LOGO Diagnostic Medical MicrobiologyLaboratory Manual Pus and wound

ﺑﺴﻢ ﺍﻟﻠﻪ ﺍﻟﺮﺣﻤﻦ ﺍﻟﺮﺣﻴﻢ 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 Specimen Collection 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 ≤ 2 h, RT ≤ 24 h, 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 q 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 Mac. Conkey 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 is suspect acid fast stain will be performed.

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 m. L of sterile nutrient broth in a sterile tissue grinder; macerate the specimen.

Procedure continue …… 4. Inoculate 0. 1 m. L of sample to a blood agar plate, in duplicate and anaerobic blood agar plate ( if indicated ), in duplicate. In addition, inoculate 0. 01 m. L 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