Bacterial growth- SECTION 1

Culture Media for the Growth of Bacteria

For any bacterium to be propagated for any purpose it is necessary to provide the appropriate
biochemical and biophysical environment. The biochemical (nutritional) environment is made
available as a culture medium, and depending upon the special needs of particular bacteria
(as well as particular investigators) a large variety and types of culture media have been
developed with different purposes and uses. 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.

Classification of Culture Media:

Bacterial culture media can be classified in at least three ways; Based on consistency, based on
nutritional component and based on its functional use.
1) Classification based on consistency:
Culture media are liquid, semi-solid or solid and biphasic.
A) Liquid media: These are available for use in test-tubes, bottles or flasks. Liquid media are
sometimes referred as “broths” (e.g nutrient broth). In liquid medium, bacteria grow uniformly
producing general turbidity. Liquid media tend to be used when a large number of bacteria have
to be grown. These are suitable to grow bacteria when the numbers in the inoculum is
suspected to be low. Inoculating in the liquid medium also helps to dilute any inhibitors of
bacterial growth. This is the practical approach in blood cultures. Culturing in liquid medium can
be used to obtain viable count (dilution methods). Properties of bacteria are not visible in liquid
media and presence of more than one type of bacteria can not be detected.
B) Solid media: Any liquid medium can be rendered by the addition of certain solidifying
agents. Agar agar (simply called agar) is the most commonly used solidifying agent. It is an
unbranched polysaccharide obtained from the cell membranes of some species of red algae.
Agar is composed of two long-chain polysaccharides (70% agarose and 30% agarapectin). Agar
is used because of its unique physical properties (it melts at 100 oC and remains liquid until
cooled to 40oC, the temperature at which it gels) and because it cannot be metabolized by most
bacteria. Hence as a medium component it is relatively inert; it simply holds (gels) nutrients that
are in aquaeous solution. Agar is available as fibres (shreds) or as powders.
C) Semi-solid agar: Reducing the amount of agar to 0.2-0.5% renders a medium semi-
solid.Such media are fairly soft and are useful in demonstrating bacterial motility and separating
motile from non-motile strains. Certain transport media such as
Stuart’s and Amies media are semi-solid in consistency. mannitol motility medium are also semi-
solid.
D) Biphasic media: Sometimes, a culture system comprises of both liquid and solid medium in
the same bottle. This is known as biphasic medium (Castaneda system for blood culture). The
inoculum is added to the liquid medium and when subcultures are to be made, the bottle is
simply tilted to allow the liquid to flow over the solid medium. This obviates the need for frequent
opening of the culture bottle to subculture.
Besides agar, egg yolk and serum too can be used to solidify culture media. While serum and
egg yolk are normally liquid, they can be rendered solid by coagulation using heat. Serum
containing medium such as Loeffler’s serum slope and egg containing media such as
Lowenstein Jensen medium and Dorset egg medium are solidified as well as disinfected by a
process of inspissation.

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2) Classification based on nutritional component:
Media can be classified as simple, complex and synthetic (or defined). While most of the
nutritional components are constant across various media, some bacteria need extra nutrients.
Those bacteria that are able to grow with minimal requirements are said to non-fastidious and
those that require extra nutrients are said to be fastidious. Simple media such as peptone water,
nutrient agar can support most non-fastidious bacteria. Complex media such as blood agar
have ingredients whose exact components are difficult to estimate. Synthetic or defined media
such as Davis & Mingioli medium are specially prepared media for research purposes where the
composition of every component is well known.

A chemically-defined (synthetic) medium is one in which the exact chemical composition is

known. A defined medium is a minimal medium if it provides only the exact nutrients (including
any growth factors) needed by the organism for growth. The use of defined minimal media
requires the investigator to know the exact nutritional requirements of the organisms in question.
Chemically-defined media are of value in studying the minimal nutritional requirements of
microorganisms, for enrichment cultures, and for a wide variety of physiological studies.

A complex (undefined) medium is one in which the exact chemical constitution of the medium
is not known. Complex media usually provide the full range of growth factors that may be
required by an organism so they may be more handily used to cultivate unknown bacteria or
bacteria whose nutritional requirement are complex (i.e., organisms that require a lot of growth
factors, known or unknown). Complex media are usually used for cultivation of bacterial
pathogens and other fastidious bacteria. Most pathogenic bacteria of animals, which have
adapted themselves to growth in animal tissues, require complex media for their growth. Blood,
serum and tissue extracts are frequently added to culture media for the cultivation of pathogens.

