What is culture medium: The food material or substances required

for growing microorganisms in vitro (outside the body) is called

culture medium. The culture media (nutrients) consist of chemicals
which support the growth of culture or microorganisms. Microbes
can use the nutrients of culture media as their food is necessary for
cultivating them in vitro.
 Functions of culture media

• Support the growth of the bacteria.

• Should be nutritive (contains the required
amount of nutrients).
• Suitable pH (neutral to slightly alkaline 7.3-
7.4).
• Suitable temperature, and suitable atmosphere.
(Bacteria grow at 370C)
 Composition of culture media
• Water
• Energy source •
• Carbon source •
• Nitrogen source •
• Mineral salts •
• Special growth factors
 Types of media.
On the basis of consistency, the culture media are of three types:
(a) Solid medium or synthetic medium:
When 5-7% agar agar or 10-20% gelatin is added the liquid broth becomes
solidified. Such media are used for making agar slants or slopes and agar stab. It
melts at 970C and solidifies at 370C.

(b) Liquid medium or broth:

In such cases no agar is added or used while preparing the medium. After
inoculation and later incubation, the growth of cells becomes visible in the form
turbidity of small mass in the broth.
(c) Semi-solid or floppy agar medium:
Such media are prepared by adding half quantity of agar (1/2 than required for
solid medium) i.e. about 0.5% in the medium. Such media are soft and are useful
in demonstrating bacterial motility and separating motile from non- motile
strains
 Classification based on the ingredients :On the basis of ingredients present,
culture media are of 4 types:

a) Simple medium: eg: Nutrient broth, Nutrient agar - NB consists of peptone,

meat extract, NaCl
b) complex medium :Complex media such as blood agar, it has ingredients that
exact components are difficult to estimate
c) Synthetic or defined medium :specially prepared media from pure chemical
substances for research purpose and composition of every component is
well known eg: peptone water – 1% peptone + 0.5% NaCl in water.
d) Enriched media : Substances like blood, serum, egg are added to the simple
medium. Itis Used to grow bacteria that are exacting in their nutritional
needs. eg: Blood agar, Chocolate agar
Blood agar contains mammalian blood(usually sheep or horse) typically at a
concentration of 5-10%, used to isolate fastidious organisms and detect
hemolysis. Chocolate agar contain red blood cells that have been lysed by
slowly heating to 80 c .and it used for growing fastidious bacteria, such as
Haemophilus influenzae.
e) Selective media : It is a media, which contains substances that prevent or
slow the growth of microorganisms other than the bacteria for which the media
is prepared for. For example
•TSI (triple sugar iron agar): slanted tube.
•EMB (Eosin Methylene blue): enteric isolation media
 Eosin methylene blue is selective for gram negative bacteria.The dye methylene blue in
the medium inhibit the growth of gram positive bacteria.
Lowenstein –Jenson medium is solid medium used for Mycobacterium tuberculosis
contain penicillin, nalidixic acid and malachite green to inhibit growth of gram positive
and gram negative bacteria, in order to limit growth to Mycobacteria species only.

f) Differential media : This media contains indicators, dyes, etc, to differentiate

microorganisms. It is designed in such a way that different bacteria can be recognized on
the basis of their colony color E.g. MacConkey agar, which contains neutral red (pH
indicator) and is used to differentiate lactose fermenter and non-lactose fermenter. (E.g.
E. coli and Salmonella.
Salmonella Shigella ( SS ) Agar : isolation and differential medium for pathogenic Gram-
negative bacilli in particular, Salmonella and Shigella. Inhibitor for Coliforms.

Triple Sugar Iron Agar (TSI): this a key medium for use in beginning the identification of a
Gram- negative bacilli of the enteric group. It contains glucose (0.1% ), Lactose (1%),
sucrose(1%). And peptone (2%) as nutritional sources. Sodium Thiosulfate serves as the
electron receptor for reduction of sulfur and production of H2S. Detects fermentation of
sucrose, lactose, glucose, as well as production of hydrogen sulfide and /or gas . Phenol
red is the PH indicator; ferric ammonium citrate is H2S indicator.
Chocolate Agar: blood agar prepared by heating blood to 95C until medium
becomes brown or chocolate in color heating the blood releases broth X and V
growth factors and also destroys the inhibitors of V factor. These factors are
required for the growth of most species of Haemophilus and also Neisseria
gonorrhoea.

g) Transport media :Media used for transporting the samples. Delicate organisms
may not survive the time taken for transporting the specimen without a transport
media. Eg: – Stuart’s medium – Buffered glycerol saline

h)Anaerobic media : These media are used to grow anaerobic organisms. Eg:
Robertson’s cooked meat medium, Thioglycolate broth medium.
 Media Preparation
The liquid medium or broth is prepared by dissolving the known amounts of
chemicals in distilled water
• The pH should be adjusted by adding N/10 HCl or 1N NaOH. The liquid medium is
dissolved into either Erlenmeyer flasks or rimless clean test tubes.
•In 15 ml capacity of test tube, 5 ml medium should be poured while in flask of 250
ml capacity, the amount of the medium should be 100 ml.
•These are then plugged with non-adsorbent cotton plugs.
•The plugged tubes or flasks should be wrapped by brown paper and placed for
sterilization by autoclaving at a pressure of 15 lbs/inch2 (at temperature 121°C), for
15 min.
•Some media can’t be sterilized by autoclaving because they contain eggs or
carbohydrates .The heat sensitive substances (protein or enzymes etc.) should be
sterilized by using membrane filters (millipore).