Culture and identification

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Culture and identification

J.Matějková, O.Melter

Ústav lékařské mikrobiologie

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Culture and media

• Culture (=grown) on solid and in liquid culture media

• Liquid culture media –

a) reinforced bacterial growth b) Biochemichal identification

• Solid media – bacterial colony

morphology, preliminary identification

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Liquid culture media

• Incubation in tubes

• General composition – nutrient broth,

source of biogenic elements – C,H,O,N,P

• Advantage – sufficient quantity and availability of nutrients

• Limitations - the individual species of bacteria can not be distinguished

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Bacterial growth patterns in liquid media

• Growth pattern in broth (A) depending on the relationship of bacteria to oxygen

• Uniform cloudiness (B)

• Sediment (C)

• Pellicle (D)

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Solid culture media

• Performed in Petri dishes

• Composition – nutrient broth + agar (a gelatinous colloidal extract of a red alga)

• Classification

- Basic – nutrient agar

- Enriched – e.g. blood agar

- Diagnostic agar – e.g. MacConkey agar (lactose fermenters grown in red colonies)

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Growth on solid culture media

• Clinical material (A) is inoculated/streaked (dilution) on solid culture medium (B) using bacteriological

loop. Usually after overnight incubation at 37C (C) visible colonies could be seen

• Typical colony morphology – size, shape, color,

surface, pigment, borders, consistency, phenomena around colonies (e.g. Hemolysis), odor

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Culture conditions

• Incubator temperature – usually 37°C (yeasts cca 20°C, Campylobacter 42°C)

• Humidity

• Nutrients, pH – source from culture media

• Optimal atmosphere - depending on the

relationship of bacteria to oxygen (aerobic, anaerobic, capnophilic 5% CO₂)

• Time of culture – usually 18-24 hours (mycobacteria up to 9 weeks)

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Biochemical identification

• Biochemical – detection of enzyme activities, e.g. saccharide feremtation

– Performed in tubes or on diagnostic strips – Today available also diagnostic kits

Biochemical identification

A. Pure bacterial culture is inoculated in liquid culture media with a substrate (e.g. saccharide), pH indicator and it is cultured

B. POSITIVE REACTION: metabolized substrate (e.g. fermented or oxidized) by microbial culture and colourful change

C. NEGATIVE REACTION – without the change

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Mass spectrometry based identification

• Based on comparing time of flight of ionized biomolecules (can be detected also e.g. carbapenemases)

(https://www.news-medical.net/life-

sciences/MALDI-in-Microbiology.aspx)

PCR based identification Usually directly from clinical materiál

(cerebrospinal fluid, stool, nasopharyngeal swab etc.)

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Serotyping (reverse agglutination)

• React antigen (usually microb surface Ag) with antibodies (Ab)

• Role – to diagnose particular serotypes (or rarely bacterial species)

• Antigens must be corpuscular – e.g. K, O, H antigens

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Serotyping procedure

• Take a single culture colony (A) and mix on the slide with the antibody (B)

• Mix the suspension and observe the

appearance of visible agglutinates (up to 30 seconds) (C), negative reactions (D)