Download Lab.13 Pus, Wound and Burns Cultures

‫بسم هللا الرحمن الرحيم‬
2014-2015
L/O/G/O
Diagnostic Medical Microbiology-Laboratory Manual
Pus and wound Culture
Aim of the test
To isolate and identify aerobic and anaerobic pathogenic
organisms from pus specimen and sensitivity test.
Types of specimen
Swabs from the infected area or aspiration from deep
wounds. Swabs in anaerobic transport media for the isolation
of anaerobes.
Criteria of specimen rejection
Inappropriate specimen transport device; mislabeled
specimen; unlabeled specimen; specimen received after
prolonged delay (usually more than 72 hours); specimen
received in expired transport media and dried samples.
Commensals bacteria may appear during bad
collection
Alpha haemolytic streptococci
Coagulase negative Staph.
Corynebacterium spp.
Propionibacterium spp.
Bacillus spp.
Pathogenic bacteria
Pseudomonas aeruginosa
Streptococcus pyogenes
Proteus spp.
Staphylococcus aureus
E.coli
Enterococcus spp.
Klebsiella spp.
Clostridium perfringens
Morganella
Fusobactrium spp.
Providencia
Peptostreptococcus spp.
Mycobacterium tuberculosis
Actinomyces israelii
Bacteroides spp.
Nocardia spp.
Fungi (Candida albicans)
Pre specimen processing
Who is authorized to order the test
Physician.
Time relapse before processing the sample
According to the type of swab ( recommended within 30 min).
Storage
Maintain specimen swab at room temperature. Do not
refrigerate.
Quantity of specimen
Sufficient amount on swab, or aspiration in transport media or
syringe ( up to 5 ml of pus ).
Pre specimen processing
Collection
Specimen
Guidelines
Time and Temp.
Device and
or minimum
vol.
Transport
Storage
Abscess (wound)
Remove surface exudate by wiping
with sterile saline or 70% ethanol.
Open


Closed
Aspirate if possible, or pass swab
deep into lesion and firmly
sample lesions advancing edge.
Note: If swabs must be used
collect two swabs 1 for culture
and 1 for Gram stain.
Aspirate abscess wall material with
needle and syringe. Aseptically transfer
all material into anaerobic transport
device.
Swab transport
system.
Anaerobic
transport
system
≤ 2h,RT
≤ 24h,RT
≤ 2h,RT
≤ 24h,RT
Pre specimen processing
Pus and wound swab Specimen collection
1. No-touching technique: remove bandage with the forceps.
2. With the forceps take a sponge, dip it in the saline and wash
the surface of the wound or ulcers.
3. Remove the swab from its covering and extend the tip of the
swab deep into the wound taking care not to touch the
adjacent skin margins.
4. Remove the stopper from the test tube with transport medium,
plunge the swab into the transport medium and replace the
stopper.
Pus swab Specimen collection
Wound swab Specimen collection
I.
The swab should be moved across the
wound surface in a zig-zag motion.
II. Returning the swab to its container
Wound Specimen collection
Pus aspiration Specimen collection
 By inserting a needle within a skin lesion, fluid or pus can
be aspirated and sent to the laboratory for examination.
Specimen processing for Open Abscess (wound)
Specimen processing for Closed Abscess (wound)
Note
Anaerobic cultures should not be routinely set up for
superficial-wound specimens or for specimens that have not
been submitted in an appropriate anaerobic container.
Specimen processing
Direct smears
gram stain to check the presence or absence and if present the
types and predominant organisms.
Culturing
Enrichment and selective media included blood agar , chocolate and
MacConkey streaked and incubated aerobically for 24 hours, and
thioglycollate broth for 24 hours.
In case of suspected anaerobic organisms (closed abscess)
another blood agar plate is streaked and incubated anaerobically for
48 hours.
Gram stain from Pus specimen
Comments
Cultures from wounds are frequently contaminated with colonized
and environmental bacteria, and swab samples often do not reflect
the true cause of infection, For this reason the most preferable
method of collecting wound specimens is aspirating purulent
material from the depths of the wound with a sterile needle and
syringe.
The wound margins should be decontaminated as much as
possible with swab and alcohol before the material is aspirated.
If Mycobacterium tuberculosis
performed.
is suspect acid fast stain will be
Burn tissue culture
For burn patients, it is important to ascertain the number of organisms
present per gram of tissue. Greater than 10^5 CFUs per gram of tissue
is considered by some clinicians to be indicative of infection, whereas
less than that number may indicate only colonization.
Procedure :
1. Cut a piece of tissue measuring several cubic millimeters,
aseptically onto a small, pre-weighed, sterile urine cup.
2. Determine the weight of the tissue by subtracting the weight of the
aluminum foil from the total weight.
3. Place the specimen and 2 mL of sterile nutrient broth in a sterile
tissue grinder; macerate the specimen.
Procedure continue ……
4. Inoculate 0.1 mL of sample to a blood agar plate, in duplicate and
anaerobic blood agar plate ( if indicated ), in duplicate. In addition,
inoculate 0.01 mL of sample using a calibrated loop to a blood agar plate
in duplicate.
5. Spread the inoculum on the plates with sterile glass spreading rod or
loop Incubate plates in 5% to 10% carbon dioxide overnight, and count
the colonies of bacteria on the plate that contains 30 to 300 CFUs if more
than 300 colonies are obtained on broth plated dilution, the factor 300 is
used as N for calculations and the result is considered greater than the
value.
6. Calculate the number of CFUs per gram of tissue with the following
formula: Number of CFUs counted
x reciprocal of value of
homogenate inoculated (10^1 or 10^2) x 2 ( volume of diluent used
for tissue homogenization) ÷ weight of tissue in grams.
Post specimen processing
Interfering factors:
Patient on antibiotic therapy.
Improper sample collection.
Result reporting:
Report Gram stain finding as an initial report.
Report the isolated and its sensitivity pattern as a final report.
Turn around time:
Gram stain result should be available half hour after specimen
receipt.
Isolation of a possible pathogen can be expected after 2-4 days.
Negative culture will be reported out 1-2 days after the receipt of
the specimen.
End of Lecture