Microbiology practical

1. General Microbiology Laboratory
Microbiology Practical 3
CULTURE MEDIA & CULTURE METHODS
Instructors: William Edema-CMLT
Cosmas Andruga

2. Aim of the practical
 Learn the different types of culture media
 Demonstrate preparation of culture media
 Demonstrate culturing methods

3. Introduction
 In natural environments, microorganisms
usually exist as mixed populations.
 However, if we are to study, characterize, and
identify microorganisms, we must have the
organisms in the form of a pure culture.
 A pure culture is one in which all organisms
are descendants of the same organism.
Techniques for obtaining pure cultures from a
mixed population will be studied in this Lab
subsequently.

4.  Bacteria have to be grown (cultured) for
them to be identified.
 By appropriate procedures they have to be
grown separately (isolated) on culture
media and obtained as pure for study.
History
 The original media used by Louis Pasteur
– urine or meat broth
 Liquid medium – diffuse growth
 Solid medium – discrete colonies.

5. Colony – macroscopically visible collection of
millions of bacteria originating from a
single bacterial cell.
 Cooked cut potato by Robert Koch –
earliest solid medium
 Gelatin – not satisfactory
- liquefy at 24oC

6. Agar
 Frau Hesse
 Used for preparing solid medium
 Obtained from seaweeds.
 No nutritive value
 Not affected by the growth of the bacteria.
 Melts at 98oC & sets at 42oC
 2% agar is employed in solid medium

7. Types of culture media
I. Based on their consistency
a) solid medium
b) liquid medium
c) semi solid medium
II. Based on the constituents/ ingredients
a) simple medium
b) complex medium
c) synthetic or defined medium
d) Special media

8. Special media
– Enriched media
– Enrichment media
– Selective media
– Indicator media
– Differential media
– Sugar media
– Transport media
– Media for biochemical reactions
III.Based on Oxygen requirement
- Aerobic media
- Anaerobic media

9. Solid media – contains 2% agar
 Distinctive colony morphology (size, shape, margins,
pigmentation, hemolysis etc.) can be appreciated.
Characteristics easy to identify
 Eg: Nutrient agar, Blood agar
Liquid media – no agar.
 Diffuse growth, no x-tics for identification
 For inoculum preparation, Blood culture, for the
isolation of pathogens from a mixture.
 Eg: Nutrient broth
Semi solid medium – 0.5% agar.
 Eg: Motility medium
Types of culture media: Based on consistency:

10. a) solid medium
b) liquid medium
c) semi solid medium
Types of culture media: Based on consistency:

11. Simple media / basal media
Most common in routine diagnostic labs:
- Eg: Nutrient Broth, Nutrient Agar
- NB consists of peptone, meat extract, NaCl,
- NB+0.5 Glucose= Glucose Broth
- NB + 2% agar = Nutrient agar
- Agar conc. reduced to (0.2-0.5%)= Semi-solid
media
Types of culture media:
Based on the constituents/ ingredients

12. Complex media
 Media other than basal media.
 They have added ingredients e.g. yeast
extract and casein hydrolysate, which consist
of a mixture of many chemical species of
unknown proportions.
 Provide special nutrients
Synthetic or defined media
 Media prepared from pure chemical
substances and its exact composition is
known; Eg: peptone water – 1% peptone +
0.5% NaCl in water

13. Enriched media
 Substances like blood, serum, egg are added
to the basal medium.
 Used to grow bacteria that are exacting in their
nutritional needs (fastidious organisms).
 E.g.: Blood agar, Chocolate agar
Blood agar Chocolate agar

14. Enrichment media
 Liquid media used to isolate pathogens from a mixed
culture.
 Media is incorporated with inhibitory substances to
suppress the unwanted organism e.g.:
– Selenite F Broth – for the isolation of Salmonella,
Shigella
– Alkaline Peptone Water – for Vibrio cholerae
Selenite F Broth Alkaline Peptone Water
Tetrathionate broth

15. Selective media
 Media which contains substances that prevent
or slow the growth of microorganisms other
than the bacteria for which the media is
prepared for.
 The inhibitory substance is added to a solid
media. E.g:
– Mac Conkey’s medium for gram negative bacteria
– TCBS – for V.cholerae
– LJ medium – M.tuberculosis
– Wilson and Blair medium – S.typhi
– Potassium tellurite medium – Diphtheria bacilli

