Culture media for culturing microorganisms

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CONTENTS
Serial
no.
Name of the content Page no.
1. Introduction
2. History of culture media
3. Characteristics of Agar
4. Classification of culture media
5. Procedures for the production of culture media
6. Conclusions
7. References

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Culture media of Microorganism
Introduction:
A culture medium is a solid or liquid preparation of nutrient or combination of nutrients used to
grow, transport, and store microorganisms. Much of the study of microbiology depends on the
ability to grow and maintain microorganisms in the laboratory, and this is possible only if
suitable culture media are available. To be effective, the medium must contain all the nutrients
the microorganism requires for growth.
Although all microorganisms require energy sources, carbon, nitrogen, phosphorus, sulphur and
various minerals, the precise composition of a satisfactory medium depends on the microbial
species one is trying to grow because nutritional requirements vary so greatly. Knowledge about
microorganism’s natural habitat often is useful in selecting an appropriate culture medium
because its nutritional requirements reflect its natural surroundings.
In preparing a culture medium for any microorganism, the primary goal is to provide a balanced
mixture of the required nutrients at concentrations that will permit good growth. Additionally,
the culturing of microorganisms requires careful control of various environmental factors which
normally are maintained within narrow limits.
Microbiological culture media, however, consist of various nutrient substances supporting the
growth of particular types of microorganisms. Some media contain solutions of inorganic salts
and may be supplemented with one or more organic compounds.
Other media are prepared from complex ingredients such as extracts or digests of plant and
animal tissues. Culture media would, thereinafter, be called ‘media’ (sing, medium) .

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Figure:set bags of culture media.
History of culture media:
The origins of microbiological culture media can be traced to the 19th Century when the science
of bacteriology was just beginning.
 Arguably the first to cultivate microorganisms on a growth medium,with a degree of
reproducibility was the French chemist and microbiologist Louis Pasteur (1922-1985).
Whilst acting as the administrator andndirector of scientific studies at the École
Normale,Pasteur fashioned a media of yeast, ash, candy sugar and ammonium salts in
1860.The object was to produce a fermentation medium. This medium contained the
basic requirements for microbial growth: nitrogen (ammonium salts), a carbon source
(sugar) and vitamins (ash).
 A wider application of materials was utilised, and consequently greater success
observed,When Robert Koch (1843-1910) discovered that broths based on fresh beef
serum or meat extracts (so-called bouillons, the term ‘broth’ for liquid culture medium
being analogous to broth or soup) produced optimal growth.Indeed, Koch’s work was so
groundbreaking that the cognomen ‘The Father of Culture Media’, oft stated in many
microbiological text books, is not misplaced.
 In 1882, Koch’s attempts at a nutrient medium were advanced.Fannie Eilshemius (1850-
1934), the wife of Dr. Walther Hesse(who was Koch’s research assistant), suggested
replacing gelatine with agar.
 Initially all types of media, starting with Koch and his researchers,produced were what is
now described as ‘natural’ or ‘empirical’ culture media. Such media is commonly used
today. This included utilising substances including milk, urine, diluted blood, vegetable
juices, meat extracts and infusions and so on. Most empirical media contain only peptone
as the major ingredient (a trypsinized or hydrolysed protein).

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Characteristics of Agar:
 Agar melts above 95°C.
 Once melted, does not solidify until it reaches 40°C.
 It used for preparing solid medium.
 It has no nutritive value.
 2%agar is employed I solid medium.
 Not affected by the growth of bacteria.
 O
 4120.21kijiuk8btained from seaweeds.
 Originally used as food thickener(Angelina Hesse).
A.Classificationofculture media used in Microbiologylaboratory on the basis
of consistency:
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:
They 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:
These media contains specific amounts of nutrients but don’t have trace of gelling agents
such as gelatin or agar. Broth medium serves various purposes such as propagation of
large number of organisms, fermentation studies, and various other tests. e.g. sugar
fermentation tests, MR-VR broth.
B.Classification of culture media based on the basis of composition:
1. Synthetic or chemically defined medium:
A chemically defined medium is one prepared from purified ingredients and therefore
whose 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.
Synthetic medium may be simple or complex depending up on the supplement
incorporated in it. A simple non-synthetic medium is capable of meeting the nutrient

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requirements of organisms requiring relatively few growth factors where as complex non-
synthetic medium support the growth of more fastidious microorganisms.
C.Classification of Bacterial Culture Media based on the basis of purpose/
functional use/ application:
Many special purpose media are needed to facilitate recognition, enumeration, and
/isolation of certain types of bacteria. To meet these needs, numerous media are available.
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 are considered as basal medium. These
media are generally used for the primary isolation of microorganisms.
Figure:Nutrient Agar
2. Enriched medium (Added growth factors):
Figure:Blood Agar

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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. 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 and enrichment media:
Selective and enrichment media are designed to inhibit unwanted commensal or
contaminating bacteria and help to recover pathogen from a mixture of bacteria. 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 of interest. Various approaches to
make a medium selective include addition of antibiotics, dyes, chemicals, alteration of
pH or a combination of these.
a) Selective medium:
Principle: Differential growth suppression
Selective medium is designed to suppress the growth of some microorganisms while
allowing the growth of others. Selective medium are agar based (solid) medium so
that individual colonies may be isolated.
Examples of selective media include:
1. Thayer Martin Agar used to recover Neisseria gonorrhoeae contains antibiotics;
vancomycin, colistin and nystatin.
2. Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contains 10% NaCl.
3. Potassium tellurite medium used to recover C.diphtheriae contains 0.04% potassium
tellurite.
4. MacConkey’s Agar used for Enterobacteriaceae members contains bile salt that inhibits
most gram positive bacteria.

