Culture Media, the birth of bacteriology

While perhaps best known to us as a cause of human disease, bacteria really should be far more famous
for their positive contributions than for their negative ones. Bacteria were first observed by Anton von
Leeuwenhoek in the late 17th century, but didn’t become the objects of serious scientific study until the
19th century, when it became apparent that some species caused human diseases. The methods devised
by Robert Koch, Louis Pasteur, and their associates during the “Golden Age” of microbiology, which
spanned from the mid-1800s to early 1900s, are still widely used today. Most of these methods involved
isolating single bacteria derived from a natural source (such as a diseased animal or human) and
cultivating them in an artificial environment as a pure culture to facilitate additional studies. During the
middle of the twentieth century, when we believed we had defeated them at their disease-causing game,
bacteria became popular subjects of empirical study in fields such as genetics, genetic engineering, and
biochemistry. With the evolution of antibiotic-resistant strains and our increased knowledge of bacterial
stealth attack strategies such as biofilms and intracellular growth, medical researchers have refocused
their attention on disease-causing bacteria and are looking for new ways to defeat them.

Growing bacteria in pure culture is still one of the most widely used methods in microbiology. Many
bacteria, particularly those that cause diseases and those used in scientific studies, are heterotrophic,
which means that they rely on organic compounds as food, to provide energy and carbon. Some bacteria
also require added nutritional components such as vitamins in their diet. An appropriate physical
environment must be created, where important factors such as temperature, pH, and the concentration of
atmospheric gases (particularly oxygen) are controlled and maintained. The nutritional needs of bacteria
can be met through specialized microbiological media that typically contain extracts of proteins (as a
source of carbon and nitrogen), inorganic salts such as potassium phosphate or sodium sulfate, and in
some cases, carbohydrates such as glucose or lactose. For fastidious bacteria (meaning, those that are
picky eaters) vitamins and/or other growth factors must be added as well.

Koch, Pasteur, and their colleagues in the 19th and early 20th centuries created media formulations that
contained cow brains, potatoes, hay, and all sorts of other enticing microbial edibles. Today,
bacteriological media formulations can be purchased in powdered form, so that all the preparer has to
do is to measure out the correct amount, add the right amount of water, and mix. After the basic formula
has been prepared, the medium is sterilized in an autoclave, which produces steam under pressure and
achieves temperatures above boiling. Once sterilized media has cooled, it is ready to be used. A
population of bacteria grown in the laboratory is referred to as a culture. A pure culture contains only
one single type; a mixed culture contains two or more different bacteria. If a bacterial culture is left in
the same media for too long, the cells use up the available nutrients, excrete toxic metabolites, and
eventually the entire population will die. Thus bacterial cultures must be periodically transferred, or
subcultured, to new media to keep the bacterial population growing.

Microbiologists use subculturing techniques to grow and maintain bacterial cultures, to examine cultures
for purity or morphology, or to determine the number of viable organisms. In clinical laboratories,
subculturing is used to obtain a pure culture of an infectious agent, and also for studies leading to the
identification of the pathogen. Because bacteria can live almost anywhere, subculturing steps must be
performed aseptically, to ensure that unwanted bacterial or fungal contamination is kept out of an
important culture.
Cultivation of microorganisms: Definition and types of culture media (chemically defined media,
complex media, selective media, differential media and enriched media); aerobic and anaerobic culture
methods; pure culture, mixed culture and contaminated culture, sterilization.

Culture media: Culture media are used in microbiological laboratories to grow different kinds of
microorganisms. A culture medium is composed of different nutrients those are essential for microbial
growth. To be cultured successfully, bacteria require the provision of nutrients in the culture medium.
There are many different formulations available to suit the differing nutritional needs of bacterial species.
The type of medium you choose will depend on the purpose of the culture. Rich, nutrient or complete
media can be helpful when trying to bulk up a pure culture and get the bacterial cells in good condition.
Minimal media on the other hand will supply only the bare necessities for survival and can be useful in
manipulating which pathways are turned on in the bacterium. Culture media contain nutrients and
physical growth parameters necessary for microbial growth. All microorganisms cannot grow in a single
culture medium and in fact many can’t grow in any known culture medium. Organisms that cannot grow
in artificial culture medium are known as obligate parasites. Mycobacterium leprae, Rickettsia,
Chlamydia, and Treponema pallidum are obligate parasites. Bacterial culture media can be classified on
the basis of composition, consistency and purpose. Media may also be classed as defined or undefined.
As the names suggests, in a defined media, all the ingredients are known. Undefined media tend to
contain complex mixtures of nutrients and chemical species in unknown proportions, such as yeast
extract. Whichever medium is chosen, this may be in liquid form as a broth culture, or agar may be added
to set the media and allow bacterial cells to be grown on a solid surface. Bacterial culture media can be
divided many ways.

On the basis of consistency of culture media can be grouped into three as follows:

(1) Solid medium: Solid medium contains agar at a concentration of 1.5-2.0% or some other, mostly
inert solidifying agent. Solid medium has physical structure and allows bacteria to grow in physically
informative or useful ways (e.g. as colonies or in streaks). Solid medium is useful for isolating bacteria
or for determining the colony characteristics of the isolate.

