Culture Media

General Characters(Cont..)
• Most important reasons for culturing bacteria
in vitro is its utility in diagnosing infectious
diseases.
• Isolating a bacterium from sites in body
normally known to be sterile is an indication
of its role in the disease process.
• Culturing bacteria is also the initial step in
studying its morphology and its identification.
General Characters(Cont..)
• Bacteria have to be cultured in order to obtain
antigens for developing serological assays or
vaccines.
• Culturing bacteria also provide a reliable way
estimating their numbers (viable count).
• Culturing on solid media is another
convenient way of separating bacteria in
mixtures.
 History
• Louis Pasteur used simple broths made up of urine or
meat extracts.
• Robert Koch realized used potato pieces to grow
bacteria.
• Wife of Walther Hesse (who was an assistant to
Robert Koch) used agar to solidify culture media.
• Before the use of agar, attempts were made to use
gelatin as solidifying agent. Gelatin had some inherent
problems; it existed as liquid at normal incubating
temperatures (35-37oC) and was digested by certain
bacteria.
 Composition of Media
• Bacteria infecting humans (commensals or
pathogens) are chemoorganoheterotrophs.
• When culturing bacteria, it is very important
to provide similar environmental and
nutritional conditions that exist in its natural
habitat.
 Characteristics of Agar Media
• Seaweeds
• Long chain polysaccharide
• No nutrients value
• Melts at 98 ⁰C
• Sets at 42 ⁰C
• Approx. 2% agar for solid media
 Common Media
• Peptone- mixture of partially digested
peptone
• Proteoses, polypeptides, amino acids
• Salts like phosphates, potassium and
Magnasium
• Growth factors
• Blood, serum, yeast extract, meat extract
 Classification of Media
• Based on
– consistency
– nutritional component
– functional use
 Classification based on consistency

• Liquid
• Semi-solid
• Solid and biphasic
 Liquid Media
• In liquid medium, bacteria grow uniformly
producing general turbidity.
• Certain aerobic bacteria and those containing
fimbriae (Vibrio & Bacillus) are known to grow
as a thin film called ‘surface pellicle’ on the
surface of undisturbed broth. Sometimes the
initial turbidity may be followed by clearing due
to autolysis, which is seen in penumococci.
 Liquid Media (Cont..)
• Long chains of Streptococci when grown in
liquid media tend to entangle and settle to the
bottom forming granular deposits.
• Liquid media tend to be used when a
largenumber of bacteria have to be grown.
These are suitable to grow bacteria when the
numbers in the inoculum is suspected to be
low.
 Semi solid media
• 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 (U-
tube and Cragie’s tube).
• Certain transport media such as Stuart’s and
Amies media are semi-solid in consistency.
 Solid Media
• Any liquid medium by the addition of certain
solidifying agents.
• Agar agar (simply called agar) is the most commonly
used solidifying agent.
• Egg yolk and serum too can be used to solidify
culture media
• 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
 Agar agar
• It melts at 95⁰C (sol) and solidifies at 42⁰C
(gel)
• No nutritive property
• Not hydrolyzed by most bacteria
• Usually free from growth promoting or growth
retarding substances
• Most commonly, it is used at concentration of
1-3% to make a solid agar medium
 Biphasic Media
• Sometimes, a culture system comprises of
both liquid and solid medium in the same
bottle. (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.
 Based on Nutritional Component
• Media can be classified as simple, complex and
synthetic (or defined).
• Minimal requirements - non-fastidious
• Extra nutrients-fastidious.
• Simple media-peptone water, nutrient agar
• Complex media such as blood agar have
ingredients whose exact components are
difficult to estimate.
• Synthetic or defined media-Davis & Mingioli
media
 Classification based on functional use or application

• Basal media
• Enriched media
• Selective/enrichment media
• Indicator/differential media
• Transport media
• Holding media.
 Basal Media
• Simple media that supports most non-
fastidious bacteria.
• Peptone water, nutrient broth and nutrient
agar considered basal medium
 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.
 Selective media
• Are designed to inhibit unwanted commensal
contaminating bacteria and help to recover
pathogen from a mixture of bacteria.
• While selective media are agar based
• 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
 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.
• Both selective media and enrichment media
serve same purpose.
 Differential media or indicator media
• 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.
• MacConkey’s agar, CLED agar, TCBS agar, XLD
agar etc.
 Transport media
• Clinical specimens must be transported to the
laboratory immediately after collection to prevent
overgrowth of contaminating organisms or
commensals.
• Such media prevent drying (desiccation) of specimen,
maintain the pathogen to commensal ratio and
inhibit overgrowth of unwanted bacteria.
• Stuart’s & Amie’s are semi-solid in consistency.
Addition of charcoal serves to neutralize inhibitory
factors.
 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. 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.
 Robertson cooked meat media
• Commonly used to grow Clostridium sps.
medium contain a 2.5 cm column of bullock heart
meat and 15 ml of nutrient broth.
• Before use the medium must be boiled in
waterbath to expel any dissolved oxygen and
then sealed with sterile liquid paraffin.
• Methylene blue or resazurin is an oxidation-
reduction potential indicator that is incorporated
in the thioglycollate medium. Under reduced
condition, methylene blue is colourless.