general presentation of bacterial cultivation (growth and multiplication) for diagnostic purposes - for medical school students

1. Culture media
growing microorganisms for
diagnostic and/or production
purposes

2. General requirements
- acellular, inert media – suitable for most bacteria and
yeasts
- cell cultures / embryonated eggs / animal models –
needed for intracellular microorganisms (ricketsiae,
chlamidiae) and viruses
- Composition of culture media – based upon knowledge
of growth requirements in order to isolate, multiply and
identify bacteria
- Exceptions: bacterial species which cannot be grown on
culture media e.g. Mycobacterium leprae (leprosy),
Treponema pallidum (syphillis)

3. General requirements (II)
• sterility
• nutriets to support microbial growth and multiplication:
– water, carbon, nitrogen, growth factors, vitamins, minerals
• pH: 7.2-7.4 suitable for most germs
– (exceptions: 6.8 for Brucella spp. and 9 for Vibrio cholerae)
• clarity (transparency) →changes induced by bacterial
growth
• aerobiosis / anaerobiosis

4. Composition of culture media
– Peptones = products of animal protein hydrolysis = source of
nitrogen – non standardised composition but suitable for all
cultivable bacteria; included in all commercially available culture
media (Merck, Oxoid, etc)
– Beef extract – obtained by boiling and dehydrating beef =
source of nitrogen (creatine, xantine, uric acid, urea) and carbon
(glycogene, lactic acid)
– Yeast extract – important source of group B vitamins
– NaCl – for adjusting osmolarity (0.9-10%)
– Additional sources of carbon: glycerine, mannitol
– PLUS: solidification agents – agar-agar = gelatin from algae
(nondigestible for bacteria, does not melt at 37°C)

5. Classification of culture media
Main classification criteria:
I. Sate of matter
II. Complexity
III. Purpose

6. Classification of culture media
(continued)
I. Depending on state of matter:
A. Liquid media
1. Broth
2. Peptoned water
B. Semisolid & solid (gelified with 5% agar)

7. Classification of culture media
(continued)
A. Liquid media:
1. Nutrient broth = powdered beef extract (peptone
content) dissolved in water – commercially available;
used to be prepared by actually boiling beaf/horse
meat
- Widely used in microbiology laboratories:
- hemoculture – blood innoculated in liquid media
- identification of isolated bacterial strains by
biochemical tests (fermentation of sugars)

8. Nutrient broth in test tubes

9. Classification of culture media
(continued)
B. Solid media
- Obtained from liquid media by adding
agar-agar (gelification)
- 1st reported use: Robert Koch 1882 –
cultivation of M. tuberculosis
- Initially gelatin was used - disadvantages:
- Digested by some bacteria
- Liquifies at 37°C – most frequently used
incubation temperature

10. 1882: Fanny Hesse – idea to use agar as
solidification agent instead of gelatin

11. Classification of culture media
(continued)
B. Solid media – Agar (continued)
1000 ml nutrient broth + 25-30 g agar-agar →melted by
boiling + pH adjustment (7.2-7.4)
Features:
- odourless, colourless, nontoxic for microbes
- Nonsoluble in cold water, soluble in boiling water; upon
cooling causes gelification

12. Classification of culture media
(continued)
B. Solid media – Agar (continued)
Advantages:
- Isolated colonies (resulting by multiplication of a single
microbe) → pure cultures can be obtained
- Morphology of bacterial colonies: shape, size, changes
induced in the medium e.g. hemolysis, colour changes,
etc.
- Counting microbes in a biological sample e.g. urinary
infections

13. E.coli colonies on agar

14. Classification of culture media
Main classification criteria:
I. Sate of matter
II. Complexity
III. Purpose

15. Classification of culture media
(continued)
II. Depending on complexity:
1. Simple media (previously described)
2. Enriched media: blood and other special nutrients may
be added to general purpose media to encourage the
growth of fastidious microbes e.g. blood agar,
chocolate agar

16. Classification of culture media
(continued)
II.2. Enriched media: Blood agar:
- 5-10% mammalian blood (sheep / horse)
- used to isolate fastidious organisms and detect
hemolytic activity:
- β-hemolysis - lysis and complete digestion of red
blood cell contents surrounding colony e.g.
Streptococcus haemolyticus
- α-hemolysis - partial lysis – incomplete hemoglobin
digestion → green or brown (due to the conversion
hemoglobin to methemoglobin) e.g. Streptococcus
viridans
- γ-hemolysis (or non-hemolytic) - lack of hemolytic
activity

