Purpose of culturing are Isolation of bacteria ( pure culture) Diagnosis of infectious diseases Properties of bacteria i.e. culturing bacteria is the initial step in studying its morphology and its identification. Maintenance of stock cultures. Estimate viable counts. Water , air, milk testing To test for antibiotic sensitivity. To create antigens for laboratory use. Vaccine preparation Sterility testing Preparation of pharmaceutical products like antibiotics, enzymes, toxins etc Certain genetic studies and manipulations of the cells also need that bacteria to be cultured in vitro. Culturing on solid media is another convenient way of separating bacteria in mixture. An artificial culture media must provide similar environmental and nutritional conditions that exist in the natural habitat of a bacterium. A culture medium contains water, a source of carbon & energy, source of nitrogen, trace elements and some growth factors. PPT prepared by: DR.C. P. PRINCE HOD & Associate Professor Department of Microbiology Mother Theresa Post Graduate & Research Institute of Health Sciences (Government of Puducherry Institution)

1. Culture Media and Bacterial
Culture methods
DR.C. P. PRINCE
HOD & Associate Professor
Department of Microbiology
Mother Theresa Post Graduate & Research Institute of Health Sciences
(Government of Puducherry Institution)

2. Cultivation/Culturing of
Bacteria
 A microbial culture, is a method of
multiplying microorganisms by letting
them to reproduce in predetermined
culture media under controlled
laboratory conditions.( Growing
Bacteria in Laboratory)
 Microbial cultures are used to determine
the type of organism, its abundance in
the sample being tested, or both.

3. Purpose of culturing
 Isolation of bacteria ( pure culture)
 Diagnosis of infectious diseases
 Properties of bacteria i.e. culturing bacteria is the
initial step in studying its morphology and its
identification.
 Maintenance of stock cultures.
 Estimate viable counts. Water , air, milk testing
 To test for antibiotic sensitivity.
 To create antigens for laboratory use.
 Vaccine preparation
 Sterility testing
 Preparation of pharmaceutical products like
antibiotics, enzymes, toxins etc
 Certain genetic studies and manipulations of the
cells also need that bacteria to be cultured in vitro.
 Culturing on solid media is another convenient way
of separating bacteria in mixture.

4. Culture Media
 An artificial culture media must provide
similar environmental and nutritional
conditions that exist in the natural habitat of a
bacterium.
 A culture medium contains water, a source of
carbon & energy, source of nitrogen, trace
elements and some growth factors.
 The pH of the medium must be set
accordingly.
 Uses:
 *Enrich the number of bacteria.
 *Select for certain bacteria and suppress
others.

5. Agar
 Solidifying agent used for preparation of solid
culture medium
 Agar, a polysaccharide extracted from marine
algae, is used to solidify a specific nutrient
solution.
 Unlike other gelling agent, it is not easily
degraded by many bacteria.
 It is not easily destroyed at higher
temperatures, and therefore it can be
sterilized by heating, the process which also
liquefies it.
 Once solidified, agar medium will remain
solid

6. Pure culture
 In the laboratory bacteria are isolated
and grown in pure culture in order to
study the functions of a particular
specie.
 A pure culture is a population of cells or
growing in the absence of other species
or types. A pure culture may originate
from a single cell or single organism, in
which case the cells are genetic clones
of one another
 Medium containing only one type of

7. Classification of Culture Media
 Bacterial culture media can be
classified in at least three ways.
 1. Consistency
 2. Nutritional component
 3. Functional use

8. Classification based on
consistency
 1. Liquid media.
 2. Solid media.
 3. Semi solid media.

9. Classification based on Nutritional
Components
 1. Simple media.
 2. Complex media.
 3. Synthetic or chemically defined
media.

10. Classification based on Functional Use or
Application
 1. Enriched media.
 2. Selective media.
 3. Differential media.
 4. Transport media.
 5. Indicator media.
 6. Anaerobic media.

11. 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.

12. 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. haemolysis on blood
 e.g. blood agar, chocolate agar, Loeffler’s
serum slope

13. Blood agar
 sterile de-fibrinated 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 oC
 Supports the growth of most delicate
organisms e.g- .Streptococcus pyogenes
 Identifying bacteria according to their
haemolytic-action on the red cells

14. 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, Haemophilus
.influenza and Streptococcus
pneumonae

15. Loeffler's serum slope
 Opaque whitish solid medium.
 The medium is solidified in hot air
inspissator at.. 75°C for 2 hr for 2
successive days
 Uses : Culture of Corynebacterium
diphtheria

16. 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.

17. Lowenstein Jensen medium
 Selective medium for
Mycobacterium tuberculosis
 Contains beaten eggs +malachite
green
 Green opaque solid medium.
 Sterilized in hot air inspissator at 75
°C for 2hr for 2 successive days

18. MacLeod's tellurite blood
agar(TBA)
 Blood agar + 0.02-0.04% K tellurite.
 Red opaque solid medium. .
 Selective medium Used for
isolation of Corynebacterium
diphtheriae .

