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1. DIAGNOSTIC MICROBIOLOGY
Dr Salem Al-Aidy
University of WASIT
College of Medicine

2. Laboratory Diagnosis of Infection
Laboratory diagnosis
doesn’t always give us a
yes or no answer,
sometimes the answer is
maybe………………………
…….
University of WASIT
College of Medicine

3. Diagnosis of Bacterial Infection
Microscopy
Antibiotic
Sensitivities
Culture
Non-cultural
techniques
University of WASIT
College of Medicine

4. Microscopy
Preparations are usually stained (e.g. Gram stain, Acid and Alcohol Fast
Bacteria stain (TB))
qAdvantages
•Rapid
•Simple to perform
qDisadvantages
•Low sensitivity (e.g. gram staining faeces, low numbers of acid fast
bacilli in sputum)
University of WASIT
College of Medicine

5. Unstained preparations
§Dark ground illumination (technique used
to observe unstained samples causing them
to appear brightly lit against a dark, almost
purely black, background.
§Used for identification of spirochetes e.g.
Treponema pallidum(syphilis)
§Wet preparation (water/saline
§Mainly ssed for identification
of fungi and protozoa
Giardia lamblia
trophozoite
University of WASIT
College of Medicine

6. Stained preparations
qGram stain
qAcid fast stain (aafb) e.g.
Mycobacterium tuberculosis
qFluorescence:
§Direct
§Indirect
GPC GPB GNB GNC
University of WASIT
College of Medicine

7. Bacterial culture
qSolid media
•Agar plates for identification (colonial morphology) and
enumeration
qLiquid media (broth)
•Enrichment
qSlopes
•Lowenstein Jensen media for TB culture
•Long term storage
LJ medium with M.tuberculosis
growth
University of WASIT
College of Medicine

8. Microbiological Media
Theory
In order to grow and reproduce microrganisms require:
qNutrients
qEnergy source
qCertain environmental conditions e.g. reduced pH
These requirements are met in the microorganisms natural habitat.
In the laboratory?
•Requirements are met by a culture medium.
University of WASIT
College of Medicine

9. Cont…
qCulture medium is basically an aqueous solution to which all the
necessary nutrients have been added.
qThere are different categories of culture media, based on the type and
quantities of nutrients present.
qTransport media plays an important role in supporting growth of
delicate pathogens from time of collection from patient to culture in
laboratory.
University of WASIT
College of Medicine

10. Categories of Microbiological Media
CategoryPurpose
Complex mediaGrowth of most heterotrophic organisms.
Defined mediaGrowth of heterotrophs, photo and
chemoautotrophs.
Selective mediaSuppress unwanted microbes,
encourage growth of required microbes.
Differential mediaDistinguish colonies of specific microbes
from others.
Enrichment mediaIncreases numbers of desired organisms
to a level they can be detected without
encouraging growth of non-desirable
microbes.
Reducing mediaGrowth of obligate anaerobes.
University of WASIT
College of Medicine

11. Nutrients
Click on the website link below to view an image of some common microbiological
media:
http://www.madsci.org/~lynn/micro/media/
Culture media can be liquid or solid:
qSolidifying agent is “agar agar“, a natural polysaccharide produced by
marine algae.
Other common nutrients:
qDigested animal proteins such as peptone and tryptone e.g. Tryptone
Soy Broth
qSoluble extracts of animal tissue such as brain and heart e.g. Heart
Infusion Broth
qSugar (usually glucose) for carbon and energy.
University of WASIT
College of Medicine

12. Click on the website link below to view an animation of media
preparation:
http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Media_Prep/Med
ia_Prep.htm
University of WASIT
College of Medicine

13. Non-cultural identification of bacteria
Biochemical identification (from pure culture).
•Samples from certain body sites often contain a number of bacteria,
it’s essential that the organism to be identified is isolated in pure
culture for the following tests.
•Tests include the ability of bacteria to utilize various sugars, produce
acid and/or gas when grown in sugar containing media (detected by a
colourchange of an indicator), ability to produce indole,H2S etc.
•The presence of certain enzymes such as
oCatalase
oGelatinase
oUrease
oLecthinase
oLipase
University of WASIT
College of Medicine

14. Commerical kits
The API identification system is a complete miniaturized set of biochemical
tests:
•Consists of a plastic strip of miniaturized tests tubes (cupules)
•Each contains a different reagent used to determine the metabolic
capabilities, and, ultimately, the bacterial genus and species.
•Different API kits for different groups of organisms e.g. API 20E for the
family Enterobacteraceae.
•Wells are inoculated with pure culture and incubated for required time.
•Manual addition of reagents is often required for some of the tests to be
interpretated.
•All wells are then observed for growth or colour changes.
University of WASIT
College of Medicine

15. API bacterial identification system
There are different API kits for different bacterial groups e.g.
API 20E for identification of enterobacteria
API 20 Strep for identification of streptococci
API 20 A for identification of anaerobes
University of WASIT
College of Medicine

16. Automated IdentificationSystems
oVitek 2 (bioMerieux) automated system for
identification of bacteria
oConsists of well cards containing 47
miniaturized biochemical tests (shown below).
oTests are innoculated with bacterial
suspension and monitored during incubation by
optical readers for changes in turbidity,
development of colour or fluorescence due to
hydrolysis of enzyme substrates.
Similar automated systems include:
oMicroscan WalkAway (Siemens)
oBD Phoenix (Becton Dickenson)
University of WASIT
College of Medicine

17. Immunological tests
oBasis of tests is the presence of antigens on the bacterial cell surface that may
be targeted by antibodies, when a specfic antibody-antigen reaction takes place
a signal may be generated.
oSimpliest form of this test is where a bacterial suspension is mixed with a
suspension of unlabelled antibody, mixture is gently agitated and observed for
visual agglutination.
•Haemagglutination (HA)
oAgglutination of red blood cells, often used to test for the presence of
antibodies directed against red cell surface antigens or carbohydrate
binding proteins or viruses in a sample.
University of WASIT
College of Medicine

