This document provides information on bacterial culture medium and culture techniques. It discusses the history of bacterial culture, the need for culture methods, types of media including solid, liquid and selective media. Common media such as nutrient agar, blood agar and MacConkey agar are described. The document also covers culture methods like streak plate, pour plate and anaerobic culture techniques. Culture media provide a controlled environment for bacterial growth, aiding identification and characterization of bacteria.

1. Bacterial Culture Medium and
Culture Technique
Manoj Mehta
Msc.clinical microbiology

2. CONTENTS
1.INTRODUCTION
2.HISTORY
3.CULTIVATION
4.EQUIPMENT
5.MEDIA CLASSIFICATION AND FUNCTION
6.FREQUENTLY USED MEDIA
7.TYPES OF CULTURE MEDIA
8.CULTURE METHODS

3. INTRODUCTION
Direct laboratory methods, such as microscopy provide preliminary
information about the bacteria involved in an infection, but bacterial growth is
usually required for definitive identification and characterization,hence the
culture method is needed.

4. HISTORY
 Louis Pasteur : which states that microorganisms are the
causes of infectious disease and used simple broths to grow;
made up of urine or meat extracts.
 Robert Koch: established that microbes can cause and realized
the importance of solid media and used potato pieces
to grow bacteria

5. Definition:
 Culture is the term given to microorganisms that are cultivated in the lab for the
purpose of identifying and studying them.
 Medium is the term given to the combination of ingredients that will support the
growth and cultivation of microorganisms by providing all the essential nutrients
required for the growth in order to cultivate these microorganisms in large
numbers to study them
 Cultivation is the process of growing microorganisms in culture by taking
bacteria from the infection site by some means of specimen collection and
growing them in the artificial environment of the laboratory.

6. Need for Bacterial Culture
• To grow and isolate all bacteria present in a clinical specimen.
• To determine which of the bacteria that grow are most likely causing
infection and which are likely contaminants or colonizers.
• To obtain sufficient growth of clinically relevant bacteria to allow
identification and characterization.
Need for Culture media
• Used to grow bacteria
• Can be used to enrich the numbers of bacteria
• Select for certain bacteria and suppress others
• Differentiate among different kinds of bacteria
• Select for certain bacteria and suppress others
 Differentiate among different kinds of bacteria

7. EQUIPMENT :
• 1.Petri dish :
2.Bunsen Burner :
3.Inoculating loop :

8. Composition of culture media
• Water
• Agar
• Casein hydrolysate
• Meat extract
• Yeast extract
• Mineral salt
• Blood / serum

9. Agar
• Frau Hesse: Idea to use agar as solidification agent instead
of gelatin because gelatin melt at 24*c
Properties of Agar
• Universally used for preparing solid medium
• Obtained from seaweed: Gelidium
• No nutritive value
• Not affected by the growth of the bacteria.
• Melts at 98°C & sets at 42°C
Concentration of Agar
• (2 – 3 )% for solidifying culture media.
• (0.2-0.5)% for Semi-solid,
• (0.05-0.1)% for anaerobes and microaerophiles

10. Basic requirements
1.Chemical Requirements( Nutritional Requirements)
• Carbon
• Nitrogen, sulfur, and phosphorous
• Trace elements
• Organic growth factor
• Vitamins (e.g. folic acid, vitamin B-12, vitamin K)
• Oxygen
2.Physical Requirements
• Temperature
• PH
• Hydrostatic Pressure
• Osmotic pressure

11. Types of culture media
1.Based on physical state
• Liquid(broth)
• Semi-solid
• Solid(agar)
• Biphasic
2. Based on presence of molecular oxygen and reducing substances
• Aerobic media
• Anaerobic media
3. Based on nutritional factors
• Simple media
• Complex media
• Special media
• Synthetic media

12.  Special type of media are further divisible into
• Enriched media
• Enrichment media
• Selective media
• Differential media
• Indicator media
• Transport media and
• Sugar media.

13. Based on their consistency
 1.Liquid media: No use of agar
• Broth
• Peptoned water
Advantages
• Present small no.of bacteria , they grow only in liquid medium e.g. blood culture.
• Specimens containing inhibitory substances like antibiotics and other antibacterial
substances get diluted by inoculation into the large volume of the fluid medium..
• widely used for biochemical tests.
• useful enrichment media like selenite F broth.
• Presumptive bacterial count in water sample is made in liquid media.
Disadvantages
• Isolation of bacteria in pure culture is not possible.
• Identification of bacteria is not possible.

14. Solid culture Medium
• Contains 1- 2% agar
 Advantages
• Distinct colony morphology
• Colony microscopically visible There is separate colony
formation.
• Identification of most bacterial species.
• Isolation of bacteria in pure culture
eg.Nutrient agar, Blood agar
Semi-solid culture media:
• Contain (0.2-0.5)% agar
• Use for the observation of bacterial motility
• and preservation of bacteria.
e.g. SIM

15. Biphasic Blood Culture Media
• Has both liquid phase and solid phase.
• Also known as Casteneda medium has
agar slope with broth medium.
• Recommended if brucellosis is
suspected.