(2) Semisolid media: Semisolid media are prepared with agar at concentrations of 0.5% or less. They
have soft custard like consistency and are useful for the cultivation of microaerophilic bacteria or for
determination of bacterial motility.

(3) Liquid (Broth) medium: Liquid medium contains specific amounts of nutrients but don’t have trace
of gelling agents such as gelatin or agar. Culture in liquid media, also known as a broth culture, gives
the bacteria present easy access to the available nutrients compared to static bacterial colonies. Gentle
agitation to keep the bacteria dispersed through the medium during incubation can aid this access further.
Liquid media will also dilute out waste products as they are formed, distributing them through the
culture. Consequently, a greater mass of bacteria may be obtained for an equivalent volume of liquid as
opposed to solid media.
On the basis of composition media can be grouped into two as follows:

(1) Synthetic or chemically defined medium: A chemically defined medium is one prepared from
purified ingredients and therefore its exact composition is known.

(2) Non synthetic or chemically undefined medium: Non-synthetic medium contains at least one
component that is neither purified nor completely characterized nor even completely consistent from
batch to batch. Often these are partially digested proteins from various organism sources. Nutrient broth,
for example, is derived from cultures of yeasts.

On the basis of purpose/functional use/application culture media can be grouped into five as
follows:

(1) General purpose media/Basic media: Basal media are basically simple media that supports most
non-fastidious bacteria. Peptone water, nutrient broth and nutrient agar (NA) are considered as basal
medium. These media are generally used for the primary isolation of microorganisms.

(2) Enriched medium (Added growth factors): Addition of extra nutrients in the form of blood, serum,
egg yolk etc. to basal medium makes 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. Blood agar is prepared by adding 5-10% (by volume) blood to a blood agar base
Chocolate agar is also known as heated blood agar or lysed blood agar.

(3) Selective media: Selective media are also available that promote or suppress the growth of certain
species, groups of species or strains with particular properties. This may be based on a strain’s ability to
utilize specific nutrients, produce certain byproducts or resistance to certain antibiotics. Selection may
be used in both broth and solid media. 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 of interest. Various
approaches to make a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH
or a combination of these. Examples of selective media include: Mannitol Salt Agar and Salt Milk Agar
used to recover S. aureus contains 10% NaCl, MacConkey Agar used for Enterobacteriaceae members
contains bile salt that inhibits most Gram positive bacteria, Cetrimide Agar used to recover Pseudomonas
aeruginosa contains cetrimide (antiseptic agent), Crystal Violet Blood Agar used to recover S. pyogenes
contains 0.0002% crystal violet.

(4) Differential/indicator media: Differential appearance: Certain media are designed in such a way
that 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. Differential
media allow the growth of more than one microorganism of interest but with morphologically
distinguishable colonies. Examples of differential media include: Mannitol Salt Agar (MSA) (mannitol
fermentation = yellow), Blood Agar (various kinds of hemolysis i.e. α, β and γ hemolysis), MacConkey
Agar (lactose fermenters, pink colonies whereas non- lactose fermenter produces pale or colorless
colonies, Thiosulfate Citrate Bile Salt Sucrose (TCBS) (Vibrio cholerae produces yellow colonies due
to fermentation of sucrose).
(5) 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.

Generalized media
Nutrient Agar: Nutrient agar is a general purpose, nutrient medium used for the cultivation of microbes
supporting growth of a wide range of non-fastidious organisms. Nutrient agar is popular because it can
grow a variety of types of bacteria and fungi, and contains many nutrients needed for the bacterial
growth.

Composition of Nutrient Agar

Peptone 0.5%- It is an enzymatic digest of animal protein. Peptone is the principal source of organic
nitrogen for the growing bacteria.
Beef extract/yeast extract 0.3%- It is the water-soluble substances which aid in bacterial growth, such
as vitamins, carbohydrates, organic nitrogen compounds and salts.
NaCl 0.5%- The presence of sodium chloride in nutrient agar maintains a salt concentration in the
medium that is similar to the cytoplasm of the microorganisms.
Agar 1.5%- It is the solidifying agent.
Distilled water 100 ml- Water is essential for the growth of and reproduction of micro-organisms and
also provides the medium through which various nutrients can be transported.
pH- It is adjusted to neutral 7.0 ±0.2 at 25 °C

Uses of Nutrients Agar: (1) It is frequently used for isolation and purification of cultures, (2) It can also
be used as a means for producing the bacterial lawns needed for antibiotic sensitivity tests. Actuality,
antibiotic sensitivity testing is typically performed on media specially formulated for that purpose.

Agar (agar agar): Agar consists of a mixture of two polysaccharides: agarose and agaropectin, with
agarose making up about 70% of the mixture. Agarose is a linear polymer, made up of repeating units
of agarobiose, a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agaropectin
is a heterogeneous mixture of smaller molecules that occur in lesser amounts, and is made up of
alternating units of D-galactose and L-galactose heavily modified with acidic side-groups, such as sulfate
and pyruvate. In and of itself, agar provides no nutrient support for bacteria. An agar-agar solution in
hot water forms a characteristic gel after setting, with a melting point between 85°C to 95°C, and a
gelling point between 32º a 45º C. This physical property makes the gel very useful as an additive when
used in many applications in the food industry. Since many scientific applications require incubation at
temperatures close to human body temperature (37°C), agar is more appropriate than other solidifying
agents that melt at this temperature, such as gelatin. Gracilaria lichenoides is the source of agar.