16. TCBS
Mac Conkey’s medium
Selective media examples

17. Potassium Tellurite media LJ media
Selective media examples

18. Indicator media
 These media contain an indicator which
changes its colour when a bacterium grows
in them.
 Eg:
– Blood agar
– Mac Conkey’s medium
– Christensen’s urease medium

19. Blood agar: shows three (3) types of hemolysis
• Erythrocytes are
incorporated into nutrient
agar medium
• Certain bacteria produce
products that lyse Red Blood
Cells
 Alpha-hemolytic- partial lysis
 Beta-hemolytic- complete lysis
 Gamma-hemolytic- no lysis

20. Urease medium

21. Differential media
 Contain indicators, dyes, etc, to differentiate
microorganisms.
 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).
– Lactose fermenters – Pink colonies
– Non lactose fermenters – colourless colonies

22.  Lactose fermenters – Pink colonies
 Non lactose fermenters – colourless colonies
Chocolate agar MacConkey agar
Examples of Lactose fermenters Escherichia coli, Enterobacter and
Klebsiella
Examples of Non-Lactose fermenters: Salmonella, Proteus species, Yersinia,
Pseudomonas aeruginosa and Shigella

23. 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
– Buffered glycerol saline – enteric
bacilli

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

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

26. Common media used in Microbiology Laboratory
 Chocolate Agar: Blood agar prepared by heating
blood to 95C until medium becomes brown or
chocolate in color.
 Heating the blood releases both 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
gonorrhea.
 MacConkey Agar: An inhibitory and differential
medium used to distinguish lactose- fermenting
Gram- negative organism from non fermentation.
Crystal violet, bile salts and neutral red are inhibitor
agent. neutral red is the PH

27. Common media used in Microbiology Laboratory
 Mannitol Salt Agar (both selective and differential)
For selective isolation of coagulase positive,
mannitol-fermenting staphylococcus.
 High salt concentration (7.5%) only permits
Staphylococcous spp. growth.
 Mannitol is a sugar alcohol fermented by certain
species
o Pathogenic Staph will ferment mannitol
o Non-pathogenic Staph will not ferment
mannitol
 If mannitol is fermented, acidic products are
formed.
o Indicated by phenol red (yellow is acidic)

28. Common media used in Microbiology Laboratory
 Mannitol Salt Agar (both selective and differential)

29. Common media used in Microbiology Laboratory
Mueller Hinton Agar:
 Rich medium that support the growth of most
microorganism.
 It is commonly used for antibiotic
susceptibility testing:
o disk diffusion antibiotic susceptibility;
o antibiotic serum level measurements;
o MBC determination.

30. Common media used in Microbiology Laboratory
 Salmonella Shigella ( SS ) Agar: Isolation and
differential medium for pathogenic Gram-negative
bacilli in particular, Salmonella and Shigella.
 It is an Inhibitor for Coliforms.
 Triple Sugar Iron Agar (TSI): this is a key medium
used in the beginning of the identification of 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 .
 Indicators: Phenol red is the PH indicator; ferric ammonium
citrate is H2S indicator.

31. Preparation of Culture Media
 When lab personnel make media, they
measure out a quantity of dry powdered
nutrient media, add water and check the pH.
– Measurements are done according to
manufacturer’s recommendation
The media is then dispensed into bottles (flask,
tube), capped and autoclaved at 121°C (15 psi)
for 20 minutes.
Once the media is autoclaved it is sterile (all
microoranism forms killed)

35. CULTURE METHODS
 Culture methods employed depend on the purpose
for which they are intended.
 The indications for culture are:
– To isolate bacteria in pure cultures.
– To demonstrate their properties.
– To obtain sufficient growth for the preparation of
antigens and for other tests.
– For bacteriophage & bacteriocin susceptibility.
– To determine sensitivity to antibiotics.
– To estimate viable counts.
– Maintain stock cultures.

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

37. STREAK CULTURE
 Used for the isolation of bacteria in pure culture
from clinical specimens.
 Platinum wire or Nichrome 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.

38. STREAK CULTURE: Procedure
Necessary Equipment

39. STREAK CULTURE: Procedure

40. Identification of bacteria
- Once a pure colony
is obtained, the next
step is to identify the
bacterial species.
This will be done in
the next practical

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

44. Antibiotic sensitivity testing

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

46. 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 stoke cultures.

47. Gelatin liquefaction Oxidation – Fermentation
medium

48. 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.

49. 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.

50. Blood culture bottles