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Laboratory Diagnosis of Bacterial InfectionLaboratory Diagnosis of Bacterial Infection

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Types of specimen

Basic Principles for Specimen CollectionBasic Principles for Specimen CollectionCollecting the correct specimenCollecting the correct specimen

Endocervical swabs for GCPernasal swabs for pertussiswhole EMU for TBSputum, not salivaBlood culture bottles, not clotted blood

Getting the specimen to the lab

Problems in delay or inappropriate storage delay in dignosis and treatment------pathogens die------contaminants overgrow

Blood culture directly into incubator------not refrigerator

CSF straight to lab

Don't put an entire surgical specimen into formalin------send a portion to microbiology in a sterile container

Collecting the specimen correctly

Take an mid-stream urine------avoids contamination with perineal flora

CSF------Avoid contamination------Avoid bloody tap

Blood cultures------Avoid contamination with skin organisms

Infection Control

Please be considerate to lab staff------Label hazardous specimens

Don't send specimens to the lab without proper packing------Leaking or blood-stained specimens are not acceptable

Factors limiting usefulness of bacteriological investigationswrong sample------saliva instead of sputum

delay in transport/ inappropriate storage------CSF

overgrowth by contaminants------blood cultures

insufficient sample/sampling error------in mycobacterial disease

patient has received antibiotics

Specimen

Etiologic diagnosis (blood,urine,stool,

cerebrospinal fluid,pus,secreta)

Examination of immune responese of the body to

an infectious agent

Direct examination of the specimen

(blood,urine,stool)

Isolation, culture and identification of the

agent. susceptibility to antimicrobial drugs

Tissue cells serum

Histochemical stain

Antibody detection

(ELISA,WB,RIA)

Microscopic examination

Light microscopy, EM,IEM

Component of microbes

Antigens(immunofluores cenece,solid-phase

radioimmunoassay and ELISA

Poison and toxicity test

Experimental animal

Nucleic acid (nucleic acid

electrophoresis,nucleic acid

hybridization,PCR,gene sequencing and gene chips

Examination of metabolite

(biochemical characteristics)

Procedures for detection of pathogens

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The main examination methods in diagnosis of bacterial infectionThe main examination methods in diagnosis of bacterial infection

※ Morphological examination

※ Isolation, culture and identification

※ Biochemical Reactions

※Antibiotic Susceptibility Test

※ Antibody detection

※ Antigens or Nucleic acids assay

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Morphological examinationMorphological examination

※ ※ Non-stained microscopic observationNon-stained microscopic observation

▲ ▲ Dark-field microscopyDark-field microscopy

▲ ▲ Observe the movement of live bacteriaObserve the movement of live bacteria

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※ ※ Stained microscopic observationsStained microscopic observations

▲ ▲ Gram stainGram stain

▲ ▲ Acid-fast stainAcid-fast stain

▲ ▲ Fluorescence stain(suchFluorescence stain(such as Auramine stain as Auramine stain))

▲ ▲ methylene bluemethylene blue stainmetachromatic granule

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Isolation & CultureIsolation & Culture

1.1.SizeSize 2. 2. ShapeShape 3. 3. ColorColor 4. 4.Surface featuresSurface features

5.5.TransparencyTransparency 6. 6. Hemolysis Hemolysis

How to describe the feature of bacterial colonies on an agar plate?

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Biochemical ReactionsBiochemical Reactions

EVERYTHING that a living organism does is the result

of the activity of an ENZYME, the SUMMATION of the

activities of all an organism's enzymes equals its

BIOCHEMICAL FINGERPRINT. That is, an organism is

the totality of its enzymes, so by determining which enzymes

are present in an unknown organism one can DESCRIBE &

IDENTIFY that organism

The theoretic basis of biochemical reaction

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Common Tests To identify Bacterial isolates

△ Indole assay

△ Methyl Red/Voges Proskauer test

△ Citrate utilization

△ H2S production （ hydrogen sulfide） △ Urea hydrolysis

△ Motility

△ Lactose fermentation

△ Sucrose fermentation

△ Glucose fermentation & gas production

△ Oxidase test

……△

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Citrate utilizationCitrate utilizationSugar FermentationSugar Fermentation HH22S TestS Test

Examples of Common Biochemical ReactionsExamples of Common Biochemical Reactions

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Antibiotic Susceptibility TestAntibiotic Susceptibility Test

The wide variation in susceptibility and high frequencies of drug resistance among strains in many bacterial species necessitates the determination of levels of resistance or susceptibility as a basis for the selection of the proper antibiotic for chemotherapy

Antimicrobial Susceptibility testing can be down by :

Minimum Inhibitory Concentration (MIC)

Disk Diffusion Method

Minimum Bactericidal Concentration (MBC)

Principle:

The tube dilution test is the standard method for determining levels of resistance to an antibiotic.