History of agar: Agar may have been discovered in Japan in 1658 by Mino Tarōzaemon, an innkeeper
in Kyoto who, according to legend, was said to have discarded surplus seaweed soup (Tokoroten) and
noticed that it gelled later after a winter night's freezing. Over the following centuries, agar became a
common gelling agent in several Southeast Asian cuisines. Agar was first subjected to chemical analysis
in 1859 by the French chemist Anselme Payen, who had obtained agar from the marine algae Gelidium
corneum. Beginning in the late 19th century, agar began to be used as a solid medium for growing various
microbes. Agar was first described for use in microbiology in 1882 by the German microbiologist
Walther Hesse, an assistant working in Robert Koch's laboratory, on the suggestion of his wife Fanny
Hesse. Agar quickly supplanted gelatin as the base of microbiological media, due to its higher melting
temperature, allowing microbes to be grown at higher temperatures without the media liquefying. With
its newfound use in microbiology, agar production quickly increased. This production centered on Japan,
which produced most of the world's agar until World War II. However, with the outbreak of World War
II, many nations were forced to establish domestic agar industries in order to continue microbiological
research. Around the time of World War II, approximately 2,500 tons of agar were produced annually.
By the mid-1970s, production worldwide had increased dramatically to approximately 10,000 tons each
year. Since then, production of agar has fluctuated due to unstable and sometimes over-utilized seaweed
populations.

Selective as well as differential medium-1

MacConkey agar: MacConkey agar is a selective and differential culture medium for bacteria designed
to selectively isolate Gram-negative and enteric (normally found in the intestinal tract) bacilli and
differentiate them based on lactose fermentation. The crystal violet and bile salts inhibit the growth of
Gram-positive organisms which allows for the selection and isolation of gram-negative bacteria. Enteric
bacteria that have the ability to ferment lactose can be detected using the carbohydrate lactose, and the
pH indicator neutral red. It contains bile salts (to inhibit most Gram-positive bacteria), crystal violet dye
(which also inhibits certain Gram-positive bacteria), neutral red dye (which turns pink if the microbes
are fermenting lactose).
Composition:
• Bacto Peptone – 17 g
• Proteose peptone – 3 g
• Lactose – 10 g
• Bile salts – 1.5 g
• Sodium chloride – 5 g
• Neutral red – 0.03 g
• Crystal violet – 0.001 g
• Agar – 13.5 g
• Water – 1 litre
• pH - 7.0±0.2

Principle of MacConkey Agar: MacConkey agar is used for the isolation of Gram-negative enteric
bacteria and the differentiation of lactose fermenting from lactose non-fermenting Gram-negative
bacteria. Pancreatic digest of gelatin (bacto peptone) and proteose peptones (meat and casein) provide
the essential nutrients, vitamins and nitrogenous factors required for growth of microorganisms. Lactose
monohydrate is the fermentable source of carbohydrate. The selective action of this medium is attributed
to crystal violet and bile salts, which are inhibitory to most species of Gram-positive bacteria. Sodium
chloride maintains the osmotic balance in the medium. Neutral red is a pH indicator that turns red at a
pH below 6.8 and is colorless at any pH greater than 6.8. Agar is the solidifying agent.
Uses of MacConkey Agar: (1) MacConkey agar is used for the isolation of Gram-negative enteric
bacteria. (2) It is used in the differentiation of lactose fermenting from lactose non-fermenting Gram-
negative bacteria. (3) It is used for the isolation of coliforms and intestinal pathogens in water, dairy
products and biological specimens.

Selective as well as differential medium-2

Mannitol salt agar: Mannitol salt agar or MSA is a commonly used selective and differential growth
medium in microbiology. It encourages the growth of a group of certain bacteria while inhibiting the
growth of others. This medium is important in medical laboratories as one method of distinguishing
pathogenic microbes in a short period of time. It contains a high concentration (about 7.5–10%) of salt
(NaCl), making it selective for most Gram-negative and some Gram-positive bacteria (Staphylococcus,
Enterococcus and Micrococcaceae) since this level of salt is inhibitory to most other bacteria. It is also
a differential medium for mannitol-fermenting staphylococci, containing carbohydrate mannitol and the
indicator phenol red, a pH indicator for detecting acid produced by mannitol-fermenting staphylococci.
Staphylococcus aureus produces yellow colonies with yellow zones, whereas other coagulase-negative
staphylococci produce small pink or red colonies with no colour change to the medium. If an organism
can ferment mannitol, an acidic byproduct is formed that causes the phenol red in the agar to turn yellow.
It is used for the selective isolation of presumptive pathogenic (pp) Staphylococcus species.

Dr. Mihir Lal Saha

Professor
Department of Botany
University of Dhaka