Bacterial Culture Media Culture medium is an environment which supplies the necessary nutrition for the growth of an organism. Culture media contains nutrients and physical growth parameters necessary for microbial growth. Organisms that cannot grow in artificial culture medium are known as obligate parasites. Mycobacterium leprae, rickettsias, Chlamydias, and Treponema pallidum are obligate parasites. Culture media generally provide sources of carbon, energy and nitrogen in the form of available carbohydrates and amino acids. Special media provide specific requirements as inorganic salts or particular growth factors. Types of Culture Media ⎯ Basic media ⎯ Enriched media ⎯ Selective media ⎯ Enrichment media ⎯ Indicator (Differential) media ⎯ Transport media 1. Basic Media These are simple media used to support the growth of microorganisms that do not have special nutritional requirements. They include nutrient broth, peptone water, and nutrient agar. i. Nutrient Broth- 1 Filtrate of cooked fresh minced meat + 1% - peptone + 0.5% NaCl. Clear yellowish fluid medium Sterilized in autoclave at- 121°C for 30 min. Base for most culture media. ii. Peptone Water Peptone + 0.5% NaCl dissolved in 1%- water Clear colorless fluid medium. Sterilized in autoclave at 121°C- for 30 min. Base for sugar media Indole production test.

1. Bacterial Culture Media
Submitted To: Dr. M. Abdul Qayyum
Submitted By: Muhammad Umer Farooq
Reg. No. 2017-BSc-Agri-94
Course Code: SES-601
Course Title: Soil Microbiology
Department of Soil & Environmental Sciences
Faculty of Agricultural Sciences
Ghazi University, Dera Ghazi Khan, Punjab, Pakistan

2. Bacterial Culture Media
Culture medium is an environment which supplies the necessary nutrition for the growth of an
organism. Culture media contains nutrients and physical growth parameters necessary for
microbial growth. Organisms that cannot grow in artificial culture medium are known as obligate
parasites. Mycobacterium leprae, rickettsias, Chlamydias, and Treponema pallidum are obligate
parasites. Culture media generally provide sources of carbon, energy and nitrogen in the form of
available carbohydrates and amino acids.
Special media provide specific requirements as inorganic salts or particular growth factors.
Types of Culture Media
⎯ Basic media
⎯ Enriched media
⎯ Selective media
⎯ Enrichment media
⎯ Indicator (Differential) media
⎯ Transport media
1. Basic Media
These are simple media used to support the growth of microorganisms that do not have
special nutritional requirements. They include nutrient broth, peptone water, and nutrient
agar.
i. Nutrient Broth- 1
Filtrate of cooked fresh minced meat + 1% - peptone + 0.5% NaCl. Clear yellowish fluid
medium Sterilized in autoclave at- 121°C for 30 min. Base for most culture media.
ii. Peptone Water
Peptone + 0.5% NaCl dissolved in 1%- water Clear colorless fluid medium. Sterilized in
autoclave at 121°C- for 30 min. Base for sugar media Indole production test.

3. iii. Nutrient Agar
Agar powder dissolved in nutrient 2-3%- broth. Yellowish semi-transparent medium.
Plates or tubes as slopes or deep agar. Sterilized in autoclave at 121°C for 30 min.
Uses:
Base of different culture media
Plates: for isolation & identification of bacteria
Slopes: for preservation of pure cultures
Deep agar: for anaerobic bacteria

4. 2. Enriched Media
Prepared by the addition of substances such as blood, serum or egg to a basic medium.
Used for cultivation of fastidious organisms that cannot grow on simple media and need
highly nutritive substances for growth. Used for culturing sterile body fluids such as blood
or CSF, where the finding of any organisms = infection due to that organism. And also, for
primary identification of microorganisms e.g. hemolysis on blood, blood agar, chocolate
agar, Loeffler’s serum.
i. Blood Agar
Sterile defibrinated sheep or human 5-10% blood + melted nutrient agar at 55°C. Red
opaque solid medium. N. agar is sterilized in autoclave at 121°C for 30 min. Blood is added
under complete aseptic condition at 45-55°C. Supports the growth of most delicate
organisms e.g. Streptococcus pyogenes. Identifying bacteria according to their haemolytic
action on the red cells.
ii. Chocolate Agar
Prepared as blood agar followed by raising. The temperature to l00 °C for 2 min to rupture
red cells and release nutrients as X and V factors. Brown opaque solid medium. Sterilized
as blood agar.
Used for the isolation of Neisseria meningitides, Hemophilus influenza and Streptococcus
pneumonae.

