Specimen Collection, Transport and Processing

8/3/2019 Specimen Collection, Transport and Processing

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SPECIMEN COLLECTION AND PROCESSING:

INDIVIDUAL SITES


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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)


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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 evenlybetween 2 bottles of medium)

The desirable blood-to-broth ratio is 1:10

Only 1 5 mL from pediatric patients


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BLOOD CULTURE METHODS

1. Broth Culture Methods

Entails inoculation of specimens to bottles or tubes containing liquidmedia

Growth is detected by:

a. visual examination of the broth for turbidity, hemolysis, gasproduction, 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 microbialgrowth in broth


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BLOODCommonly 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 clottingb. 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 mostclinically significant bacteremias


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2. Biphasic Agar-Broth Culture

Consists of a conventional blood culture broth bottle with anattached chamber containing agar media on a paddle.

The bottle is tipped, allowing the blood-broth mixture toenter the chamber and flow over the agar media.


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3. Lysis-centrifugation blood culture system

Consists of a tube containing reagents that inhibitcoagulation and the complement cascade, lyse

blood cells, and provide a cushion for themicroorganisms during centrifugation.


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CSF Collection


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CSF Collected to diagnose meningitis and viral encephalitis

After introduction of the needle into the subarachnoid space, the fluid iscollected 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


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Processing CSF for Routine Bacterial Culture

Centrifugation (if 1 mL or more of specimen isreceived) for at least 15 minutes at 1500 g.

Preparation of smear for Gram stain or acridineorange stain and culture.

Blood agar, chocolate agar, and enriched broth areinoculated for culture


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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 savedand stored at 4C or below if future antigen detection testsare anticipated.


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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.


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OTHER BODY FLUIDS

Pericardial

Peritoneal

Synovial

Pleural

Precautions for collection and testing are thesame for CSF


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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 culturebottles at the bedside.


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EAR SPECIMENS

Middle ear fluid aspirates should be delivered promptly to the laboratoryfor culture

Tympanocentesis should be performed with aseptic technique

In the event that the tympanic membrane has already ruptured, fluidremaining in the ear canal may be absorbed on a swab.

Fluid is processed by performing a Gram stain and inoculation of mediasuitable for growth of the middle ear pathogens


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EYE Specimens

A. Conjunctival Specimens Obtained from the superior and inferior tarsal conjunctiva

by using a swab moistened with broth or a sterile platinumspatula.

Swab is rolled on the surface of blood and chocolate agarplates.

B. Corneal Specimens

Obtained using the Kimura spatula and inoculation of theof appropriate media using C streaks.


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EYE Specimen Collection


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GI TRACT

Stool is the preferred specimen over rectal swabs.

Transported in sterile, plastic cups with screw-capped lids.

Stool should not be contaminated with urine orbarium sulfate.

Should be placed in a Cary-Blair medium if not

processed immediately.


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GI TRACT

Specimens are directly plated to appropriate mediato allow recovery of salmonella,. Shigella andCampylobacter spp.

For detection of Campylobacter jejuni/coli, thesample can be filtered, the filtrate planted onBrucella sheep blood agar or chocolate agar.


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GENITAL TRACT

A. Vaginal secretions

Most common pathogens are Gardnerella vaginalis,Candida spp, Trichomonas vaginalis.

A wet-mount preparation is the most valuable diagnostictest.

The slide is examined for

a. clue cells (epithelial cells covered with small

coccobacillary bacteria) - non-specific vaginosis

b. pseudohyphae candidiasis

c. motile trichomonads


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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 transportspecimen, 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


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UPPER RESPIRATORY TRACT Throat specimens are collected by having the patient open

his/her mouth widely, depressing the tongue to improvevisibility, and inserting the swab so that the tip makescontact with exudative, inflamed regions of the posteriorpharynx and tonsils.


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LOWER RESPIRATORY TRACT

Expectorated sputum is the most common specimen.

The patient coughs deeply and expectorates into a sterilecontainer.

Or obtained by bronchoscopy, suction throughtracheostomy or endotracheal tubes, bronchoalveolarlavage, direct lung aspiration, or biopsy


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UPPER RESPIRATORY TRACT

Specimens include swabs of the nasopharynx, throat, andsinus 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 andpuncture of the cartilage surrounding the sinus cavity.


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Bronchoscopy


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URINARY TRACT

Acceptable methods of urine collection include midstreamclean-catch, catheterization, and suprapubic aspiration.

All urine specimens should be processed within two hoursafter collection.

Urine may be refrigerated up to 24 hours; may be collectedin sterile Vacutainer tubes containing boric acid-sodiumformate transport media.


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Suprapubic Urine Collection


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WOUND SPECIMENS

Any material collected from a wound should be examinedwith a Gram-stained smear.

Specimens from superficial wounds are frequently collectedby swabs.

All wound specimens should be cultured on blood agar,enteric agar, and anaerobic blood agar and incubated for 48hours at 25C-37C


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Specimen Collection, Transport and Processing

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