17. Blood agar plates
Left: Staphylococcus; Right: Streptococcus

18. Classification of culture media (continued)
II.2. Enriched media (continued): Chocolate agar
- variant of blood agar in which red blood cells have been
lysed by slow, gradual heating to 80°C in order to
provide additional growth factors contained in red blood
cells
- !Does not contain chocolate!! The name is suggestive for
the brownish colour resulted after red blood cell lysis
- used for growing fastidious respiratory bacteria e.g.
Haemophilus influenzaze, Neisseria meningitidis

19. Attention!
Enriched media are non-selective – i.e.
they contain additional substances aiming
to a better growth & multiplication
≠
Enrichment media are selective i.e.
content adjusted to favour certain germs
and inhibit others (see below)

20. Left: enriched, nonselective; Right: enrichment, selective

21. Classification of culture media
Main classification criteria:
I. Sate of matter
II. Complexity
III. Purpose

22. Classification of culture media (continued)
III. Depending on purpose:
1. Selective & enrichment media
2. Diagnostic media
3. Special media

23. Classification of culture media (continued)
III.1. Selective & enrichment media
- Favour the growth and multiplication of certain bacteria
while suppresing other species
- Very useful for polymicrobial biological products when
attempting to isolate pure cultures
- Used for inoculation of biological products (primary
isolation)
- Composition & cultivation conditions (temperature,
aero/anaerobiosis, etc) adjusted according to the known
growth characters & requirements of the suspected
microbe

24. Classification of culture media (continued)
III.1. Selective & enrichment media (continued)
Liquid selective media and/or cultivation condition –
examples:
- Nutrient broth + acid sodium selenite – Salmonella spp
- Peptone water – Vibrio cholerae – the alkaline pH (9)
inhibits other species
- Temperature: +4°C – inhibits the growth of most bacteria
EXCEPT Listeria spp

25. Classification of culture media (continued)
III.1. Selective & enrichment media (continued)
Solid selective media – same principles, same inhibition
criteria
Chemical inhibitors: antibiotics (chosen depending on the
known natural sensitivity of bacteria) e.g. Vancomycin
added when trying to isolate gram negative anaerobic
bacteria (gram positive anaerobic bacteria are
vancomycin sensitive and their growth will be inhibited)

26. Classification of culture media (continued)
III. Depending on purpose:
1. Selective & enrichment media
2. Diagnostic media
3. Special media

27. Classification of culture media (continued)
III.2. Diagnostic media
- Contain indicator systems demonstrating metabolic
characters of certain microbial species (fermentation of
sugars, production of H2S, etc)
E.g. Fermentation of sugars:
nutrients + sugar + pH indicator – in case fermentation
occurs the colour will change indicating the presence of
a bacteria which ferments that sugar
- Identification relies on performing a number of tests and
analyzing the ”profile” which is further matched to known
metabolic & growth characters of bacteria

28. III.2. Diagnostic media (continued)
Mannitol Salt Agar (Chapman) - selective medium with a
high salt concentration for the isolation, growth and
enumeration of Staphylococcus species: organisms that
use mannitol turn the medium colour to yellow

29. Chapman agar – mannitol acidification

30. III.2. Diagnostic media (continued)
Mannitol Salt Agar (Chapman) (continued)
- both selective and differential:
- selective for organisms which survive & grow in high salt
concentration (Staphylococcus species) in contrast
to Streptococcus species, whose growth is inhibited by this high
salt agar → discriminate between Staphylococcus and
Streptococcus
- Content of mannitol allows selection of mannitol fermenting
bacteria (turn the colour of the medium from red to yellow) and
differentiate between fermenting (yellow) and non-fermenting
(red) species

31. Staph. aureus - mannitol fermentation (left side, left plate)
Staph.epidermidis - no mannitol fermentation (right side, left plate)
Streptococcus pneumoniae – plate on the right

32. Commercially available Kits for biochemical
tests (API galleries)

33. Staphylococcus spp – biochemical tests

34. Streptococcus spp – biochemical tests

35. Classification of culture media (continued)
III. Depending on purpose:
1. Selective & enrichment media
2. Diagnostic media
3. Special media

36. Classification of culture media (continued)
III.3. Special media
- Specially designed for certain species
E.g.
- Lowenstein-Jensen for M. tuberculosis
- Tynsdale for C. diphtheriae
- Bordet-Gengou for Bordetella pertussis

37. What if bacteria do not grow?
Troubleshooting
• Wrong culture medium
• Wrong quantities of ingredients
• Wrong pH
• Contamination (improper cleansing and/or sterilization of
plates, tubes, flasks)
• Impaired incubation conditions (power failures during
overnight incubation)
• Improper sample collection/transportation
• Lack of proper quality control of culture media (reference
strains)