Preparation of Microbial Media

Aim:
To prepare microbial media for growth and isolation of bacteria.

Theory:
Much of the study of microbiology depends on the ability togrow and
maintain microorganisms in the laboratory, and this ispossible only if
suitable culture media are available. A culturemedium is a solid or liquid
preparation used to grow, transport,and store microorganisms. To be
effective, the medium must
contain all the nutrients the microorganism requires for growth.Specialized
media are essential in the isolation and identificationof microorganisms,
the testing of antibiotic sensitivities,water and food analysis, industrial
microbiology, and other activities.Although all microorganisms need
sources of energy,carbon, nitrogen, phosphorus, sulphur, and various
minerals, theprecise composition of a satisfactory medium will depend
onthe species one is trying to cultivate because nutritional
requirementsvary so greatly. Knowledge of a microorganismsnormal
habitat often is useful in selecting an appropriate culturemedium because
its nutrient requirements reflect its natural surroundings.Frequently a
medium is used to select and grow specificmicroorganisms or to help
identify a particular species. In such cases the function of the medium also
will determine itscomposition.
Synthetic or Defined Media
Some microorganisms, particularly photolithotrophic autotrophs such as
cyanobacteria and eukaryotic algae, can be grown on relatively simple
media containing CO2as a carbon source (often added as sodium
carbonate or bicarbonate), nitrate or ammonia as a nitrogen source,
sulfate, phosphate, and a variety of minerals. Such a medium in which all
components are known is a defined medium or synthetic medium.
Many chemoorganotrophic heterotrophs also can be grown in defined
media with glucose as a carbon source and an ammonium salt as a
nitrogen source. Not all defined media are as simple as the examples in
but may be constructed from dozens of components. Defined media are
used widely in research, as it is often desirable toknow what the
experimental microorganism is metabolizing.
Complex Media
Media that contain some ingredients of unknown chemical composition
are complex media. Such media are very useful, as a single complex

medium may be sufficiently rich and complete to meet the nutritional

requirements of many different microorganisms. In addition, complex
media often are needed because the nutritional requirements of a
particular microorganism are unknown, and thus a defined medium cannot
be constructed. This is the situation with many fastidious bacteria, some
of which may even require a medium containing blood or serum. Complex
media contain undefined components like peptones, meat extract, and
yeast extract. Peptones are protein hydrolysates prepared by partial
proteolytic digestion of meat, casein, soya meal, gelatin, and other protein
sources. They serve as sources of carbon, energy, and nitrogen. Beef
extract and yeast extract areaqueous extracts of lean beef and brewers
yeast, respectively. Beef extract contains amino acids, peptides,
nucleotides, organic
acids, vitamins, and minerals. Yeast extract is an excellent source of B
vitamins as well as nitrogen and carbon compounds. Three commonly
used complex media are (1) nutrient broth, (2) tryptic soy broth, and (3)
MacConkey agar.
If a solid medium is needed for surface cultivation of microorganisms,
liquid media can be solidified with the addition of 1.0 to 2.0% agar; most
commonly 1.5% is used. Agar is a sulfated polymer composed mainly of
D-galactose, 3,6-anhydro-L-galactose, and D-glucuronic acid. It is usually
extracted from red algae. Agar is well suited as a solidifying agent
because after it has been melted in boiling water, it can be cooled to
about 40 to 42C before hardening and will not melt again until the
temperature rises to about 80 to 90C. Agar is also an excellent hardening
agent because most microorganisms cannot degrade it. Other solidifying
agents are sometimes employed. For example, silica gel is used to grow
autotrophic bacteria on solid media in the absence of organic and to
determine carbon sources for heterotrophic bacteria by supplementing the
medium with various organic compounds.
Types of Media
Media such as tryptic soy broth and tryptic soy agar are called general
purpose media because they support the growth of many microorganisms.
Blood and other special nutrients may be added to general purpose media
to encourage the growth of fastidious heterotrophs. These specially
fortified media (e.g., blood agar) are called enriched media.
Selective media:
It favors the growth of particular microorganisms. Bile salts or dyes like
basic fuchsin and crystal violet favour the growth of gram-negative
bacteria by inhibiting the growth of gram-positive bacteria without
affecting gram-negative organisms. Endo agar, eosin methylene blue
agar, and MacConkey agar three media widely used for the detection of E.

