SPECIMEN COLLECTION AND PROCESSING: INDIVIDUAL SITES

BLOOD
Skin Disinfection and Specimen Collection Use 2-step disinfection process - Wipe first with 70% alcohol - Apply Povidone-Iodine to the site - Remove residual iodine with a second application of 70% alcohol (optional)

BLOOD
Timing of Blood Cultures Prior to administration of antimicrobial agents Before, during or immediately after a fever spike Volume of Blood Cultured Specimens from adults should contain 20-30 mL (split evenly between 2 bottles of medium) The desirable blood-to-broth ratio is 1:10 Only 1 5 mL from pediatric patients

BLOOD CULTURE METHODS 1. Broth Culture Methods Entails inoculation of specimens to bottles or tubes containing liquid media Growth is detected by: a. visual examination of the broth for turbidity, hemolysis, gas production, or colonies adhering to the walls of the vessel.

b. microscopic examination of stained broth smears

c. subculture of broth to agar media

d. instrument-assisted detection of CO2 produced during microbial growth in broth

BLOOD Commonly used media: Tryptic soy broth Trypticase soy broth Columbia broth Brain-heart infusion broth Commercially prepared media are bottled under vacuum with CO2; - contains 0.025% sodium polyanethol sulfonate (SPS) to: a. prevent clotting b. neutralize the bactericidal effect of human serum, c. inhibit complement activity and phagocytosis d. inactivates aminoglycosides 7 days has been considered the traditional time for detection of most clinically significant bacteremias

BLOOD CULTURE METHODS 2. Biphasic Agar-Broth Culture Consists of a conventional blood culture broth bottle with an attached chamber containing agar media on a paddle. The bottle is tipped, allowing the blood-broth mixture to enter the chamber and flow over the agar media.

BLOOD CULTURE METHODS

3. Lysis-centrifugation blood culture system Consists of a tube containing reagents that inhibit coagulation and the complement cascade, lyse blood cells, and provide a cushion for the microorganisms during centrifugation.

CSF Collection

CSF
Collected to diagnose meningitis and viral encephalitis After introduction of the needle into the subarachnoid space, the fluid is collected sequentially into 3 separate tubes (ideally 1 mL/tube) Tube 1 for chemical and immunologic testing Tube 2 - for microbial testing Tube 3 for hematology/cytology Tests ordered on CSF are considered STAT

Processing CSF for Routine Bacterial Culture

Centrifugation (if 1 mL or more of specimen is received) for at least 15 minutes at 1500 g. Preparation of smear for Gram stain or acridine orange stain and culture. Blood agar, chocolate agar, and enriched broth are inoculated for culture

Processing CSF for Routine Bacterial Culture Plates are incubated in 5%-10% CO2 and examined daily for 3 days. The broth is incubated and examined daily for turbidity. If possible, a portion of each CSF specimen should be saved and stored at 4C or below if future antigen detection tests are anticipated.

In bacterial meningitis CSF is usually purulent (>100 cell/mm3), containing PMNs Glucose level is usually less than half the serum level. In fungal/viral menigitis Cell count is lower; mostly mononuclear Glucose levels are usually normal or reduced.

OTHER BODY FLUIDS

Pericardial Peritoneal Synovial Pleural

Precautions for collection and testing are the same for CSF

OTHER BODY FLUIDS

A volume of 1-5 ml is adequate for isolating most bacteria. To transport the fluid, the air is expelled from the syringe, the needle is removed, and the syringe is tightly capped with a sterile rubber stopper. The sample may be directly inoculated to blood culture bottles at the bedside.

EAR SPECIMENS
Middle ear fluid aspirates should be delivered promptly to the laboratory for culture Tympanocentesis should be performed with aseptic technique In the event that the tympanic membrane has already ruptured, fluid remaining in the ear canal may be absorbed on a swab. Fluid is processed by performing a Gram stain and inoculation of media suitable for growth of the middle ear pathogens

EYE Specimens
A. Conjunctival Specimens Obtained from the superior and inferior tarsal conjunctiva by using a swab moistened with broth or a sterile platinum spatula. Swab is rolled on the surface of blood and chocolate agar plates.

B. Corneal Specimens Obtained using the Kimura spatula and inoculation of the of appropriate media using C streaks.

EYE Specimen Collection

GI TRACT
Stool is the preferred specimen over rectal swabs. Transported in sterile, plastic cups with screwcapped lids. Stool should not be contaminated with urine or barium sulfate. Should be placed in a Cary-Blair medium if not processed immediately.

GI TRACT
Specimens are directly plated to appropriate media to allow recovery of salmonella,. Shigella and Campylobacter spp. For detection of Campylobacter jejuni/coli, the sample can be filtered, the filtrate planted on Brucella sheep blood agar or chocolate agar.

GENITAL TRACT A. Vaginal secretions Most common pathogens are Gardnerella vaginalis, Candida spp, Trichomonas vaginalis. A wet-mount preparation is the most valuable diagnostic test. The slide is examined for a. clue cells (epithelial cells covered with small coccobacillary bacteria) - non-specific vaginosis b. pseudohyphae candidiasis c. motile trichomonads

GENITAL TRACT B. Endocervical and Urethral Specimens Urethral specimens are obtained using a flexible shafted swab inserted 1-2 cm into the urethral orifice. Endocervical specimens are obtained after the cervix is visualized with the aid of a speculum moistened only with warm water. Direct fluorescent antibody staining for C. trachomatis; to transport specimen, 2 sucrose-phosphate and sucrose glutamate phosphate containing antibiotics (gentamicin, vancomycin, nystatin) are used Gram stain; direct inoculation of a selective agar medium (Thayer Martin) to isolate N. gonorrheae

UPPER RESPIRATORY TRACT

Throat specimens are collected by having the patient open his/her mouth widely, depressing the tongue to improve visibility, and inserting the swab so that the tip makes contact with exudative, inflamed regions of the posterior pharynx and tonsils.

LOWER RESPIRATORY TRACT

Expectorated sputum is the most common specimen.

The patient coughs deeply and expectorates into a sterile container. Or obtained by bronchoscopy, suction through tracheostomy or endotracheal tubes, bronchoalveolar lavage, direct lung aspiration, or biopsy

UPPER RESPIRATORY TRACT Specimens include swabs of the nasopharynx, throat, and sinus cavities Nasopharyngeal specimens are collected using a flexible, wire-shafted swab composed of calcium alginate fibers. Sinus aspirates are collected with a needle and syringe and puncture of the cartilage surrounding the sinus cavity.

Bronchoscopy

URINARY TRACT Acceptable methods of urine collection include midstream clean-catch, catheterization, and suprapubic aspiration. All urine specimens should be processed within two hours after collection. Urine may be refrigerated up to 24 hours; may be collected in sterile Vacutainer tubes containing boric acid-sodium formate transport media.

Suprapubic Urine Collection

WOUND SPECIMENS Any material collected from a wound should be examined with a Gram-stained smear. Specimens from superficial wounds are frequently collected by swabs. All wound specimens should be cultured on blood agar, enteric agar, and anaerobic blood agar and incubated for 48 hours at 25C-37C

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