Culture and identification of

infectious agents, Lecture 25

Dr. Alvin Fox

Key Terms
Isolation (culture)
Agar plate
plate/colonies
Liquid media
Identification & taxonomy
Family
Genus
Species
Type
Strain

After culture
Biochemical (physiological) tests
Genetic tests
- Polymerase chain reaction (PCR)
- Sequencing
- DNA-DNA homology/arrays
- Restriction enzymes (digests)
Chemical
- fatty acid/protein profiling
Immunological
Direct detection (i.e. without culture)
PCR
Antigen detection
Staining (e.g. Gram stain)
2
Serology (antibody detection)

Taxonomy
Defines common traits among strains for
a bacterial species
Usually genetic
Allows development of diagnostic kits
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Species versus strains

- selecting discriminating features

Classification

Strain: one single isolate or line

Type: sub-set of species
Species: related strains
Genus: related species
Family: related genera

Both terms define the species name

Streptococcus pyogenes

Genus part

Species part

So Streptococcus pyogenes and Streptococus agalactiae

would be two species in the same genus
In a report
On first usage: Streptococcus pyogenes
On second usage: S. pyogenes
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Identification of infectious agents

in the diagnostic laboratory

Aids treatment
Helps antibiotic selection
General hospital laboratory
physiological tests
More fully equipped laboratories
Genetic tests
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Steps in isolation and identification

Step 1: Streaking culture plates

colonies on incubation (e.g 24 hr)
size, texture, color, hemolysis
oxygen requirement

Sheep blood agar plate culture

Bacillus cereus.

Bacillus anthracis

CDC/Dr. James Feeley

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Mixed colonies

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Isolation and identification

Step 2: Colonies Gram stained
cells observed microscopically

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Gram negative

Gram positive
Heat/Dry
Crystal violet stain
Iodine Fix
Alcohol dede stain
Safranin stain
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Gram stain morphology

Shape
cocci (round)
bacilli (rods)
spiral or curved (e.g. spirochetes)
Single or multiple cells
clusters (e.g. staphylococci)
chains (e.g. streptococci)
Gram positive or negative
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Step 3:
Isolated bacteria are speciated
Generally using physiological tests

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Clinical Microbiology
Laboratory Bench

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Step 4:
Antibiotic susceptibility testing

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Antibiotic susceptibility testing

Susceptible

Not susceptible

Bacterial
lawn
No
growth

Growth

Antibiotic disk
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Molecular
differentiation
Genomics

Gene characterization
PCR
Sequencing
Restriction digestion
Hybridization (probes, arrays)
% guanine-cytosine

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16S rRNA Sequencing

Differentiates bacterial species
Development of clinical tests based
on sequence (e.g. PCR)

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Real-time PCR
ds DNA

Cycle one
Dye

Cycle two

Cycle 30
2 30

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DNA-DNA hybridization
Strain 1
Heat

0% Homology

Strain 2

100% Homology

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Profiles
Long chain fatty acids
- structural (e.g. cell membrane)

Short chain
- metabolic
- volatiles
- Fatty acids/alcohols
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 Protein profiling: defining a species by

characteristic (low molecular weight)
proteins
Proteomics: defining all proteins
expressed by a species under specific
growth conditions

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Bacterial DNA sequences can be

amplified directly from human body
fluids

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Laboratory diagnosis without

culture
In general, when speed is of essence

Of additional importance:
The organism grows poorly/slowly
The organism cant be cultured
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Microscopy

spinal fluids (meningitis)

sputum (tuberculosis)
sensitivity poor

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Streptococcal Agglutination Test

Streptococcal
antigenic extract
Antibody

Latex beads

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Serologic identification
antibody response to the infecting agent
several weeks after an infection has occurred

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Epidemics
associated with particular strains/types
state or federal laboratory system

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