18. Cont..
To view an animation of a haemagglutination test, click on the website link
below:
http://www.wellcome.ac.uk/stellent/groups/corporatesite/@msh_publishing_gr
oup/documents/web_document/wtd039602.swf
•Enzyme-linked immunosorbent assay (ELISA)
To view an animation of how ELISA works, click on the website link below:
http://www.sumanasinc.com/webcontent/animations/content/ELISA.html
University of WASIT
College of Medicine

19. •Latex particle agglutination
oSame principle as haemagglutination, but using smaller, antigen-coated
latex particles improves sensitivity and reagent longevity.
oAntibodies can also be absorbed to the latex particles by binding to the Fc
region of antibodies, leaving the Fab region free to interact with antigens in
the patients serum.
Cont…
Great variation in sensitivity and specificity of these different tests.
Most tests will detect all antibody classes however some assays can be adapted
to detect one specific class only i.e.. IgM, IgG or IgA.
University of WASIT
College of Medicine

20. Non-cultural identification from other
sample types
qAntigen detection
•Latex agglutination
•ELISA
qAntibody detection
•ELISA
qMolecular methods
•PCR
•DNA and RNA Probes
qSpectroscopy methods
•MALDI-TOF
Tests can be
performed
on various
sample types
e.g. pneumococcal
capsular
antigen detected
by latex agg.
from sputum.
University of WASIT
College of Medicine

21. Antibiotic sensitivity testing
qDisc diffusion methods (Stokes)
•Predict the usefulness of different antibiotics against a bacterial isolate.
•Performed on solid media
University of WASIT
College of Medicine

22. qMIC (Minimum Inhibitory
Concentration)
•Minimum concentration of antibiotic
required to inhibit growth of the organism.
•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 (highest dilution)
of antibiotic preventing appearance of
turbidity is considered to be the minimal
inhibitory concentration (MIC).
Cont…
University of WASIT
College of Medicine

23. qE-test
•Method for measuring MICs of antimicrobial agents against bacteria
and is based on diffusion of a pre-formed antibiotic gradient from a
plastic strip.
•Biomerieux Etest® is a predefined, stable gradient of 15 antibiotic
concentrations on a plastic strip.
Cont…
•Using innovative dry chemistry
technology, Etest is used to determine
the on-scale Minimum Inhibitory
Concentration (MIC) of antibiotics,
antifungal agents and anti-mycobacterial
agents.
University of WASIT
College of Medicine

24. oOver 100 antimicrobials are now available in the product range for testing of
aerobic bacteria and fastidious organisms such as Pneumococci, Haemophilus, H.
pylori, Meningococci, Gonococci, Anaerobes, Fungi and Mycobacteria.
http://www.biomerieux-diagnostics.com/servlet/srt/bio/clinical-
diagnostics/dynPage?doc=CNL_CLN_PRD_G_PRD_CLN_22
University of WASIT
College of Medicine

25. Diagnosis of viral infection
qElectron microscopy
•Rapid diagnosis
qAntigen detection
qAntibody detection
qCulture
•Cell culture –detection of cytopathic effects (laborious technique)
qMolecular methods
•PCR
University of WASIT
College of Medicine

26. Diagnosis of Fungal Infection
qSolid agar (Sabarouds agar)
qLower incubation temperature (250C)
qLonger culture time (up to 4 wks).
qMicroscopy
Wet preparation of sample –
e.g. Candida albicans from a
vaginal swab.
Can observe budding and
hyphae.
University of WASIT
College of Medicine

27. Diagnosis of Protozoal Infection
qVery fastidious growth requirements:
§Certain media and/or medium components may be toxic.
§Type of glass used for the culture container determine success or failure of
culture.
qMicroscopic observation of
§Motile trophozoites (active growth and feeding stage)
§Cysts (dormant stage, ability to survive harsh conditions)
in faeces or duodenal aspirates e.g. Gardia lamblia
Multiple examinations are often required to diagnose the infection
because of the irregular shedding of parasites.
University of WASIT
College of Medicine

28. qDiamond's medium (liquid medium) is the gold
standard method for detecting T. vaginalis.
•Costly
•Takes 2-7 days –can delay treatment
qInPouch™TV
•The InPouch™TV culture system combines a
two-chambered bag that allows one to perform
a wet mount using the upper chamber and a
culture using the lower chamber, in one self-
contained system.
•The sensitivity,costand time required to
perform the InPouchsystem is comparable to
Diamond's medium.
University of WASIT
College of Medicine

29. Overview of microbiological diagnosis of
infection
§Patient assessed by clinician (PRE-ANALYTICAL)
§Specimen is collected and transported to laboratory
§Microscopy
§Culture (ANALYTICAL)
§Antibiotic sensitivity tests
§Non-cultural methods
§Reporting results
§Specimen storage (POST ANALYTICAL)
University of WASIT
College of Medicine

30. Processing a sample in the microbiology
laboratory (bacteriology)
1.Sample arrives with accompanying form giving clinical history, symptoms and
patient details.
2.Sample is cultured onto appropriate agar –usually a combination of broad
based, differential and selective agar depending on sample type and possible
organisms present.
3.Agar plates are incubated overnight and examined for growth the following day.
4.Colonial morphology is noted and suspect colonies are gram stained.
5.Based on results of gram stain, colonies undergo further biochemical tests to
identify bacteria.
6.Suspect colonies are tested for antibiotic sensitivity.
7.Final results are generated and sent to clinician.
University of WASIT
College of Medicine

31. University of WASIT
College of Medicine