6 laboratory diagnosis of bacterial infection
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Transcript of 6 laboratory diagnosis of bacterial infection
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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?