Culture Media

Culture media are mediums that provide essential nutrients and minerals to
support the growth of microorganisms in the laboratory.

Microorganisms have varying nature, characteristics, habitat, and even

nutritional requirements; thus, it is impossible to culture them with one type of
culture media. However, there are also microorganisms that can’t grow on a
culture media at all in any condition – these are called obligate parasites.

Culturing microorganisms is essential for diagnosing infectious diseases,

obtaining antigens, developing serological assays for vaccines, genetic studies,
and identification of microbial species.

Furthermore, it’s also essential for isolating pure cultures, storing culture stock,
studying biochemical reactions, testing microbial contamination, checking
antimicrobial agents and preservatives effect, testing viable count, and testing
antibiotic sensitivity.

This article will focus on the composition, classification, and types of culture
media used in microbiology labs to study a spectrum of microbial forms.

Nature and Classification of Culture Media

Growing microorganisms in the lab involve mimicking the organisms’ natural

habitat or environment, and this is possible in the laboratory by formulating
culture media that meets their requirements. Therefore, many culture media
were developed by scientists according to the microbial species to be cultured.

The basic media contains a source of carbon & energy, nitrogen source, growth
factors, and some trace elements. Some commonly used media components
include peptone, agar, water, casein hydrolysate, malt extract, meat extract, and
yeast extract. In addition, the pH of the medium should be set accordingly.

However, some additional components or nutrients are added to the media when
growing specific microorganisms.

Culture media can be classified in three ways: based on their consistency,

nutritional component, and applications.
Nature Of Culture Media
1. Solid media: In these media, the agar which is an unbranched long
chain of polysaccharides is added in the concentration of 1.5-2.0%.
Most commonly, 1.3% agar is used to prepare solid media in labs.
The agar-containing media solidifies at 37 ºC. Sometimes, in the
place of agar, some other inert solidifying agents are used, such as
gellan gum.

Solid media are used to grow microorganisms in their full physical

form, prepare bacterial pure cultures, or isolate bacteria to study
colony characteristics. The bacterial growth on solid media varies in
appearance as mucoid, round, smooth, rough, filamentous, irregular,
and punctiform. The media is not hydrolyzed by microorganisms and
is free from growth-inhibiting substances. Examples of solid media
are blood agar, nutrient agar, McConkey agar, and chocolate agar.

2. Semisolid media: This media has 0.2-0.5% agar concentration, and

due to the reduced agar concentration, it appears as a soft, jelly-like
substance. It’s mainly used to study the motility of microorganisms,
distinguish between motile and non-motile bacterial strains (through
U-tube and Cragie’s tube), and cultivate microaerophilic bacteria –
bacteria on this media appear as a thick line. Examples of semi-solid
media are: Hugh and Leifson’s oxidation fermentation medium,
Stuart’s and Amies media, and Mannitol motility media.

3. Liquid media: These media do not contain any traces of solidifying

agents, such as agar or gelatine, and large growth of bacterial
colonies can be observed in the media. Liquid media are also called
broths, they allow for uniform and turbid growth of bacterial strains
when incubated at 37ºC for 24hrs. The media is used for the profuse
growth of microorganisms and fermentation studies. Examples
include Tryptic soy broth, phenol red carbohydrate broth, MR-VP
broth, and nutrient broth.

Classification of Culture Media

1. Simple Media and Basal Media: These are routinely used simple media
having carbon and nitrogen sources that boost the growth of many
microorganisms. They are also known as general-purpose media and are
considered non-selective media.
 The basal media do not require enrichment sources for the growth of non-
fastidious bacteria and are suitable for growing Staphylococcus and
Enterobacteriaceae. They are generally used to isolate microorganisms in
labs or in sub-culturing processes. Examples are nutrient broth, nutrient
agar, and peptone water.

2. Enriched Media: This media is prepared by adding additional substances

like blood, serum, or egg yolk in the basal medium. It’s used to grow
fastidious microorganisms as they require additional nutrients and
growth-promoting substances.

Examples are chocolate agar, blood agar, and Loeffler’s serum slope.
Chocolate media is used to grow N. gonorrhea while blood agar (which is
prepared by adding 5-10% blood by volume to a blood agar base) is used
to identify hemolytic bacteria.

