organisms per ml of the
sample tested.

Table.10.1: Summary of methods for Measuring Bacterial Growth. Method Applications

Method Applications Expression of Growth

Direct cell count Enumeration of bacteria in Number of cells/ml

vaccines and cultures
Electronic enumeration Same as for direct cell count Number of cells per ml

Plate count Enumeration of bacteria in Colony forming units per ml


Membrane filter
Turbidimetric measurement
Nitrogen determination
Dry weight
Measurements of biochemical
activity Eg.Acid production by
cultures.
milk, water, foods, soil,
cultures, etc.
Same as plate count Colony forming units per ml
Microbiological assay, Optical density (absorbance)
estimation of cell crop in
broth, cultures or aqueous
suspensions
Measurement of cell crop Mg nitrogen per ml
from culture suspensions to be
used for research in
metabolism
Same as for nitrogen Mg dry weight of cells per ml
determination
Microbiological assays Milliequvalents of acid per ml
of culture

Maintenance of Bacterial Culture: Storing bacterial culture alive for future use is called
maintenance of bacterial culture. It is the preservation of bacterial culture. The preserved culture
is called stock culture collection. Some cultures can be preserved for 80 years.

Different methods are employed for maintenance of bacterial cultures. They are:
1. Periodic transfer to freshmedia.
2. Maintaining with mineral oil.
3. Maintenance in formaldehyde .
4. Lyophilization.
5. Preservation by liquid nitrogen at very low temperature.
6. Soredelli's method of preservation.
7. Storage in sterile soil
8. Storage in silica gel

1. Periodic Transfer to Fresh Media: The culture can be stored alive by transferring the
culture to fresh medium at regular interval. The transfer can be done once in a month. For better
results, the medium should favour slow rate of growth. The medium used for this method is
called nutrient agar. Nutrient agar is composed of peptone, beef extract, NaCl, agar and distilled
water. The culture is maintained in agar slant. Agar slant is an agar nutrient containing test tube
kept in a slanting position.

2. Maintaining with Mineral Oil: Bacterial culture can be stored alive for 2 years in mineral
oil. The culture is kept in agar slant. Mineral oil is poured into the agar slant. The oil level must
be above the tip of the slanted surface. The oil covered slant is stored at 5°C.

3. Maintenance in Formaldehyde: Agar plate cultures can be preserved by placing a drop of

formaldehyde on the inner side of lid. It is stored in a refrigerator at 4°C.
4. Lyophilization: Lyophilization or freeze-drying can be used to preserve many kinds of
bacteria that would be killed by ordinary drying. In this process a dense cell suspension is placed
in small vials and frozen at -60°C to -78°C. The vials are then subjected to rapid dehydration
under high vacuum. This results in minimum damage to delicate cell structures. The vials are
then sealed off under a vacuum and stored in a refrigerator. By this method one can preserve a
culture for more than 30 years. This method has the following advantages:
1. It requires minimum storage space.
2. Hundreds of lyophilized cultures can be stored in a small area.
3. Culture vials can be sent conveniently through the mail.
4. The cultures are revived by opening the vials, adding liquid media and transferring the
rehydrated medium to a suitable growth medium.

5. Preservation by Liquid Nitrogen at very Low Temperatures: A dense cell suspension is

taken in a medium containing a cryoprotective agent such as glycerol or dimethyl sulfoxide
which prevents cell damage due to ice crystal formation. The cell suspension is sealed into small
vials and then frozen at a controlled rate to -50°C. The vials are then stored in liquid nitrogen at
-150°C to -196°C. This method is useful for species that cannot be preserved by lyophilization.
However, this method is highly expensive.

6. Soredelli's Method of Preservation: In this method, the cultures are incubated on a solid
medium for a suitable time. When good growth is obtained, a heavy inoculum from the solid
growth is emulsified in a loopful of horse serum and is deposited on the inner wall of a small
tube which can be inserted into another tube. A small amount of phosphorus pentoxide is placed
at the bottom of the outer tube with the help of a glass rod and a funnel.
The small tube containing the culture is inserted into the larger tube in such a way that the inner
tube is placed above the phosphorus pentoxide. The outer tube is constricted, attached to a
vacuum pump after cooling, and a vacuum is applied for 5 minutes. The tube is then sealed at the
constriction and the culture can be stored at the room temperature away from light.

For a small number of samples and for a small laboratory, preservation by lyophilization, liquid
nitrogen is not ideal and feasible. We can use Soredelli's method in such cases.

7. Storage in Sterile Soil: Soil is sterilized. Bacteria are added to sterile soil. The mixture is
dried at room temperature and stored in the refrigerator. Spore forming bacteria and fungi are
stored in this method for about 80 years.

8. Storage in Silica Gel: Silica gel is powdered, sterilized and cooled. Bacterial cells are mixed
with silica gel powder and stored at low temperature. Bacteria and yeast can be stored for about
2 years in this method. Significance are:
1. The stored microbes can be used for laboratory experiments.
2. It is used for research work.
3. It is used as a test agent.
4. It can be used as a reference strain for taxonomic studies.
5. It is used for the assay of vitamins, amino acids, etc.