Serial dilutions of the antibiotic are made in a liquid medium which is inoculated with a standardized number of organisms and incubated for a prescribed time.

The lowest concentration of antibiotic preventing appearance of turbidity is considered to be the minimal inhibitory concentration (MIC).

Different concentrations of Tetracycline in Nutrient broth:

Conc. in mcg/ml （ microgramme）0.1 0.2 0.4 0.8 1.6 3.1

6.3 12.5

Tetracycline, generally considered a bacteriostatic antibiotic, for this bacterium, has an MIC of 1.6 mcg/ml

1.Minimum Inhibitory Concentration (MIC) :

2. Disk-diffusion Method (Kirby-Bauer Method):

The disk-diffusion method (Kirby-Bauer) is more suitable for routine testing in a clinical laboratory where a large number of isolates are tested for susceptibility to numerous antibiotics.

An agar plate is uniformly inoculated with the test organism

A paper disk impregnated with a fixed concentration of an antibiotic is placed on the agar surface.

Growth of the organism and diffusion of the antibiotic commence simultaneously resulting in a circular zone of inhibition in which the amount of antibiotic exceeds inhibitory concentrations.

The diameter of the inhibition zone is a function of the amount of drug in the disk and susceptibility of the microorganism.

This test must be rigorously standardized since zone size is also dependent on:

inoculum size, medium composition, temperature of incubation, excess moisture and thickness of the agar.

Zone diameter can be correlated with susceptibility as measured by the dilution method.

Further correlations using zone diameter allow the designation of an organism as "susceptible", "intermediate", or "resistant" to concentrations of an antibiotic which can be attained in the blood or other body fluids of patients requiring chemotherapy.

Staphylococcus aureus (MRSA)

Note the yellowish pigmentation of the bacterial lawn, and the lack of inhibition by the Oxacillin disk

Streptococcus pneumoniae (Pneumococcus):

The brownish tint of the blood agar plate outside the zones of bacterial inhibition is caused by alpha-haemolysis.

Pseudomonas aeruginosa:

The greenish tint of the lawn and plate in general is caused by the diffusible pigment made by the Pseudomonas aeruginosa itself.

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Serological AssaysSerological Assays

※ ※ Detection antibody in the patient’s serumDetection antibody in the patient’s serum

※ ※ A current infection should beA current infection should be

△ △ IgM positiveIgM positive

△ △ A 4-fold or greater rise on antibody titer A 4-fold or greater rise on antibody titer

■ ■ the convalescent sample is usually taken 10-14 the convalescent sample is usually taken 10-14

days after the acute sample. days after the acute sample.

※ ※ Major drawbacksMajor drawbacks

■ ■ A single IgG antibody titer is difficult to interpret,A single IgG antibody titer is difficult to interpret,

of course, In certain diseases, a single titer of of course, In certain diseases, a single titer of

sufficient magnitude can be used as presumptive sufficient magnitude can be used as presumptive

evidenceevidence

■ ■ Some exceptionsSome exceptions

Molecular Biology TechniquesA- Genetic probes (DNA or RNA probes): Detection of a segment of DNA sequence (gene) in unknown organism using a labeled probe

Probe: consists of specific short sequence of labeled single- stranded DNA or RNA that form strong covalently bonded hybrid with specific complementary strand of nucleic acid of organism in question

B- Polymerase chain reaction (PCR): Amplification of a short sequence of target DNA or RNA Then It is detected by a labeled probe

C- Plasmid profile analysis: Isolation of plasmids from bacteria and determination of their size and number compared with standard strains by agarose gel electrophoresis

Microbes and humans

Very few microbes are always pathogenic

Many microbes are potentially pathogenic

Most microbes are never pathogenic

Microbes and humans

How do we know that a given pathogen causes a specific disease?

Diagnosis and effective treatment of infection depends not just on isolating an organism, but in establishing a plausible link between the laboratory findings, recognised syndromes and the patient's clinical condition

potential pathogen isolated from or

detected in clinical samples

recognised syndromese.g. meningitis

pneumonia

patient's clinic condition

Exercises:

1. What are the basic principles for specimen collectionbasic principles for specimen collection?

2. What’s antibiotic susceptibility test and its useantibiotic susceptibility test and its use?

3.What are the main examination methods in diagnosis of main examination methods in diagnosis of bacterial infectionbacterial infection?