Diagnosis of Bacterial Infection

Bacterial Cultivation

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 Outline
 General procedure of bacteriological Dia
gnosis
 Culture medium
• concept
• categories
• bacterial growth patterns
 Inoculation and transfer techniques

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General Procedure of Bacteriological Diagnosis

Specimens collection

General rules
The specimens should be representative of the infectious
process;
Sufficient material;
Avoid the contamination of specimens;
Be sent to the lab immediately in an appropriate method
and examined ASAP.
Be collected before using antimicrobial agents, e.g. antibio
tics.

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General Procedure of Bacteriological Diagnosis

specimens
Morphologic identification
microscopy & staining

Isolation, identification

Biochemical tests
Antigen detection
EIA, ELISA, IF test, agglutination test
Serological diagnosis (Ab titer)
convalescent phase / acute phase≥4

Molecular Biological Diagnosis(hybridization, PCR, RT-PCR,etc)

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 Experiment 1

Bacterial Cultivation

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Requirements for bacterial growth
 Nutrients
H2O, C-source, N-source,
Inorganic salts, Growth factors
 Temperature

 pH
 Gas

incubator  Temperature, gas

culture medium  Nutrients, pH
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 Culture medium
• is the mixture of various nutrients that is suitabl
e for the growth of microorganisms.

 Types of Culture Media

based on the function and chemical components
based on the physical state

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Based on the function and the chemical components:

 Basic Medium
--contains the basic nutrients for the most bacterial growth;
--the base of other kind of media.
--e.g. broth.
 Nutrient Medium/Enriched Medium
Additional or special nutrients (e.g., serum, growth
factors, trace elements) are added to support some
fastidious bacterial growth.
e.g. blood agar.

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 Selective Medium
the medium that can prevent the certain bacterial
growth while permitting others.
e.g. SS agar

 Differential Medium
Some special substrates and indicators are added into the
media in order to produce a visual differentiation
when several bacteria grow on the same kind of medium.
e.g. EMB agar (Eosin-methylene blue agar).

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E.coli on EMB S.dysenteriae on EMB

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 Citrate slant

Double sugar iron slant

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 Anaerobic Medium
a medium for the cultivation of certain anaerobes. The medi
um contains reducing agent, such as non-saturation fatty ac
id.

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Based on the physical state
 Liquid medium:
• Without agar.
• for the proliferation of bacteria.
 Solid medium:
• 1.5-2.5% agar.
• for the isolation and identification of bacteria
• e.g., slant, Petri dishes/plates.
 Semisolid medium:
• 0.3-0.5% agar.
• for the observation of bacterial motility and preservation of bacteri
a.

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 Bacterial growth patterns
 In liquid medium:
Superficial growth;
Turbidity/diffuse;
Precipitate growing;
(sediment)
 In solid medium:
Confluent growth / Smear;
Colony:
a cluster of microorganisms growing
on a solid medium. It is directly visible
and arises from a single cell.

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 In semi-solid medium:
• Only grow along the line of inoculation
• Grow diffusely

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 General procedure of bacteriological Diagnosis
 Culture medium
• concept
• categories
• bacterial growth patterns
 Inoculation and transfer techniques

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Inoculation and Transfer Techniques
 Streak-plate technique

 Slant inoculation
 Liquid medium inoculation
 Semisolid medium inoculation

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 Streak-plate technique
four-area streak plate technique
1/10 II
I I 1/5

Rotate plate 90

Flame loop

Flame loop
Rotate 90

Rotate 90
III
1/4
IV
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Slant inoculation

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Liquid medium inoculation

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 2 students/group

Inoculation method Culture media Bacterial strains

Streak plate 2 plates (1plate/student) A mixed liquid
of S.aureus & E.coli
Slant inoculation 2 slants (1slant/student) 1 E. coli slant
Liquid medium 1 liquid medium 1 E. coli slant
inoculation 1 liquid medium 1 B. subtitis slant
Semisolid medium 1 semisolid medium 1 Proteus slant

inoculation (Stabbing)1 semisolid medium 1 S.dysenteriae slant

Mark your freshly inoculated plate/tube;
NOTE!!! Shake up the mixed liquid of bacterial strains before using it.
Prepare Gram Stain & Acid-fast Stain.