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Figure: MacConkey’s Agar
5. Pseudosel Agar (Cetrimide Agar) used to recover P. aeruginosa contains cetrimide
(antiseptic agent).
6. Lowenstein Jensen Medium used to recover M. tuberculosis is made selective by
incorporating malachite green.
7. Wilson and Blair’s Agar for recovering S. typhi is rendered selective by the addition of
dye brilliant green.
8. Selective media such as TCBS Agar used for isolating V. cholerae from fecal specimens
have elevated pH (8.5-8.6), which inhibits most other bacteria.
b) Enrichment culture medium:
Enrichment medium is used to increase the relative concentration of certain
microorganisms in the culture prior to plating on solid selective medium. Unlike
selective media, enrichment culture is typically used as broth medium. Enrichment
media are liquid media that also serves to inhibit commensals in the clinical
specimen. Selenite F broth, tetrathionate broth and alkaline peptone water (APW) are
used to recover pathogens from fecal specimens.

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Figure: Culture media for common bacteria.
4. Differential/ indicator medium:
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:
1. Mannitol salts agar (mannitol fermentation = yellow)
2. Blood agar (various kinds of hemolysis i.e. α, β and γ hemolysis)
3. Mac Conkey agar (lactose fermenters, pink colonies whereas non- lactose fermenter
produces pale or colorless colonies.
4. 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.
 Cary Blair transport medium and Venkatraman Ramakrishnan (VR) medium are used to
transport feces from suspected cholera patients.

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 Sach’s buffered glycerol saline is used to transport feces from patients suspected to be
suffering from bacillary dysentery.
 Pike’s medium is used to transport streptococci from throat specimens.
6. Anaerobic media:
Anaerobic bacteria need special media for growth because they need low oxygen content,
reduced oxidation –reduction potential and extra nutrients.
Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin
K. Such media may also have to be reduced by physical or chemical means. Boiling the
medium serves to expel any dissolved oxygen. Addition of 1% glucose, 0.1%
thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can render a
medium reduced. Before use the medium must be boiled in water bath to expel any
dissolved oxygen and then sealed with sterile liquid paraffin.
Robertson Cooked Meat (RCM) medium that is commonly used to grow Clostridium
spps contains a 2.5 cm column of bullock heart meat and 15 ml of nutrient broth.
Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast extract and
casein hydrolysate.
Methylene blue or resazurin is an oxidation-reduction potential indicator that is
incorporated in the medium. Under reduced condition, methylene blue is colorless.
7. Assay media:
These media are used for the assay of vitamins, amino acids and antibiotics. E.g.
antibiotic assay media are used for determining antibiotic potency by the microbiological
assay technique.
Other types of medium includes-
 Media for enumeration of Bacteria,
 Media for characterization of Bacteria,
 Maintenance media etc.
Procedure for the preparation of the liquid media:
To begin with, you will need a functional autoclave. You will use it to sterilize the media at high
temperatures under pressure.After tightly securing the machine, heat it to a pressure of 15psi and
temperature of 121ºC for 15 minutes
You will also need the following:
 Distilled water,
 Clean 500 ml measuring cylinder,

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 Clean conical flask,
 Electronic weighing scale,
 Sterile media, and
 Clean test tubes.
How to make culture media: 8-step-process:
1. Weigh 6.5 grams of the sterile nutrient broth and transfer into the clean conical flask. The
manufacturer recommends a dilution of 13 g/l but we need to make only 500 ml of the
media.
2. Add 500 ml of distilled water into the measuring cylinder and transfer into the conical
flask to dilute the media.
3. Put the conical flask with the media solution from step 2 into an autoclave basket. Ensure
you properly secure the mouth of the flask with cotton wool before lowering it into the
autoclave. Secure the autoclave and start the sterilization. It will take some time to attain
sterilization temperatures. However, once you achieve the recommended
temperature/pressure combinations, hold it there for the recommended 15 minutes. That
time is adequate under these conditions to sterilize the media.
4. Allow the autoclave to cool down.
5. Put the petri dishes into a hot air oven at 80ºC for one hour to sterilize them.
6. Remove the conical flask containing the now sterile media from the autoclave. Pour 15
ml into each petri dish and seal.
7. Make sure your working bench is not only clean but also always sterile. Wipe the
surfaces with 7% alcohol and keep the UV lamp on.
8. Label the petri dishes and store in a refrigerator for later use.
Conclusions:
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 Wilson and 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.

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References:
1. Loeffler, F. (1884) Mittheil. Kaiserl. Gesunheitsante. 2. cited in Brock, T. (1998) Robert
Koch: a life in medicine and bacteriology. Science Technical Publications. Madison, WI,
USA.
2. Hungate RE (1969) A roll tube method for cultivation of strict anaerobes: Methods in
Microbiology Academic Press, London, UK 3B:117-132.
3. Collard, P.J. (1976). The development of microbiology, Cambridge: Cambridge
University Press
4. Levine, M. and Schoenlein, H. W. (1930).A Compilation of culture media. Williams and
Williams:Baltimore USA
5. Development, manufacture and control of microbiological culture media. Published by
Oxoid, Basingstoke UK

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