L/O/G/O 2014-2015 Diagnostic Medical Microbiology-Laboratory
Manual
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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.
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 ).
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Specimen Collection Time and Temp. Guidelines Device and or
minimum vol. TransportStorage Abscess (wound) Swab transport
system. 2h,RT 24h,RT Open Remove surface exudate by wiping with
sterile saline or 70% ethanol. 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. Closed Aspirate abscess wall material with needle and
syringe. Aseptically transfer all material into anaerobic transport
device. Anaerobic transport system 2h,RT 24h,RT Pre specimen
processing
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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.
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Pus swab Specimen collection
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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
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Wound Specimen collection
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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.
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Specimen processing for Open Abscess (wound)
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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.
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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.
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Gram stain from Pus specimen
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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 is suspect acid fast stain
will be performed.
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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.
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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.
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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.