19. Modified Thayer-Martin agar
 Chocolate agar + vancomycin +
colistin +nystatin
 Brown opaque-solid medium.
 Selective medium for Used for
Isolation of Neisseria from non-
sterile specimens.

20. 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.
 Selective medium Used for
isolation of Vibrio cholerae.

21. Deoxycholate citrate agar
(DCA)
 Na deoxycholate and citrate + Agar
+ lactose + neutral red indicator.
 Reddish semi transparent solid
medium.
 Selective medium Used for
isolation of Shigella and
Salmonella.

22. XLD Media
 Agar + lactose + phenol red
indicator + ferric citrate +
desoxycholate + xylose + lysine
+sucrose + yeast extract
 Reddish semi transparent solid
medium.
 Selective medium Used for
isolation of Shigella and Salmonella

23. ENRICHMENT MEDIA
 Fluid media that contain substances
which favour the growth of wanted
organisms on the expense of others.
 Usually used as a preliminary step for
isolation of pathogens before
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

24. INDICATOR (DIFFERENTIAL MEDIA)
 These are media to which dyes or
other substances (Indicators()are
added to differentiate
microorganisms.
 Indicators change colour when acid
is produced following fermentation
of a specific carbohydrate e.g.
MacConkey's agar medium.

25. MacConkey's agar medium
 Peptone, agar, lactose, bile salt
and neutral red indicator.
 Reddish transparent solid medium
 Detects lactose fermenting and non
lactose fermenting bacteria

26. 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 colour of the medium and
or the production of gas.
 They include peptone water sugars,
litmus milk, and gelatin media.

27. TRANSPORT MEDIA
 Semisolid media that contain ingredients
to prevent the overgrowth of commensal
& ensure the survival of aerobic and
anaerobic pathogens when specimens
cannot be cultured immediately.
 Examples:
 1- Cary-Blair medium for preserving
enteric pathogens.
 2- Stuarts and Amies transport medium
for ensuring the viability of gonococci
 3- Thioglycollate broth and deep agar for-
3 anaerobic organisms

28. CULTURE MEDIA FOR
ANAEROBES
 Media for anaerobes is the same as
media for aerobes except that:
 1. They are richer in organic
constituents .
 2. Contain reducing agents (cysteine
& haemin).
 3. Contain a redox indicator .
 The inoculated media are incubated in
anaerobic environment using
anaerobic gas pack .

29. Robertson's cooked meat
medium
 Anaerobic enrichment media
 cooked minced meat to which broth
is added
 Anaerobiosis is achieved through
(reducing substances in the
meat).e.g. haemin and glutathione
 Sterilized in autoclave at 121°C-for
30 min

30. Anaerobic GasPak System
 A method for the exclusion of
oxygen from a sealed jar used for
incubation of anaerobic cultures in
a non-reducing medium .

31. Robertson's cooked meat
medium

33. Anaerobic Culture Methods
 Production of a vacuum
 Displacement of Oxygen with other
gases
 Absorption of Oxygen by chemical or
biological methods
 By using reducing agents

34. McIntosh & Filde’s Jar

37. Anaerobic GasPak System

38. Displacement of Oxygen

39. Anaerobic Glove Chamber

40. Anaerobic Glove Chamber

41. Nutrient broth

42. L-J medium

43. TCBS

44. XLD Agar

45. MacConkey's agar medium

46. Robertson's cooked meat
medium

47. Culture Methods
 Streak culture
 Lawn culture
 Stroke culture
 Stab culture
 Pour plate method

48. Streak culture
 Used for the isolation of bacteria in pure
culture from clinical specimens.
 Platinum wire is used.
 One loop full 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.

49. The streak-plate method to
obtain pure cultures

50. Streak culture

51. Lawn Culture
 Provides a uniform surface growth of the
bacterium.
 Lawn cultures are prepared by flooding
the surface of the plate with a liquid
suspension of the bacterium
 Uses
 – For bacteriophage typing.
 – Antibiotic sensitivity testing.
 – In the preparation of bacterial antigens
and vaccines.

52. Lawn Culture

53. Stroke Culture
 • Stroke culture is made in tubes
containing agar slope / slant.
 Uses:
 Provides a pure growth of bacterium
for slide agglutination and other
diagnostic tests.

54. Stroke Culture

55. 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.

56. Pour Plate Culture
 1 ml of the innoculum is added to the
molten agar.
 Mix well and pour to a sterile Petri
dish.
 Allow it to set.
 Uses:
 – Gives an estimate of the viable
bacterial count in a suspension.
 – For the quantitative urine cultures

59. Thanks