16. • Based on their nutritional factors
 Simple media / basal media:
• Most common in routine diagnostic laboratories
E.g: Nutrient Broth, Nutrient Agar
• NB consists of peptone, meat extract, NaCl, water
• NB + 0.5% Glucose = Glucose Broth
• NB + 2% agar = Nutrient agar
 Uses: to prepare enriched media
• To maintain stock culture of control bacterial strains.
• To subculture pathogenic bacteria from selective/ differential medium prior to
performing biochemical or serological test e.g. nutrient broth, nutrient agar.
• Reduced conc.of agar to 0.2-0.4% enables motile bacteria (6% agar inhibits
swarming growth of Proteus spp.)

17. Commonoly used media in lab
• Nutrient Agar
• Contain 2% agar
• commonly used
• Concentration can be increased to 6%
to prevent swarming
• Can be reduced to 0’5%

18. GREEN PIGMENTED COLONIES
NONPIGMENTED COLONIES
RED PIGMENTED COLONIES
YELLOW PIGMENTED COLONIES
Used to isolate separate colonies for studying
1.colony morphology.
2.pigmentation.
3.biochemical identification tests

19. Enriched media
• Substances like blood, serum, egg are added to the basal medium.
• Used to grow bacteria that are exacting in their nutritional needs.
• Eg: Blood agar, Chocolate agar,Lowenstein-Jensen’s medium
Chocolate
agar
Blood agar

20. Blood Agar
• Used to cultivate a wide variety of moderately fastidious bacterial
organisms.
• Blood agar base can be enriched by the addition of 5% to 10%
defibrinated sheep, rabbit, or human blood.
• Provides enrichment for the growth of the bacterial organisms, but also
allows for the detection and characterization of hemolytic activity.

22. • Heated blood agar
• Prepared by heating blood agar at 75-80 degrees
in a water bath for about 2o mins or until
it becomes chocolate brown in colour.
• Used for the growth of Haemophilus influenzae,
Neisseria, S. pneumoniae etc.
CHOCOLATE AGAR

23. Lowenstein-Jensen medium
• Used to cultivate Mycobacterium spp.
• Composition
• Malachite green (2%)
• Glycerol
• Asparagine
• Coagulated eggs
• Mineral salt solution
• Potassium dihydrogen phosphate
• Magnesium sulfate
• Sodium citrate

24. Brain heart infusion broth
• composition
• Calf brain infusion
• Beef heart infusion
• Protease peptone
• Dextrose
• Sodium chloride
• Sodium phosphate ( sodium
polyanethol sulphate)
• Enriched medium suitable for the
cultivation of nonfastidious and
moderately fastidious microorganisms
and used as a blood culture medium.

25. Enrichment media
• Liquid media used to isolate pathogens from a mixed culture.
• Media is incorporated with inhibitory substances to suppress
the unwanted organism → increase in numbers of desired
bacteria
Eg: Selenite F Broth –for the isolation of salmonella,Shigella
Tetrathionate Broth ( inhibit coliforms)
Alkaline Peptone Water – for Vibrio cholerae

26. Selective media
• The inhibitory substance is added to a solid media
• Increase in number of colonies of desired bacterium
Eg:
• Desoxycholate citrate medium for dysentery bacilli
• Mac Conkey’s medium for gram negative bacteria
• Thiosulfate citrate bile salts sucrose (TCBS) agar– for V. cholerae
• LJ medium – M. tuberculosis

27. Indicator media
• contain an indicator which changes its colour when a bacterium
grows in them
• Eg:
Wilson-Blair medium – S. typhi forms black colonies
McLeod’s medium (Potassium tellurite)– Diphtheria bacilli
Wilson-Blair Medium McLeod’s medium

28.  Differential media
 Media to which substances are added to diffential
differentiate bacteria.
Eg: MacConkey agar: Distinguish between lactose
fermenters and non lactose fermenters bacteria's
• Lactose fermenters – Pink colonies
• Non lactose fermenters – colourless colonies
 TCBS agar differentiates sucrose forment blue colonies
of vibrio species

29. MacConkey Agar
• Is a selective, differential, primary
plating medium.
Composition :
• Peptne
• Lactose
• Agar
• Neutral red
• Bile salt ( Sod. taurocholate)
(Omission of Sod. chloride prevents the
swarming of proteus colonies.)

30. Cysteine Lactose Electrolyte Deficient (CLED) agar
• CLED agar is now used by most laboratories to isolate urinary pathogens.
• Composition
• Cysteine
• Lactose
• Bromothymol blue
• Agar
• peptone
• No electrolyte so to prevent the swarming growth of Proteus spp.

31.  Sugar media
• Media containing any fermentable substance
• Eg: glucose, arabinose, lactose, starch etc.
• Media consists:
1% of the sugar in peptone water + Indicator
• Contain a small tube (Durham’s tube) for the detection of gas by the
bacteria

32.  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 – non nutrient soft agar gel
containing a reducing agent & charcoal
used for Gonnococci
• Buffered glycerol saline – enteric bacilli

33.  Anaerobic media
• These media are used to grow anaerobic organisms.
• Eg: Robertson’s cooked meat medium, Thioglycolate medium.