3) Classification based on functional use or application:

These include basal media, enriched media, selective/enrichment media, indicator/differential
media, transport media and holding media.
A) Basal media are basically simple media that supports most non-fastidious bacteria.
Peptone water, nutrient broth and nutrient agar considered basal medium.
B) Enriched media: Addition of extra nutrients in the form of blood, serum, egg yolk etc, to
basal medium makes them enriched media. Enriched media are used to grow nutritionally
exacting (fastidious) bacteria. Blood agar, chocolate agar, Loeffler’s serum slope etc are few of
the enriched media.
C) Selective and enrichment media. A selective medium is one which has a component(s)
added to it which will inhibit or prevent the growth of certain types or species of bacteria and/or
promote the growth of desired species. While selective media are agar based, enrichment
media are liquid in consistency. Both these media serve the same purpose. Any agar media can
be made selective by addition of certain inhibitory agents that don’t affect the pathogen. Various
approaches to make a medium selective include addition of antibiotics, dyes, chemicals,
alteration of pH or a combination of these. Example: bile salts inhibit growth of most gram-
positive bacteria and some gram-negative bacteria, but enteric bacteria adapted to life in animal
gut can grow well. Include bile salts in some media such as EMB, MacConkey agar to select for
enterics.
D) Enrichment media are liquid media that also serves to inhibit commensals in the clinical
specimen. Selenite F broth, tetrathionate broth and alkaline peptone water are used to recover
pathogens from fecal specimens.
E) Differential media or indicator media: Certain media are designed in such a way that

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different bacteria can be recognized on the basis of their colony colour. Various approaches
include incorporation of dyes, metabolic substrates etc, so that those bacteria that utilize them
appear as differently coloured colonies. Such media are called differential media or indicator
media. Exmples: MacConkey’s agar, CLED agar, TCBS agar, XLD agar etc. . Ex: MacConkey
agar has color indicator that distinguishes presence of acid. Bacteria that ferment a particular
sugar (e.g., glucose in culture media) will produce acid wastes on plates, turn pH indicator red.
Bacteria that cannot ferment the same sugar will grow but not affect pH, so colonies remain
white.
F) Transport media: Clinical specimens must be transported to the laboratory immediately after
collection to prevent overgrowth of contaminating organisms or commensals. This can be
achieved by using transport media. Such media prevent drying (desiccation) of specimen,
maintain the pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria. Some of
these media (Stuart’s & Amie’s) are semi-solid in consistency. Addition of charcoal serves to
neutralize inhibitory factors. Cary Blair medium and Venkatraman Ramakrishnan medium are
used to transport feces from suspected cholera patients. Sach’s buffered glycerol saline is used
to transport feces from patients suspected to be suffering from bacillary dysentery.
Preparation and preservation
Care must be taken to adjust the pH of the medium before autoclaving. Various pH indicators
that are in use include phenol red, neutral red, bromothymol blue, bromocresol purple etc.
Dehydrated media are commercially available and must be reconstituted as per manufacturers’
recommendation. Most culture media are sterililized by autoclaving. Certain media that contain
heat labile components like glucose, antibiotics, urea, serum, blood are not autoclaved. These
components are filtered and may be added separately after the medium is autoclaved. Certain
highly selective media such as Blair’s medium and TCBS agar need not be sterilized. It is
imperative that a representation from each lot be tested for performance and contamination
before use. Once prepared, media may be held at 4-5˚C in the refrigerator for 1-2 weeks.
Certain liquid media in screw capped bottles or tubes or cotton plugged can be held at room
temperature for weeks.
Preserving cultures
a. Preserving cultures is important for:
i. scientific reasons
ii. identification
iii. vaccine production
iv. industrial use
b. Methods of preserving cultures include:
i. Refrigeration at 4˚C can be effective for short periods.
ii. Stabs -Cultures are stabbed deeply into agar using a inoculating needle. The
stabs are incubated until visible cultures form, then sealed and stored at room or
lower temperature
iii. Slants Cultures may be streaked onto the surface of the solid medium in a slant
tube.The slants are incubated until visible culture formation then sealed and
stored at room or lower temperature.
iv. Lyophilization -Lyophilization is the freeze-drying of cultures. Cultures are first
frozen and then dried under high vacuum. To revive cultures they are rehydrated
by broth. Lyophilization can be an effective long term method of storage

Freezing -Broth cultures are mixed with various ingredients (e.g., glycerol) to limit damage upon
freezing and then frozen to temperatures ranging from -50˚C to -95˚C. To revive cultures they
are thawed, pelletted, and resuspended into broth. Freezing can be an effective long term
method of storage

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Microbial Growth curve
A microbial lab culture typically passes through 4 distinct, sequential phases of growth that form
the standard bacterial growth curve.

