BACHELOR OF BIOMEDICAL

SCIENCES
BASIC MICROBIOLOGY
BMC123
BASIC MICROBIOLOGY-2

CHAPTER 7:BACTERIAL
CULTIVATION
 Learning Outcomes

At the end of the sessionstudents should be

able to
• discuss the various culture media and
methods used in microbiology
• explain the significance of culture
methods in relation in clinical microbiology
• describe the different culture methods
used in microbiology
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CULTURE MEDIA

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• Culture Media- Any solid material that
have all nutrients that promote growth of
bacteria which can be seen as
colonies .
• Colonies represent clone of cells
originating from single parent cell

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Basic constituents of culture media

Water: Source of hydrogen and oxygen.

Electrolytes: Sodium chloride or other

electrolytes

Peptone: Complex mixture of partially

digested
proteins
Serves as the nitrogen source.

Meat extract: contains protein degradation

products, inorganic salts, carbohydrates
and growth factors.
Commercially available meat extract is
known as Lab - Lemco BIOMEDICAL SCIENCE, FACULTY OF MEDICINE
Blood or serum: used for enriching culture
media
Agar: prepared from sea weed.
does not provide any nutrition to
bacteria
acts as a solidifying agent.

CLASSIFICATION OF CULTURE MEDIA

Based on physical state:
1. Liquid media
2. Semisolid media
3. Solid media BIOMEDICAL SCIENCE, FACULTY OF MEDICINE
 Based on oxygen requirement of
organism
1. Aerobic media
2. Anaerobic media

Based on nutritional factors

1. Simple media
2. Complex media
3. Synthetic media
4. Special media

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 SPECIAL MEDIA
 Enriched media
 Enrichment media
 Selective media
 Differential media
 Indicator media
 Transport media
 Sugar media

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 Simple media
 Consists of only basic necessities for
growth
 Can be solid or liquid
Eg:
1. Nutrient broth( peptone, meat extract,
Nacl, distilled water)
2. Nutrient agar( N. broth+ agar)
3. Peptone water
Complex media
 All media other than simple media
 Have added ingredients
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 Synthetic or Defined Media
• Media prepared from pure chemical
substances and the exact composition
of the medium is known.
• Ex- Peptone water- 1% peptone and
0.5%Nacl in water

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BIOMEDICAL SCIENCE, FACULTY OF MEDICINE
 SPECIAL MEDIA
ENRICHED MEDIA

 Basal medium added with some

nutrients such as blood, serum or egg

 Eg: 1. Blood agar

2. Loeffler’s serum slope
3. Chocolate agar

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 BLOOD AGAR

5-10% sheep blood is added to nutrient agar

Used for growing a number of bacteria
To demonstrate haemolysis.
Specific eg: Streptococcus

LOEFFLER’S SERUM SLOPE

 Serum is added for enriching the medium
Used for growing Corynebacterium
diphtheriae
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BIOMEDICAL SCIENCE, FACULTY OF MEDICINE
 ENRICHMENT MEDIA
Liquid media incorporated with certain
chemical substance which enhance the
growth of pathogenic organisms and
suppress the unwanted bacteria

Eg: 1. Tetrathionate broth ( for salmonella)

2. Selenite ‘F’ broth (Salmonella & Shigella)
3. Alkaline peptone water (V. cholerae)

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 SELECTIVE MEDIA

 Purpose: Same as enrichment media

 But a solid media
Eg:
1. Lowenstein- Jensen’s medium for
Mycobacteria
2. TCBS for Vibrio cholerae

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 DIFFERENTIAL MEDIA

Contains substances which help to distinguish

differing characteristics of bacteria
Eg: MacConkey’s medium

MacConkey’s Agar:
 Contains peptone, lactose, agar, sodium taurocholate
and neutral red.
 Due to fermentation of lactose, there is acidic pH
which forms pink colonies in the presence of neutral
red indicator.
 The lactose fermenters (LF) form pink coloured
colonies whereas non lactose fermenters (NLF)
produce colourless or pale colonies.
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Mac Conkey’s Blood agar
agar

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 INDICATOR MEDIA

Contains an indicator which changes

colour when bacterium grows in them.

Eg:
1.Wilson and Blair medium
Salmonella typhi grow as black colonies.
2.MacConkey’s Agar
Lactose fermenters form pink coloured
colonies
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← Mac Conkey’s
agar
Eosin
Methylene Blue
medium (EMB)→

Salmonella
Shigella agar

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 TRANSPORT MEDIA
Used in the case of delicate organism (eg:
Gonococci) which may not survive the time
taken for transit
OR
May be over grown by non pathogenic
bacteria (eg: Vibrio cholerae)

Eg:
Stuart’s transport media for Gonococci
Buffered glycerol saline for Shigella

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 Sugar Media
• Sugar media consists of 1% of sugar in
peptone water along with an
apppropriate indicator.
• A small tube ( Durhams tube ) is kept
inverted in the sugar tube to detect gas
production

