- Infection of soft tissues associated with pus formation
- Pus consists PMNs, bacteria, fibrin and other body fluids
- May be endogenous or exogenous
Endogenous: source of infection commensal flora, for example, abdominal surgical wound infection from normal flora of bowel
Exogenous: source of infection outside the body, for example burn infection
- most commonly isolated pathogens from wounds, abscesses, burns, and draining sinuses.
Gram positive Gram negative
S aureus Pseudomonas aeruginosa
S pyogenes Proteus spp
Enterococcus spp E. coli
Anaerobic streptococci Bacteriodes spp
Other streptococci Klebsiella spp
Clostridium perfringens Pasteurella spp
ULCER MATERIAL AND SKIN SPECIMENS
Gram positive Gram negative
Staphylococcus aureus E coli
Streptococcus pyogenes Proteus
Enterococcus spp Pseudomonas aeruginosa
Anaerobic streptococci Yersinia pestis
Erysipelothrix spp Vincent’s organisms
S aureus , H. influenzae
Group A Streptococci Coliform bacteria
M tuberculosis (Bone TB)
Collection and transportation of sample
- should be collected by a medical officer or an experienced nurse
Collection time: best collected when abscess is incised and drained, or when wound ruptured naturally
Special precaution: should be collected before an antiseptic dressing is applied.
1.Using sterile needle aspirate from a drainage in to a tube up to 5 ml of pus.
2.Transfer to a leak-proof sterile container.
When pus not discharged
1.Use a sterile cotton-wool swab to collect a sample from the infected site.
(Sample may get desiccated and bacteria may become trapped in swabs)
- Immerse the swab in a container of Amies transport medium
- If possible two swabs are taken, one for microscopy, next for culture
- If it can be arranged, use cooked meet broth, inoculate swab directly into broth immediately after collection
- Without delay transport to lab and should be processed immediately
Examination for pyogenic and anaerobic pathogens
Special request for:
mycobacteria, actinomycetes , diphtheria , anthrax bacillus and fungi
- Pus from Actinomycetes infection contain granules, Small yellow granules
Appearance of pus Presumptive organism
Creamy and thick Staphylococcus aureus
Straw colored Watery Streptococcus pyogens
Fishy smell Proteus spp
Blue pigmentation, musty odor Pseudomonas spp
Putrid offensive smell Anaerobic infection
Actinomycetes Yellow colored granules
Fungal infection Black or brown granules
- Gram positive cocci could be S. aureus or streptococci
- Gram negative rods that could be Proteus species, E. coli or other coliforms, P. aeruginosa
- Gram positive large rods with square ends could be C. perfringens or B. anthracis
- Large numbers of pleomorphic bacteria (streptococci, Gram positive and Gram negative rods of various size and fusiform bacteria), associated with anaerobic infections.
- Gram positive yeast cells with pseudohyphae , suggestive of Candida albicans
- Vincent’s organisms, if tropical ulcer is suspected, Gram negative spirochetes (Borrelia vincenti ) and Gram negative fusiform rods
- Ziehl-Neelsen smear when tuberculosis or M. ulcerans disease is suspected
- Potassium hydroxide preparation when ringworm or other superficial fungal infection is suspected
- inoculate on MA, BA, Cooked meat medium or thioglycolate broth
Blood agar to isolate S. aureus and streptococci.
MacConkey agar to isolate Gram negative rods.
On Cooked meat medium to isolate anaerobs at the bottom of tube
- Incubate blood agar plate at 35–37°C in a carbon dioxide atmosphere (candle jar) one aerobic next anaerobic incubation.
- MacConkey agar plate aerobically.
- Cooked meat medium at 35–37 °C for up to 72 hours. Subculture at 24 h, and if indicated at 48 h and 72 h
- Anaerobic culture
- Suspected anaerobic infection, specimen is often foul-smelling, or the Gram smear shows an ‘anaerobic mixed flora’, inoculate a second blood agar and incubate it anaerobically for up to 48 hours.
- plate may be made selective by adding neomycin to final concentration of 50–70 g/ml
- Majority of facultative anaerobic Gram negative rods will be inhibited.
- metronidazole disc (5 μg) may be added to the anerobic blood plates, majority of anaerobes show a zone of inhibition, whereas aerobes grow up to the disc
Examine for relative number and types of colonies
If no colonies are isolated, report no growth
- FEW CONSIDERATIONS
- Pure growth of recognized pathogens is reported as significant
- Scanty growth of skin commensals like CONS, usually disregarded and not reported
- Few colonies of E. coli potentially isolated from wound likely to contaminated with fecal bacteria is considered as not significant
- Pure growth of commensal type organisms isolated from deep site aspirate considered significant
IDENTIFICATION OF ISOLATES
Gram stain, Coagulase for Stahylococci
Lancefield grouping of beta hemolytic streptococci
Biochemical test for coliforms and anaerobs
- Anaerobic blood agar culture and cooked meat culture
Cooked meat medium: Grows rapidly in with H2S production (gas bubbles in turbid medium) and reddening but no decomposition of the meat.
Anaerobic blood agar: colonies are usually seen after 48 h incubation, most strains produce a double zone of haemolysis (inner zone of clear haemolysis, outer zone of partial haemolysis.
CMM: decomposition of meat with blackening of the meat (foul-smelling proteolytic reaction).
Anaerobic blood agar: non-haemolytic grey colonies
- PeptostreptococcusCooked meat medium: production of large amounts of hydrogen sulphide gas.
Anaerobic blood agar: small nonhaemolytic white colonies
How to confirm organism are anaerobic?
When there is a mixed growth and colonies appear on the anaerobic plate and not present on the aerobic plate, confirm that the organisms are anaerobes by sub inoculating the colonies on three plates of blood agar and incubating one aerobically, one anaerobically, and the third in a carbon dioxide atmosphere (candle jar).