1.       Lag Phase - In the lag phase, the number of cells doesn't increase. 
However, considerable metabolic activity is occurring as the cells prepare to grow. This
lag in division is associated with a physiological adaptation to the new environment, by
the cells, prior to their resumption of division. That is, cells may increase in size during
this time, but simply do not undergo binary fission.

2.      Log Phase (logarithmic or exponential phase) - cell numbers increase

exponentially; during each generation time, the number of cells in the population
increases by a factor of two). This phase of growth is called logarithmic or
exponential because the rate of increase in cell number is a multiplicative
function of cell number. The number of microbes in an exponentially increasing
population increases slowly at first, then extremely rapidly.  Organisms in a tube
of culture medium can maintain log growth for only a limited time, as nutrients
are used up, metabolic wastes accumulate, microobes suffer from oxygen
depletion.
3.    Stationary Phase – the death rate equals the growth rate.The number of cells
doesn't increase, but changes in cells occur: cell become smaller and
synthesize components to help them survive longer periods without growing
(some may even produce endospores); the signal to enter this phase may have
to do with overcrowding (accumulation of metabolic byproducts, depletion of
nutrients, etc.).
4.    Death Phase - In this phase, cells begin to die out.  Death occurs
exponentially, but at a low rate.  Death occurs because cells have depleted all

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 the nutrients. The metabolic byproducts become toxic to the cells so they lyse. 

Continuous Culture of Microbes

In the lab, cultures usually pass through all growth phases - not in nature.  In nature, nutrients
continuously enter the cell's environment at low concentrations, and populations grow
continually at a low but steady rate.  The growth rate is set by the concentration of the scarcest
or limiting nutrient, not by the accumulation of metabolic byproducts - in nature there is always
some other microbe that can use these metabolic byproducts for their own metabolism.  In the
lab, we have to continually replace the media.
 Measuring Numbers of Microbes
 
A.  Indirect Measurements-measure a property of the mass of cells and then estimate
the number of microbes
 
1.    Turbidity – Can hold tube up to the light and look for cloudiness as evidence of
growth (difficult to detect slight growth).  A spectrophotometer can measure how
much light a solution of microbial cell transmits; the greater the mass of cells in
the culture, the greater its turbidity (cloudiness) and the less light that will be
transmitted.  Disadvantages:  Not sensitive in terms of numbers of bacterial cells
& not useful for detecting minor contamination.
2.    Metabolic Activity  - 3 ways:
a.       The rate of formation of metabolic products, such as gases or acids, that a
culture produces.
b.      The rate of utilization of a substrate, such as oxygen or glucose.
c.       The rate of reduction of certain dyes.  Ex. methylene blue becomes
colorless when reduced.
      B.  Direct Measurements - Give more accurate measurements of numbers of microbes.
1.    Direct Counts - Coulter Counter - electronic counter; rapid & accurate only if
bacterial cells are the only particles present in the solution. [gives a total count -
live & dead cells].
3.    Plate Count – Bacterial colonies are viewed through the magnifying glass
against a colony-counting grid; called a Quebec colony counter which gives a
viable count
4.    Filtration - A known volume of liquid or air is drawn through a membrane filter by
vacuum.  The pores in the filter are too small for microbial cells to pass through. 
Then the filter is placed on an appropriate solid medium and incubated.  The
number of colonies that develop is the number of viable microbial cell in the
volume of liquid that was filtered. It gives a viable count.
Isolating pure cultures
Bacteria in natural circumstances are almost always found as mixtures of many species. For
most purposes, it is necessary to isolate the various organisms in pure culture before they can
be identified and studied. To obtain separated colonies from a mixed culture, various isolation

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methods can be used.

One is the streak plate method, in which a sample of mixed bacteria is streaked several times
along one edge of a Petri dish containing a medium such as nutrient agar. A loop is flamed and
then touched to the first area to retrieve a sample of bacteria. This sample is then streaked
several times in the second area of the medium. The loop is then reflamed, touched to the
second area, and streaked once again in the third area. The process can be repeated in a fourth
and fifth area if desired. During incubation, the bacteria will multiply rapidly and form colonies
(Figure 1 ).

