Based on the application or function media can be divided into Supportive media (General purpose media) and special media. Special media includes a number of media like Enriched media, Enrichment media, Selective media, Differential media, Indicator media, Sugar media and Transport media. However, sometimes a single media may fulfil more than one functions. For example; Blood agar can act as both Enriched and Differential media. Eosin Methylene Blue (EMB) agar can act as both Selective and Differential media. MacConkey (MAC) agar can act as Selective, Indicator and Differential media. Mannitol Salt agar can act as both Selective and Differential media. 1. Supportive media (General purpose media) This media support the growth of most of the organisms and do not contain any added inhibitors. Basal media used in microbiology lab like Peptone water, Nutrient Agar and Nutrient Broth can be considered as supportive media for growing non-fastidious organisms. Media such as Tryptic Soy Broth and Tryptic Soy Agar are also examples of GPM as they sustain the growth of many microorganisms. Blood or other nutrients can be added to the GPM to support the growth of fastidious organisms, then they are called Enriched media. 2. Special Media 2.1. Enriched Media Enriched media contains special ingredients like blood, serum or egg added to the basal media. It is used to cultivate microorganisms that are more exacting in their nutritional needs.ie; to grow fastidious organisms. Eg: Blood agar, Chocolate agar, Egg media and Brain Heart Infusion Broth. a) Blood agar:
Blood agar contains 5% mammalian blood
added to the basal media. Blood is added after autoclaving the media. It is
enriched medium as it contains many nutrients like protein, carbohydrate,
lipid, iron, a number of growth factors and vitamins that can support the
growth of many fastidious organisms including aerobic and anaerobic bacteria. Vitamin
K, cysteine and hemin in blood support the growth of anaerobic organisms. Blood
agar acts as both enriched and differential medium. Blood agar is a
differential media as it distinguishes haemolytic and non- haemolytic bacteria.
Hemolytic organisms produce hemolysin, a protein that cause hemolysis or
breakdown of Red Blood Cells (RBC). Sheep blood is preferred as hemolysis is
more clearly defined on it. Hemolysis
appears as a clear zone if there is complete lysis of RBC (β hemolysis) or
greenish halo around the colony if there is partial lysis of RBC (α hemolysis).
Blood agar also help to differentiate among Gram positive cocci.
Eg: Streptococcus
pyogenes and Staphylococcus aureus cause β hemolysis. Streptococcus
viridans cause α hemolysis.
a)
Chocolate
Agar: Used to grow fastidious organisms like Neisseria gonorrhoeae, N. meningitidis, H.
influenza and Pneumococci. It is
called chocolate agar due to its chocolate brown appearance. The brown colour
is the result of heating red blood cells and lysing them before adding to the
media. Heating a mixture of sheep blood and nutrient agar releases haemoglobin,
hemin or X factor and Nicotinamide adenine dinucleotide (NAD or V factor) which
gives the brown colour.
b)
Brain
heart infusion broth: It is highly nutritious buffered
liquid media for cultivating fastidious organisms. It is prepared using non-
enzymatic infusion from calf brain and cow heart with added peptone and
dextrose.
1.1.
Enrichment Media
In samples containing mixed population
of microorganisms the bacteria to be isolated is often overgrown by the
unwanted microbes. Usually
non-pathogenic or commensal bacteria tend to overgrow the pathogenic ones. Enrichment
media are liquid media containing special nutrients that stimulate the growth of
particular organisms that might not be otherwise present in sufficient amount
for isolation and identification. For eg: In fecal samples S typhi may not be sufficiently numerous for identification and
usually overgrown by E coli. If
cultivated on the medium Selenite
cystine (SC) broth with trace element selenium greater number of S typhi could be obtained on incubation
and selenium is inhibitory to coliforms. Such media which contain substances
that have a stimulatory effect on wanted organisms are called enrichment media.
