1. Proper specimen collection is essential for accurate laboratory diagnosis of bacterial infections, as the wrong sample, delay in transport, or contamination can limit test usefulness. 2. Common examination methods for diagnosing bacterial infections include morphological analysis, isolation and culture of pathogens, biochemical reactions, antibiotic susceptibility testing, and detection of antigens or nucleic acids. 3. Antibiotic susceptibility testing determines the sensitivity of isolated bacteria to different antibiotics, which helps clinicians select the proper treatment. Methods include minimum inhibitory concentration and disk diffusion tests.

1. 1
Laboratory Diagnosis of Bacterial InfectionLaboratory Diagnosis of Bacterial Infection

2. 2
Types of specimen

3. Basic Principles for Specimen CollectionBasic Principles for Specimen Collection
Collecting the correct specimenCollecting the correct specimen
Endocervical swabs for GC
Pernasal swabs for pertussis
whole EMU for TB
Sputum, not saliva
Blood culture bottles, not clotted blood

4. 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

5. 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

6. 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

7. Factors limiting usefulness of bacteriological
investigations
wrong 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

8. 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

9. 9
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

10. 10
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

11. 11
※※ Stained microscopic observationsStained microscopic observations
▲▲ Gram stainGram stain
▲▲ Acid-fast stainAcid-fast stain
▲▲ Fluorescence stain(suchFluorescence stain(such as Auramine stainas Auramine stain))
▲▲ methylene bluemethylene blue stain
metachromatic granule

12. 12
Isolation & CultureIsolation & Culture
1.Size 2. Shape 3. Color 4.Surface features1.Size 2. Shape 3. Color 4.Surface features
5.Transparency 6. Hemolysis5.Transparency 6. Hemolysis
How to describe the feature of bacterial colonies on an agar plate?

13. 13
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

14. 14
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
△ ……

15. 15
Citrate utilizationCitrate utilizationSugar FermentationSugar Fermentation HH22S TestS Test
Examples of Common Biochemical ReactionsExamples of Common Biochemical Reactions

16. 16
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)

17. 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).

18.  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) :

19. 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.

20.  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.

21. Staphylococcus aureus (MRSA)
 Note the yellowish pigmentation of the bacterial lawn, and the
lack of inhibition by the Oxacillin disk

22. Streptococcus pneumoniae (Pneumococcus):
 The brownish tint of the blood agar plate outside the zones of
bacterial inhibition is caused by alpha-haemolysis.

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

24. 24
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 titerA 4-fold or greater rise on antibody titer
■■ the convalescent sample is usually taken 10-14the 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 ofof course, In certain diseases, a single titer of
sufficient magnitude can be used as presumptivesufficient magnitude can be used as presumptive
evidenceevidence
■■ Some exceptionsSome exceptions

25. Molecular Biology Techniques
A- 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

26. Microbes and humans
Very few microbes are always
pathogenic
Many microbes are potentially
pathogenic
Most microbes are never
pathogenic

27. Microbes and humans

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

29. 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 syndromes
e.g. meningitis
pneumonia
patient's clinic condition

30. 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 ofmain examination methods in diagnosis of
bacterial infectionbacterial infection?