A second isolation method is the pour plate method. In this method, a sample of bacteria is
diluted in several tubes of melted medium such as nutrient agar. After dilution, the melted agar
is poured into separate Petri dishes and allowed to harden. Since the bacteria have been diluted
in the various tubes, the plates will contain various dilutions of bacteria, and where the bacteria
are most diluted, they will form isolated colonies (Figure 1 ).

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Figure 1Two processes for isolating bacteria from a mixed culture. (a) The streak plate
technique. (b) The pour plate technique.

The third method is the Spreading a plate. Spreading a plate is an additional method of
quantifying microorganisms on solid medium. Instead of embedding microorganisms into agar,
as is done with the pour plate method, liquid cultures are spread on the agar surface using a
sterile swab or a sterile glass rod. A suspended inoculum is palced at the cemtre of the solid
media and spread using a sterile swab or glass rod evenly on the plate. An advantage of
spreading a plate over the pour plate method is that cultures are never exposed to 45C melted
agar temperatures which can kill the bacteria.
Incubation of Inoculated Media

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Inoculated media should be incubated as soon as possible. A delay in incubation can affect the
viability of pathogens especially anaerobes, pneumococci, meningococci, gonococci, and
Haemophilus influenzae. It can also increase the risk of plates becoming contaminated from
small insects and dust. Uninoculated and inoculated media must be protected from sunlight.
Microorganisms require incubation at the temperature and in the humidity and gaseous
atmosphere most suited to their metabolism. The length of time of incubation depends on how
long an organism takes to develop the cultural characteristics by which it is recognized.

Temperature of incubation
The temperature at which a microorganism grows best is referred to as its optimum
temperature. The temperature below which growth stops (not necessarily resulting in death) is
called the minimum temperature, and that above which growth stops and death occurs is called
the maximum temperature. The temperature selected for routine culturing is 35–37°C.
Growth Factors
Microbes can exist in a great many environments because they are small, easily
dispersed, need only small quantities of nutrients, are diverse in their nutritional
requirements.
 
A.   Physical Factors
1.    pH – bacteria can classified as:
a.       acidophiles (acid-loving) – grow best at a  pH of 1 to 5.4; Ex.
Lactobacilllus (ferments milk)
b.      neutrophiles – exist from pH to 5.4 to 8.5; most bacteria that cause
human disease are in this category.
c.       alkaliphiles (base loving) – exist from pH to 7.0 to 11.5; ex. Vibrio
cholerae (causes cholera)
 
2.    Temperature – bacteria can be classified as:

a.     psychrophiles (cold-loving) 15oC to 20oC; some can grow at 0oC.

b.    mesophiles  - grow best between 25oC and 40 C; human body temp
is 37oC.

c.     thermophiles (heat-loving) – 50oC to 60oC; found in compost heaps

and in boiling hot springs.
 
3.    Moisture – only the spores of sport-forming bacteria can exist in a
dormant state in a dry environment.
 
B.    Oxygen Requirements
1.    strict or obligate anaerobes – oxygen kills the bacteria; ex. Clostridium

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 tetani
2.    strict or obligate aerobes – lack of oxygen kills the bacteria; ex.
Pserdomonas
3.    facultative anaerobes – can shift their metabolism (anaerobic if oxygen
is absent or aerobic if oxygen is present); ex. E. coli, Staphylococcus
4.    aerotolerant – the bacteria don’t use oxygen, but oxygen doesn’t harm
them; ex. Lactobacillus
5.    microaerophiles – like low oxygen concentrations and higher carbon
dioxide concentrations; ex. Campylobacter
 
C.    Nutritional (Biochemical) Factors – Nutrients needed by microorganisms
include:
o Carbon – carbon containing compounds are needed as an energy source
(ex. glucose) and for building blocks.
o Nitrogen - needed for amino acids and nucleotides; some can synthesize
all 20 amino acids; others have to have some provided in their medium.
o Sulfur – needed for amino acids, coenzymes,
o Phosphorus – needed for ATP, phospholipids, and nucleotides
o Vitamins – a vitamin is an organic substance that an organism requires in
small amounts and that is typically used as a coenzyme; many bacteria
make their own, but some are required in the medium; microbes living in
the human intestine manufacture vitamin K, needed for blood clotting, and
some of the B vitamins, thus benefiting their host

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