3. Selective Media: This media allows the growth of certain microbes while
inhibiting the growth of others. It’s an agar-based medium that is used to
isolate microorganisms in labs. The selective growth of microbes is
decided by adding substances like antibiotics, dyes, bile salts, or by pH
adjustments.

4. Enrichment Media: It’s a liquid medium, used to increase the relative

concentration of certain microbes before culturing them on a solid
medium plate. It’s used as a broth medium and inhibits the growth of
commensal species of microorganisms (those who live in close
association with each other) in the clinical specimen. It’s also used in
isolating fecal and soil microorganisms. Examples are selenite F broth
which is used to isolate Salmonella typhi from a fecal sample,
tetrathionate broth, and alkaline peptone water.

5. Differential or indicator media: It contains certain indicators like dyes

or metabolic substrates in the medium composition which gives different
colors to colonies of different microbial species when they utilize or react
with these components.
It allows the growth of more than one microorganism, however, the
bacterial colonies are differentiated based on their color when a chemical
change occurs in the indicator, such as neutral red, phenol red, methylene
blue. Examples are:[1]
 Blood agar: In blood agar, three types of blood cell lysis or hemolysis
are observed: alpha, beta, and gamma hemolysis.[5] It allows the growth
of many microorganisms, however, their ability to lyse blood cells
differs, and this helps to distinguish the bacterial colonies. For
example, S. pyogenes completely lyse blood cells (beta hemolysis), thus
causing total clearing of the media around its colonies. S.
pneumoniae partially lyse red blood cells, resulting in a greenish-colored
medium, while gamma hemolytic microorganisms like Enterococcus
faecalis, Staphylococcus saprophyticus, and Staphylococcus epidermidis,
can’t lyse red blood cells, thus causing no color change in the medium.[6]

 Mannitol salts agar: The fermentation of mannitol by Staphylococcus

aureus causes the media to change to yellow, however, coagulase-
negative staphylococci that can’t cause fermentation to appear in pink. [7]

 MacConkey agar: It differentiates the gram-negative bacteria based on

their lactose metabolism. The lactose fermenting bacteria, such
as Escherichia coli, Klebsiella spp, Citrobacter, and Enterobacter forms
pink-red colonies, while lactose non-fermenters, like Salmonella,
Shigella, Proteus, Providencia, Pseudomonas, and Morganella form pale
or colorless colonies.[8]

 Thiosulfate citrate bile salts sucrose (TCBS) agar: The media contain
sucrose, which is utilized by ferment microbes and helps to distinguish
them from non-ferment microorganisms. Based on this characteristic,
different colored bacterial colonies are formed on the media that help to
identify and distinguish them from each other. For example, V.
cholerae ferment the sucrose and form slightly flattened yellow colonies
having opaque centers and translucent peripheries. Whereas, V.
parahaemolyticus can’t ferment the sucrose and forms green to blue-
green colonies.[9]

6. Transport media: Transport media are useful for clinical specimens

which are required to be transferred immediately to labs to maintain the
viability of potential pathogens and to prevent overgrowth of commensals
or contaminating microorganisms. Some of them are semi-solid in
consistency, and examples include:

 Sach’s buffered glycerol saline: It’s used to transport feces from patients
suspected to be suffering from bacillary dysentery.
 Cary Blair transport and Venkatraman Ramakrishnan media: Fecal
samples collected from suspected cholera patients are transported using
these media.
 Pike’s medium: A throat specimen containing Streptococci is transported
using this medium.

7. Anaerobic media: This media is for anaerobic bacteria which require

low oxygen levels, extra nutrients, and reduced oxidation-reduction
potential. It is supplemented with hemin and vitamin K nutrients and
oxygen is removed by boiling it in a water bath and sealing it with
paraffin film.[2]
Examples are: Thioglycollate broth and Robertson Cooked Meat (RCM)
medium which is commonly used to grow Clostridium spp.

8. Assay media: It’s used for amino acids, vitamins, and antibiotics assays.
For example, antibiotic assay media is used to determine the antibiotic
potency of microorganisms.

9. Storage media: It’s used to store microorganisms for a longer period,

examples are chalk cooked meat broth and egg saline medium.[2]