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The accumulation of pus, either within an
abscess or exuding from a sinus tract or from
a mucocutaneous surface, is one of the
cardinal indicators of local sepsis.
Redness, pain and swelling
Liable to contamination from body surface &
Contaminants relatively low numbers
Infection occurs if contaminants evades the
Multiplication of commensal may be
Virulence and resistance determines infection
Source of infection is outside the body of the
Decubitus pressure ulcers
Animal or Human bites
Caused by organisms that have been leading
a commensal existence elsewhere in the
Surgical or post operative sepsis.
pyogenes, pneumococcus and coliform
Anaerobic organisms involved in soiled deep
or lacerated wounds and devitalized tissues
Gas gangrene and tetanus
Ill, bed ridden patient
Anaerobic conditions because of tissue
Most of these lesions are located near the
anus or on the lower extremities
Infection with bowel flora
B. fragilis, Clostridia sp, Enteric bacteria,
S. aureus and P. aeruginosa.
Pus or exudate
Fragments of excided tissue removed at
wound toilet or Curettings
Physician should be urged that when a
special investigation is required, they should
state this clearly on the request form.Thus
the routine investigation is usually confined
to a search for the common pyogenic
bacteria and anaerobic pathogens and does
not include an examination for
mycobacteria, actinomyces, nocardia,
diphtheria, anthrax or fungi
Staphylococci - thick creamy pus
Strep. Pyogenes – straw colored & watery
Proteus – fishy smell
Pseudomonas – sweet,musty odour & a blue
Anaerobes – offensive, putrid smell
Actinomycosis – sulphur granules
Mycetoma – black or brown granules
Amoebic abscess – anchovy sauce
Presence of relative numbers of polymorphs and
Morphology and arrangement
Wet film – fungi or motile bacteria
- fluid aspirated from inflamed joint
resembling septic arthritis may show
uric acid crystals
- Dark ground microscopy
Ziehl Neelsen or Fluorescent staining – AFB
Immunofluorescent staining – Clostridia species
Hematoxylin & Eosin – viral inclusions
Blood agar – aerobic
MacConkey agar or CLED Agar
Cooked Meat Broth
PNPG Blood Agar
Culture plates are examined after overnight
incubation at 37º C
Relative number and type of colonies noted
If there is no growth, the plates should be
reincubated for another 24 h.
If A. Israeli or Bacteroides suspected, plates
incubated for 7 days
If turbid, the broth should be subcultured
Difficulty in culture of slow growing
anaerobes that are highly sensitive to oxygen.
Their still invisible growth may be killed by
exposure to air during examination.
Inoculated on two anaerobic plates
Difficult in mixed cultures
Scanty growth of CONS, diptheroids are not reported
E.Coli from perineal wound etc are not reported.
Physician informed in case of Clostidium perfringens.
In chronic superficial lesions, the presence of mixed
commensal bacteria can be disregarded as
Pure growth of commensal type organism grown from
deep sites(eg.pleural fluid) should be reported with
sensitivities, unless the number of the organism is so
small as to indicate they are contaminants.
Numerous or predominant organism is
Relative number of colonies may not reflect the
number of organism in lesion.
Variations such as relative speed of growth of
different species under the cultural conditions
used, the presence of traces of antibacterial
drugs , and the greater tendency of delicate
pathogens to die during transport
Colonies in subculture from broth bears no
relation to the number of organism in lesion.
Discussion with the physician.
Significance of isolates
Predict the likelihood of burn wound sepsis
Probability of wound healing
Tissue weighed, homogenized, diluted serially,
and inoculated into multiple agar plates.
S.pyogenes are clinically significant, no matter
what the quantity of bacteria present.
>105 CFU/g is considered significant in burns
Single biopsy of a wound will not give an
accurate picture of the microbial flora of chronic
Buchanan et al
0.1(10-1) & 0.01(10-2 ) ml of sample in blood
agar in duplicate
The number of CFUs per gram of tissue is
calculated using the formula:
Number of CFUs counted * Reciprocal of
volume of homogenate inoculated(10-1or 10-2 )
* volume of diluent used for tissue
homogenization /weight of tissue