Culture media

growing microorganisms for diagnostic and/or production

purposes

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)

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

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)

Classification of culture media

Main classification criteria:

I. Sate of matter

II. Complexity

III. Purpose

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)

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)

Nutrient broth in test tubes

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

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

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

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

E.coli colonies on agar

Classification of culture media

Main classification criteria:

I. Sate of matter

II. Complexity

III. Purpose

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

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

Blood agar platesLeft: Staphylococcus; Right: Streptococcus

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

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)

Left: enriched, nonselective; Right: enrichment, selective

Classification of culture media

Main classification criteria:

I. Sate of matter

II. Complexity

III. Purpose

Classification of culture media (continued)

III. Depending on purpose:

1. Selective & enrichment media

2. Diagnostic media

3. Special media

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

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

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)

Classification of culture media (continued)

III. Depending on purpose:

1. Selective & enrichment media

2. Diagnostic media

3. Special media

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

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

Chapman agar – mannitol acidification

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

Staph. aureus - mannitol fermentation (left side, left plate) Staph.epidermidis - no mannitol fermentation (right side, left plate)

Streptococcus pneumoniae – plate on the right

Commercially available Kits for biochemical tests (API galleries)

Staphylococcus spp – biochemical tests

Streptococcus spp – biochemical tests

Classification of culture media (continued)

III. Depending on purpose:

1. Selective & enrichment media

2. Diagnostic media

3. Special media

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

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)