Clinical Microbiology and
Isolation of pathogens from clinical specimens: infected
tissues or fluids are collected, ex. blood, urine, feces, sputum,
cerebrospinal fluid, pus.
What steps should be taken to properly obtain clinical
General purpose media: ex. blood agar, supports the growth
of most aerobic and facultatively aerobic organisms.
Enrichment media: the addition of growth factors allows
metabolically fastidious to grow, ex. Neisseria gonorrhoeae.
Selective media: enhance the growth of certain organisms
while retarding the growth of others.
Differential media: specialized media that allow ID of
organisms based on their appearance on the media.
Bacteremia: presence of bacteria in the
blood, uncommon in healthy individuals.
Septicemia: blood infection resulting from
the growth of a virulent organism entering
the blood from a focus of infection, results
in fever and chills and possibly septic
shock including organ failure.
Common, causal agents often identical to
normal flora, ex. E. coli. Enteric bacteria
account for 90% of UTIs.
Significant UTI = 105+ organisms per ml of
Dipstick tests test for nitrate production,
etc. related to large #’s of organisms in the
Samples must be collected and preserved
properly and processed ASAP.
Processing includes inoculation into a
variety of selective media for isolation of
Intestinal parasites are ID’ed
microscopically rather than by culture.
Wounds and Abscesses
Types of wounds include animal or human
bites, burns, cuts, penetration of foreign
Wounds must be carefully sampled and
results carefully processed since normal
flora are likely to be present.
Genital specimens (ex. for gonorrhea) and
anaerobic specimens must be specially
processed in order to preserve the causal
agents for proper diagnosis.
Growth on Selective and
Unknown pathogens are first cultured on primary
isolation media and then subcultured onto selective and
differential media for further diagnosis.
Eosin-methylene blue (EMB) = selective (for gram neg.
bacteria) and differential (for diff. genera of gram neg.
Biochemical tests measure the presence or absence of
enzymes involved in the catabolism of substrate(s) in the
100’s of tests have been developed for clinical use,
though only ~20 are used routinely. (see Table 24.3)
Some commercial kits contain a battery of tests that ID
and individual species, ex. S. aureus, M. tuberculosis.
Testing Cultures for Antimicrobial
Minimum Inhibitory Conc. (MIC) – broth
(or antibiotic) dilution assay highest
dilution (lowest conc.) of antibiotic that
completely inhibits growth.
Kirby-Bauer disk diffusion method (see
Safety in the Clinical Lab
1. Access to lab restricted.
2. Decontamination procedures.
3. Personnel must be adequately vaccinated.
4. All clinical specimens should be considered
5. No mouth pipetting.
6. Animals handled only by trained lab personnel.
7. Lab personnel must wear appropriate safety
equipment: lab coat, sealed shoes, rubber gloves,
masks, eye protection, etc.
8. All clinical specimens should be treated as if they
Immunoassays detect and measure specific immune
responses to unique molecular portions of pathogens, and
are used especially on those pathogens (ex. viruses) that are
difficult to grow.
New pathogen first infects individual phagocytized
phagocytes present Ag from pathogen to T helper cells T
helper cells recruit and stimulate other phagocytes and B cells
B cells produce Ab (primary Ab response), occurs within 5
days, but Ab do not reach peak quantities for several weeks.
After 1st Ag exposure specific immunity via T and B cells
develops. After 2nd Ag exposure, stimulation of these specific
immune cells causes a rapid and strong immune response,
which quickly destroys the pathogen. Thus, the immune
system has memory, which is characterized by and increase
in Ab titer (used to track infections).
Ab Titers, Skin Tests,
& Diagnosis of Infectious Disease
Serological tests are set up by making dilutions of patient
serum and determining the highest dilution at which the Ag-
Ab rxn. occurs.
2 situations: 1. Must see a rise in Ab titer to conclude active
infection (having Ab present could mean prior infection or
immunization and not current infection). 2. The mere
presence of Ab is sufficient to indicate infection, ex. HIV.
Not all infections result in production of systemic Ab, ex.
Skin testing: ex. tuberculin text = intradermal injection of a
soluble extract of Mycobacterium tuberculosis pos.
inflammatory rxn. at site of injection = current infection or
previous exposure to M. tuberculosis.
Polyclonal and Monoclonal Ab
Antiserum (serum containing Ab) contains a mixture of
different Ab directed at the numerous determinants on
an Ag. This mixture of serum Ab is called polyclonal Ab
and, though they protect the host, they do not provide
reproducible results for use in clinical diagnostic testing.
Only a few Ab are directed toward each Ag determinant.
Ab produced by a single B cell (or in vitro clones of a
single B cell) = monoclonal Ab. Monoclonal Ab do
provide reproducible results for clinical testing ex. for
immunological typing of bacteria, pregnancy testing and