5. iii. Loeffler's serum
Parts of sheep or horse serum + part glucose broth Opaque whitish solid medium. Serum
is sterilized by filtration & glucose. broth by Koch's steamer. The medium is solidified in
hot air inspissator at 75°C for 2 hours for 2 successive days.
Uses: Culture of Corynebacterium diphtheria.
3. Selective Media
Solid media that contain substances (e.g. bile salts or other chemicals, dyes, antibiotics)
which inhibit the growth of one organism to allow the growth of another. Used when
culturing a specimen from a site having a normal microbial flora to prevent unwanted
contaminants overgrowing a pathogen. They include the following media:
i. Lowenstein Jensen medium
3 +parts beaten eggs + 1-part water, malachite green. Green opaque solid medium.
Sterilized in hot air inspissator at 75 °C for 2hour for 2 successive days.
Used for Isolation of Mycobacterium tuberculosis.
ii. MacLeod's tellurite blood agar

6. Blood agar + 0.02-0.04% K tellurite. Red opaque solid medium. Sterilized as blood agar.
Used for isolation of Corynebacterium diphtheriae from contaminated materials.
iii. Modified Thayer-Martin agar
Chocolate agar + vancomycin + colistin + nystatin. Brown opaque solid medium. Sterilized
as Chocolate agar.
Used for Isolation of Neisseria from non-sterile specimens.
iv. Thiosulphate citrate bile sucrose agar (TCBS)
Alkaline agar + sucrose + thiosulphate + citrate and bromothymol blue indicator. Greenish
transparent solid medium. Sterilized in autoclave at 121°C for 30 min.
Used for isolation of Vibrio cholerae.
v. Deoxycholate citrate agar (DCA)
Agar + lactose + neutral red indicator. Na deoxycholate and citrate + Reddish
semitransparent solid medium. Sterilized in autoclave at 121°C for 30 min.
Used for isolation of Shigella and Salmonella.

7. vi. XLD Media
Agar + lactose + phenol red indicator + ferric citrate + deoxycholate + xylose + lysine +
sucrose + yeast extract. Reddish semitransparent solid medium. Sterilized by boiling.
Used for isolation of Shigella and Salmonella.
4. Enrichment Media
Fluid media that contain substances which favor the growth of wanted organisms on the
expense of others. Usually used as a preliminary step for isolation of pathogens before

8. subculturing on solid selective media. Examples are: Selenite broth for isolation of
Salmonella and Shigella species from faeces Tetrathionate broth for isolation of Salmonella
from faeces. Alkaline peptone water for isolation of Vibrio cholerea.
5. Indicator (Differential) Media
These are media to which dyes or other substances (Indicators) are added to differentiate
microorganisms. Indicators change color when acid is produced following fermentation of
a specific carbohydrate e.g. MacConkey's agar medium.
MacConkey's agar medium
Peptone, agar, lactose, bile salt and neutral red indicator. Reddish transparent solid
medium. Sterilized in autoclave 121°C for 30 min. Supports the growth of most Gram-
negative bacilli, especially the enterobacteriaceae, but inhibits the growth of Gram-positive
organism and some fastidious Gramnegative bacteria, such as Haemophilus and Neisseria.
6. Identification Media
These include media to which substrates or chemicals are added to help identify bacteria
isolated on primary cultures. i.e. organisms identified must be first isolated in pure culture.
Organisms are mainly identified by a change in the color of the medium and or the
production of gas. They include peptone water sugars, litmus milk, and gelatin media.
i. Peptone water sugar media
Peptone water + 1% tested sugar + 1%- Andrade's indicator
Durham tube is an inverted tube to visualize-gas bubbles produced from sugar fermentation.
Yellowish transparent. Sterilized in Koch's steamer for 20 min on-three successive days
(tyndallisation)

9. Used to test the biochemical activity of bacteria on carbohydrates.
ii. Litmus Milk
Steamed fresh milk for 1 hour (cream is removed) + litmus solution. Mauve Sterilized in
Koch's steamer for 20 min on three successive days (tyndallisation)
Used to test the saccharolytic activity of bacteria for identification of enterococci bleaching.
iii. Gelatin
Gelatin sheets dissolved in nutrient broth 10-15%. The medium is solid below 24°C. Above
this temperature it melts into yellowish fluid. Sterilized in Koch's steamer for 20 min on
three successive days (tyndallisation)
Used to test the proteolytic activity of different organisms.
7. Transport Media
Semisolid media that contain ingredients to prevent the overgrowth of commensals & ensure
the survival of aerobic and anaerobic pathogens when specimens cannot be cultured
immediately.
Examples:
1- Cary-Blair medium for preserving enteric pathogens.
2- Amies transport medium for ensuring the viability of gonococci Thioglycollate broth and
deep agar for- 3 anaerobic organisms.
Culture Media for Anaerobes
Media for anaerobes is the same as media for aerobes except that:
i. They are richer in organic constituents.
ii. Contain reducing agents (cysteine & haemin).
iii. Contain a redox indicator.
The inoculated media are incubated in anaerobic environment using anaerobic gas pack.
Reference
1. https://en.wikipedia.org/wiki/Growth_medium

10. 2. https://courses.lumenlearning.com/microbiology/chapter/media-used-for-bacterial-
growth/
3. https://www2.slideshare.net/MostafaMahmoud76/bacterial-culture-media-74315094