1 Diagnosis of Bacterial Infection Bacterial Cultivation.
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Transcript of 1 Diagnosis of Bacterial Infection Bacterial Cultivation.
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Diagnosis of Bacterial Infection
Bacterial Cultivation
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Outline
General procedure of bacteriological Diagnosis
Culture medium • concept• categories• bacterial growth patterns
Inoculation and transfer techniques
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Specimens collection
General Procedure of Bacteriological Diagnosis
General rulesThe 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. antibiotics.
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specimens
General Procedure of Bacteriological Diagnosis
Isolation, identification
Biochemical tests
EIA, ELISA, IF test, agglutination test
Antigen detection
Molecular Biological Diagnosis(hybridization, PCR, RT-PCR,etc)
Serological diagnosis (Ab titer)convalescent phase / acute phase≥4
Morphologic identificationmicroscopy & staining
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Experiment 1
Bacterial Cultivation
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Requirements for bacterial growth
Temperature
Nutrients
H2O, C-source, N-source, Inorganic salts, Growth factors
pH Gas
incubator Temperature, gas
culture medium Nutrients, pH
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Culture medium
• is the mixture of various nutrients that is suitable for the growth of microorganisms.
Types of Culture Mediabased 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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Double sugar iron slant
Citrate 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 acid.
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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 techniquefour-area streak plate technique
IV
III 1/5 I1/10
Rotate plate 90
Flame loopRotate 90
Rotate 90III
1/4
Flame loop
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Slant inoculation
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Liquid medium inoculation
2 students/group
Inoculation method Culture media Bacterial strainsStreak 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 slantMark your freshly inoculated plate/tube;
Shake up the mixed liquid of bacterial strains before using it.
Prepare Gram Stain & Acid-fast Stain.
NOTE!!!