coli and related bacteria in water supplies and elsewhere, contain

dyesthat suppress gram-positive bacterial growth. MacConkey agaralso
contains bile salts. Bacteria also may be selected by incubationwith
nutrients that they specifically can use. A medium containingonly
cellulose as a carbon and energy source is quite effectivein the isolation of
cellulose-digesting bacteria. The possibilities for selection are endless, and
there are dozens of specialselective media in use.
Differential media are media that distinguish between differentgroups of
bacteria and even permit tentative identificationof microorganisms based
on their biological characteristics.Blood agar is both a differential medium
and an enriched one. It distinguishes between hemolytic and
nonhemolytic bacteria. Hemolytic bacteria (e.g., many streptococci and
staphylococci isolatedfrom throats) produce clear zones around their
colonies becauseof red blood cell destruction. MacConkey agar is both
differential and selective. Since it contains lactose and neutral reddye,
lactose-fermenting colonies appear pink to red in color and are easily
distinguished from colonies of non-fermenters.
Thus the media prepared for growth of micro-organisms are:
1. MacConkeys Agar:
It is a selective and differential medium for cultivation of enteric micro
organism from a variety of clinical specimens. The selective action for this
medium is attributed to neutral red and bile salt which are inhibitory to
more species of Gram positive bacteria. Gram negative bacteria usually
grow well on the medium and can be differentiated by their ability to
ferment the lactose.
Lactose fermenting strains grow as pink colonies and may be surrounded
by zone of a acid precipitated bile salt. The pink colour is due to
production of acid by lactose absorption of neutral red and subsequent
colour change of a dye when the pH of the medium falls below 6.8.
Lactose non-fermenting strains such as shigella and salmonella are
colourless and transparent and they do alter the appearance of the
medium.
COMPOSITION:
Peptone
Sodium Taurocholate
Lactose
Sodium chloride
Neutral red
Distilled water
pH

20 gm/lit
5 gm/lit
10 gm/lit
5 gm/lit
2% (w/v) in 50% ethanol
1000 ml
7.2

ORGANISMS USED FOR MacConkeys Agar:

Enterobacter aerogenes
pink colony
Escherishia coli
pink colony
Klebsiella pneumoniae
pink colony
Proteus species
colourless colony
Salmonella species
colourless colony
Shigella species
colourless colony
Staphulococcus aureus
GETS INHIBITED
Incubation period: 370C for 1-2 days

2. Potato dextrose agar:

PDA is common microbiological growth media made from potato infusion
and dextrose. It is most widely used medium for growing fungi and
bacteria which attack living plants or decaying dead plant matter.
Potato infusion can be made by boiling 200gms unpeeled slice potatoes in
1 litre distilled water for 30 minutes and then decanting or staining the
broth through cheesecloth. Distilled water is added such that the total
volume of suspension is 1 litre. 20gms dextrose and 20gms agar powder
is then added and the medium is then sterilised by autoclaving at 15
pounds per square inch (100kpa) for 15 minutes. Common organisms
cultured on PDA are yeasts such as Candida albicans and Saccaromyces
cerevisiae and molds such as A. niger.
PDA is a general medium for yeasts and molds that can be supplemented
with acid or antibiotic to inhibit bacterial growth .It is recommended for
plate count methods. The nutritionally rich base potato infusion
encourages mold sporulation and pigment production is some
decmatophytes.
PRINCIPLE of the procedure:
PDA is composed of dehydrated potato infusion and dextrose that
encourage luxuriant fungal growth- agar is added as a solidifying agent.
10% Factaric acid is used to lower the pH of this medium.
COMPOSITION:
Potatoes peeled and sliced 200 gms
Dextrose
20 gms
Distilled water
1000 ml
Agar
40 gms

Boil the potatoes for 30 minutes in water.

Decant out the extract and make up the volume to 1 litre with water
then add the agar constituents.