34. CULTURE METHODS
 Culture methods employed depend on the purpose for
which they are intended.
 Purposes:
Isolation
Properties of bacteria
To create antigens for laboratory
use
To test for Antibiotic sensitivity
Estimate viable counts
Maintain stock cultures

35.  Culture methods include:
• Streak culture
• Lawn culture
• Stroke culture
• Stab culture
• Pour plate method
• Liquid culture
• Anaerobic culture methods

36.  STREAK CULTURE
• Used for the isolation of bacteria in pure
culture from clinical specimens.
• Platinum wire is used.
• One loopful of the specimen is transferred
onto the surface of a well dried plate.
• Spread over a small area at the periphery.
• The inoculum is then distributed thinly over
the plate by streaking it with a loop in a
series of parallel lines in different segments
of the plate.
• On incubation, separated colonies are
obtained over the last series of streaks.

37.  LAWN CULTURE
oProvides a uniform surface growth
of the bacterium.
oUses
• For bacteriophage typing.
• Antibiotic sensitivity testing.
• In the preparation of bacterial antigens and vaccines.
oLawn cultures are prepared by flooding the surface of the plate with a
liquid suspension of the bacterium.
Antibiotic sensitivity testing

38. Mueller Hinton agar
• Beef infusion
• Casein hydrolysate
• Starch
• Agar
• Useful in antibiotic susceptibility
testing and also for testing starch
hydrolysis.

39. Properties of MHA
• Constituents of media is defined.
• Support growth of all types of pathogens.
• No enrichment or selective in nature.
• pH well adjusted at 7.3,isotonic and depth 4mm so antibiotics can
diffuses in media.
• Agar broth version same formula.
• Constituents are not antagonist to any drug.
• Starch is included in the medium for two reasons—it may protect the
organisms against toxic substances and also can serve as an energy
source for some bacteria.

40.  STROKE CULTURE
• Stroke culture is made in tubes containing agar
slope.
• Uses
• Provide a pure growth of bacterium for slide
agglutination and other diagnostic tests.

41.  STAB CULTURE
• Prepared by puncturing a suitable medium – gelatin or glucose
agar with a long, straight, charged wire.
• Uses
• Demonstration of gelatin liquefaction.
• Oxygen requirements of the bacterium under study.
• Maintenance of stock cultures.

42.  LIQUID CULTURES
• Liquid cultures are inoculated by touching with a charged loop or
by adding the inoculum with pipettes or syringes.
 Uses
• Blood culture
• Sterility tests
• Continuous culture methods
 Disadvantage
• It does not provide a pure culture
from mixed inocula

43. ANAEROBIC CULTURE METHODS
• Anaerobic bacteria differ in their requirement and sensitivity to
oxygen.
• Cl. tetani is a strict anaerobe
• Methods:
• Production of vacuum
• Displacement of oxygen with other gases
• Chemical method
• Biological method
• Reduction of medium

44.  POUR PLATE CULTURE
• Agar medium is melted (15 ml) and cooled to 45oC.
• 1 ml of the inoculum is added to the molten agar.
• Mix well and pour to a sterile petri dish.
• Allow it to set.
• Incubate at 37oC, colonies will be distributed throughout the depth
of the medium.
 Uses
• Gives an estimate of the viable bacterial count in a suspension.
• For the quantitative urine cultures.

45.  Displacement of oxygen with other gases
• Displacement of oxygen with hydrogen, nitrogen, helium or CO2.
• Eg: Candle jar
Inoculated plates are placed inside a large airtight container
And a lighted candle kept in it before the lid is sealed
The burning candle is expected to use up all the oxygen inside
Before it is extinguished ,but some oxygen is always left behind.
The candle jar provides a concentration of carbondioxide which
stimulates the growth of most bacteria.

46. Chemical method
• Alkaline pyrogallol absorbs oxygen.pyrogallic acid added to a solution
of sodium hydroxide in a large test tubes placed inside an airtight jar
povides anaerobic environment but small amount of carbonmonoxide,
which is formed during reaction,may be inhibitory to some bacteria
• Instead of alkaline pyrogallol,anaerobic environment has been
produced within jars with a mixture of Chromium and Sulphuric acid
McIntosh – Fildes’ anaerobic jar
• Consists of a metal jar or glass jar with a metal lid
which can be clamped air tight.
• The lid has 2 tubes – gas inlet and gas outlet
• The lid has two terminals – connected to electrical supply.
• Under the lid – small grooved porcelain spool, wrapped
with a layer of palladinised asbestos.

47.  Biological method
• Absorption of oxygen by incubation with aerobic bacteria,
germinating seeds or chopped vegetables.
 Reduction of oxygen
• By using reducing agents – 1% glucose, 0.1% Thioglycolate