Other Egs: Tetrathionate broth to
promote growth of Typhoid and Paratyphoid bacilli in samples
containing coliforms; Selenite F Broth
for Dysentery bacilli.
1.2.
Selective media
Selective
media are solid media that contain nutrients to promote the growth of wanted
organisms and also contain substances that inhibit or suppress the growth of
unwanted organisms. Incorporation of substances like bile salts or dyes such as
basic fuchsin and crystal violet allow the growth of Gram negative organisms
while they inhibit the Gram positive ones. MacConkey agar, Eosin Methylene Blue
Agar are examples of selective media which are widely used for detection of E coli in water supplies. These media
will not allow the growth of Gram positive organisms.
a) MacConkey Agar
is made up of peptone, lactose, agar, neutral red, taurocholate, crystal violet
and bile salts. Crystal violet and bile salts act as selective agents and
inhibit the growth of G +ive bacteria
and allow G –ive growth.
b)
Eosin Methylene Blue Agar contains two dyes, Eosin Y and
Methylene Blue that inhibit G +ive bacteria.
c) Mannitol Salt Agar allows the selective growth of halophiles. 7.5% NaCl act as selective agent promoting the growth of halophiles and inhibiting non-halophiles.
d) Sulfadiazine and Polymyxin Sulfate (SPS)
Agar: It is used to isolate Clostridium
botulinum which causes food poisoning. This media promote the growth
of Clostridium
botulinum while inhibiting the growth of most other Clostridium species. It
contains the antibiotics sulfadiazine and Polymyxin sulphate as selective
agents.
e)
Desoxycholate Citrate Agar for isolating Dysentery bacilli
f)
Thiosulphate Citrate Bile Sucrose Agar
(TCBS) for isolation of Vibrio
species from fecal samples. Alkaline pH of the medium promote the growth of Vibrio species and high concentrations of sodium thiosulfate and sodium
citrate to inhibit the growth of Enterobacteriaceae.
g)
Thayer Martin Media used to isolate N.gonorrhoeae from clinical specimens contain antimicrobials like
vancomycin, colistin, nystanin in Chocolate agar. On incubation in the presence
of 3-10% CO2, this media will give colonies of N.gonorrhoeae and inhibit other commensals.
h)
Lowenstein-Jensen Media used for the isolation of Mycobacterium species. This media
contains mineral salts, asparagine, glycerol, malachite green and hen’s egg. Malachite
green in the medium act as the inhibitor for unwanted organisms.
1.3.
Indicator media
Indicator
media contains an indicator that changes its colour when bacterium grows in
them. Blood agar can be considered
as an indicator media. In some indicator media, a pH indicator will be
incorporated. As the organisms grow, pH of the medium will be changed due to
acid production and it causes the indicator to change its colour. Example is
MacConkey agar. The presence of the pH indicator neutral red makes it an
indicator media. The indicator changes the colonies of lactose fermenters into red
or deep pink in colour and leaves the non-fermenters colourless or translucent.
Lactose fermenters catabolise lactose by fermenting it and release acidic waste
products. These acidic waste diffuse into the media, changing the colour of
indicator. Neutral indicator is red or orange in acidic pH (<6.8) and colourless
when pH is over 6.8. The indicator imparts pink or red colour to colonies of
the lactose fermenters.
Other
Examples:
Wilson
& Blair Medium for isolation of S typhi. This media contains the indicator Sulphite. S typhi reduces Sulphite to Sulphide in
the presence of Glucose and produce colonies with black metallic sheen.
McLeod’s
Medium or Potassium Tellurite Agar used to isolate C.diphtheriae. Potassium Tellurite in
the medium is reduced to metallic tellurium by C.diphtheriae to produce black colonies.
Thiosulphate
Citrate Bile Sucrose Agar (TCBS) for isolation of Vibrio species. Bromothymol blue is the
indicator in the medium (Yellow in acidic, green in neutral and blue in
alkaline conditions). Organisms that ferment sucrose appear as yellow colonies
while the non-fermenters appear as green colonies. Hence act as an indicator
medium also.