Result obtained would be:

Yeasts will grow as creamy to white colonies
Molds will grow as filamentous colonies of various colours.
Limitation of the procedure:
Due to nutitional variation some strains may be encountered grow poorly
or fail to grow on this medium.
Incubation period:
(22-25)0C/ (30-32)0C for 2-7 days or longer
3. Luria Bertani medium:
Luria Bertani medium subsequently became the medium of choice for
growth of E.coliand other related enteric species.
COMPOSITION:
Peptone
10gms
Yeast extract10gms
NaCl10gms in 950ml deionized water
Adjust pH of medium to 7.0 using 1N NaOH and bring volume upto 1
Litre.
Autoclave on liquid cycle for 20 minutes at 15 psi.Allow solution to
cool to 550C and add antibiotic if needed(50g/ml of ampicillin).
Store at room temperature or 40C.
LB medium is a rich medium that is commonly used to culture members of
the enterobacteriaceae as well as for coliphage plaque assays. LB is used
extensively in rDNA work and other molecular biology procedures. Often
an antibiotic is added to the medium for the selection of cells that contain
a specific genetic element such as plasmid and transposon,or a gene
disruption via an antibiotic resistance cassette.It is a nutrient rich media
commonly used to culture bacteria in the laboratory. The addition of agar
to LB results in the formation of a gel that bacteria can grow on;as they
are unable to digest the agar but can gather nutrition from the LB within.
The addition of an antibiotic to this gel allows the selection of only those
bacteria with the specific antibiotic resistance usually conferred by a
plasmid carrying the antibiotic resistant gene.

LB media formulation have been an industry standard for the cultivation of

E.coli. These media have been widely used in molecular microbiology
application for the preparation of plasmid DNA and recombinant proteins.
It continues to be one of the most common media used for maintaining
and cultivating laboratory recombinant strains of E.coli for physiological
studies. Peptides and peptones are provided by tryptone, vitamins and
certain trace elements are provided by yeast extract.Sodium ions for
transport and osmotic balance are provided by sodium chloride. Tryptone
is used to provide essential amino acids to growing bacteria and yeast
extract is used to provide organic compound helpful for bacterial growth.

25x M9 minimal media:

M9 medium is a minimal growth medium used for bacterial cultures. It has
the advantage of being cheap and has very low auto fluorescence and has
very low absorbance.M9 medium can be supplemented to produce higher
growth rates or to allow growth of strains that require additives. Eg:
thiamine and casamino acids.
COMPOSITION: (gms/lit)
25x M9 salt solution
Na2HPO4
188
KH2PO4
75
NH4Cl
25
NaCl
12.5

+800ml D/W =1 litre

1M Cacl2
1M MgSO4
0.1% Thiamine
20% Glucose
The above solution was autoclaved at 121 oC for 15 minutes. To prepare
the M9 medium the individual components were mixed aseptically in the
following order.
For 500ml M9 medium:
1X
D/w
1M CaCl2
1M MgSO4
0.1% Thiamine

400ml
0.05ml
5.0ml
5ml

2X

0.1ml
10ml
10ml

25XM9 salt
Glucose

20ml
5ml

40ml
10ml

ALGAE AGAR:
Agar-agar and gelatine are products made primarily from the algae
Geladium gracelaria (red seaweeds) best known as solidifying components
of bacteriological culture media. Agar is isolated from algae as amorphous
and translucent product sold as powder, flakes or bricks. Agar is insoluble
in cold water. It absorbs as much as 20 times its own weight it dissolves
readily in boiling water, a dilute solution is still liquid at 42 0C but solidifies
at 370C into a firm gel. In the natural state agar occurs as a complex cell
wall constituent containing a complex carbohydrate with sulfate and
calcium.

Protozoa
Entaemoeba media:
Composition:
gm/litre
Liver infusion from
272.00 gms
Proteose peptone
5.50 gms
Sodium beta glycerophosphate
3.00
Sodium chloride
2.70
Agar
11.00
Final pH at 25o 7+ 0.2
Procedure
Suspense 33gms in 1000ml of d/w
Heat to boiling
Dispense in tubes and sterilise by autoclaving
Allow tubes to solidify in slanted position, cover about half of the slant
with fresh sterile horse serum saline mixture (1:6) and add a 5mm loopful
of rice powder which has been sterilise in an oven at 160oC for 1 hr.

Reference:
Hi manual Media and microbiology journal, 2nd edition
Introduction to Microbiology, Prescott, MacGraw Hill production, 2002