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 BIPHASIC MEDIA
Containing both liquid and solid media in the
same bottle
Eg: Brain heart infusion agar and broth in the
same bottle
Mainly used for blood culture
Blood is inoculated into liquid medium and
bottle incubated in upright position
For inoculation of agar, bottle is tilted at
intervals so that the broth flows over the agar
slant
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Colonies formed on the agar slant are studied
and identified

ADVANTAGES
Minimize materials and manipulations required
for processing the specimen
Reduces contamination
Reduce risk of infection to
lab workers

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 Anaerobic Media
• Used for the cultivation of anaerobic
bacteria

Eg: 1. Robertson’s cooked meat media :

A liquid medium with finely divided
meat pieces, which acts as reducing

agents
2. Thioglycollate broth
(can also be used as transport media for anaerobes

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• INDICATIONS FOR BACTERIAL CULTURE

• 1.Isolate bacteria in pure culture from the

clinical specimens & their identification by
various tests.
• 2.Determine antibiotic susceptibility
• 3.Prepare antigens for serodiagnosis of
infective diseases
• 4.Maintain stock cultures
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• Demonstrate their proprieties
• To estimate viable counts
• Type isolate by methods such as
bacteriophage & bacteriocin suceptiability

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• METHODS OF CULTURE

• Streak culture
• Stroke culture
• Stab culture
• Lawn culture
• Pour plate culture
• Liquid culture
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• STREAK CULTURE

• Routine method employed for bacterial isolation

in pure culture
• A platinum or nichrome wire loop of 2-4 mm
diameter is used
• Confluent growth occurs at the primary
inoculum
• Well separated colonies on the final streaks
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• The loop should be flamed & cooled
between the different sets off streaks.
• On incubation growth confluent at the site
of original inoculum but becomes thinner &
well separated colonies at the final
streaks

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BIOMEDICAL SCIENCE, FACULTY OF MEDICINE
• STAB CULTURE

• Employed to
• Oxygen requirement of the bacterium

• Maintain stock cultures for the preservation

of bacteria
• For demonstration of gelatin liquefication.
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BIOMEDICAL SCIENCE, FACULTY OF MEDICINE
• STROKE CULTURE
• Uses
• Providing a pure growth of bacterium for
slide agglutination and other diagnostic
tests

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• LAWN CULTURE ( Carpet Culture)

• Employed in
• Antibiotic susceptibility testing

• Bacteriophage typing

• Preparation of bacterial antigens and vaccines

where large amount of bacterial growth is required.

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• Provides a uniform suface growth of the
bacterium.
• Prepared by flooding the surface of the plate
with a liquid culture or suspension of the
bacterium ,pipetting the excess
inoculum.OR
• The surface of the plate may be inoculated
by applying a swab soaked in bacterial
culture.
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BIOMEDICAL SCIENCE, FACULTY OF MEDICINE
 Pour Plate
• Tubes with 15 ml of the agar medium
melted & left to cool in a water bath at 45-
50. Appropriate dilutions of the inoculum
are added in 1ml vol of the agar, mixed
well& contents of the tube poured into
pertidish& allowe to set.
• After incubation colonies seen distrubuted
through the depth of the medium.

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• Colonies counted by colony counters.
• Gives an estimate of the viable bacterial
count in a suspension.

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 Sweep Plate
• The edges of the petridishes containing
the medium are rubbed over the fabric
• With the medium facing it.
• The dust particles stirred up from the cloth
get settled on the medium & colonies
develop after incubation.
• Counts are made.

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 Liquid Culture
• Done in tubes,bottles by touching with a
charged loop or by adding inoculum.
• Adopted for blood culture & sterility test
where the concentration of bacteria is
small
• Preferred when large yields required
• Demerit- Does not provide a pure culture
from the mixed inocula

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• ANAEROBIC CULTURE METHODS
• Production of vacuum

• Displacement of oxygen

• Displacement and combustion of oxygen

• Absorption of oxygen by chemical or biological methods

• By reducing agents

• Anaerobic chamber

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 1.Cultivation In Vaccum
• Attempted by incubating cultures in a
vacuum dessicator.
• Demerit- Some oxygen always remains
behind.

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REFERENCES
• Tortora JG, Funke BR & Case CL 2014,
Microbiology: An Introduction (12th edition). Los
Angeles: Benjamin/Cummings Publishing Co.
•  Joanne.M.Willey,Linda.M.Sherwood,
Christopher.J.Woolverton; 2010; Prescott, Harley
and Klen’s Microbiology(8th Edn.); McGrawHill.
•  Geo F Brooks, Karen C Caroll, et al., 2010;
Jawetz, Helnick and Adelberg’s Medical
Microbiology (25th Edn.);McGrawHill.
•  Greenwood & Slack & Barer & Irving, 2012,
Medical Microbiology (18th Edn.); Churchill
Livingstone/Elservier.

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 THANK YOU

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make an effort in your life makes all the
difference.”

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