1.4.
Differential media
Differential
media helps to distinguish among different groups of microorganisms and even
permit identification of organisms based on their biological characteristics. In
some cases, indicator in the differential media changes its colour when a
particular biochemical reaction occurs. Many of the media we mentioned yet also
falls under differential media.
Blood
agar which is an enriched media, indicator media, is an example of differential
media too. It helps to differentiate haemolytic and non-haemolytic organisms.
ie; they differentiate organisms based on their ability to produce
hemolysin. MacConkey agar is also a differential media as
it helps to differentiate between lactose fermenters and non-lactose
fermenters. Neutral red indicator changes its colour in acidic pH and impart
red colour to lactose fermenters. Most of the commensals of intestine including
E coli are good lactose fermenters
and appear pink to red in colour whereas most of the pathogens like Salmonella and Shigella are non-lactose fermenters and appear colourless.
Mannitol
salt agar, selective media for halophiles act as differential media too. It
helps to differentiate pathogenic and non-pathogenic Staphylococci. Pathogenic
ones like Staphylococcus aureus
release acidic by-products as they make use of mannitol as carbon or energy
source. Acidic products cause the phenol
red indicator in the medium to change into yellow colour (Phenol red indicator:
Yellow in acidic condition and red in alkaline conditions).
EMB
agar which is a selective media also serve as the differential media. Eosin Y
and Methylene blue dyes in EMB agar react with acidic products released by some
G –ive bacteria when they use lactose or sucrose in the medium as carbon and
energy sources. Fecal bacteria such as E
coli produce large amounts of acidic products and have green metallic sheen
on EMB agar. Help to differentiate E
coli.
SPS
(Sulfite
Polymyxin Sulfadizine) Agar, selective media for Clostridium botulinum also serve as differential media. Clostridium botulinum produce black
colonies on SPS Agar because of the production of hydrogen sulphide by the
organism from the sulphur containing additives.
1.5.
Sugar media
Sugar
media contains fermentable sugars in it. It can be monosaccharides like
pentoses (eg: Arabinose, Xylose) or hexoses (eg: Dextrose, mannose),
disaccharides (eg: Saccharose, lactose), trisaccharides (eg: Raffinose) or
polysaccharides (eg Starch). Sugar media has two functions. Primary function is
that the media will act as readily available source of energy, provided the
organisms are able to utilize the sugar present in the media. Second function
is that it is helpful in the identification and classification of organisms.
Sugar
media consists of 1% sugar in peptone water along with an indicator. Durham
tube is kept inverted in the media to detect gas production. Indicator changes
its colour if the inoculated organism ferments the sugar. Fermentation could be
with or without gas production. Gas production is indicated as bubble formation
in Durham tube.
1.6.
Transport media
Special
media devised to transport the microorganisms are called transport media. They
are usually used to transport either fastidious or delicate organisms that
cannot withstand the time taken for transport of specimen from the site of
collection to the laboratory. Transport media are essentially buffered
solutions containing carbohydrates, peptones and other nutrients (excluding the
growth factors) which would preserve the viability of
bacteria during transport without allowing their multiplication.
Examples: Glycerol saline for Enteric
bacilli, Stuart’s
medium for Gonococci, Venkatraman Ramakrishnan (VR) medium for Vibrio cholera.
|
Examples
of multipurpose media |
|
|
Blood Agar |
Enriched /indicator/
differential media |
|
MacConkey agar |
Selective/ indicator/
differential media |
|
Eosin Methylene Blue Agar |
Selective /differential
media |
|
Mannitol Salt Agar |
Selective /differential
media |
|
Sulfadiazine and Polymyxin Sulfate
(SPS) Agar |
Selective/ differential
media |
|
Thiosulphate Citrate Bile Sucrose Agar
(TCBS) |
Selective/ indicator
media |
