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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

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III 1/5 I1/10

Rotate plate 90

Flame loopRotate 90

Rotate 90III

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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!!!