BACTE LEC 1.3 SPECIMEN COLLECTION AND TRANSPORT.pdf

BACTERIOLOGY SPECIMEN COLLECTION & TRANSPORT LECTURE SPECIMEN COLLECTION collect before antibiotic therapy, ensure aseptic collection quantity must be sufficient, must be placed in sterile containers Ideally, specimens should be transported to the lab within 2 hrs of collection Aerobes swab, ne...

BACTERIOLOGY SPECIMEN COLLECTION & TRANSPORT LECTURE SPECIMEN COLLECTION collect before antibiotic therapy, ensure aseptic collection quantity must be sufficient, must be placed in sterile containers Ideally, specimens should be transported to the lab within 2 hrs of collection Aerobes swab, needle aspiration Anaerobes needle aspiration and tissue Urine, CSF, serous fluid, stool, sputum sterile containers throat, nose, eyes, wound and abscess Use sterile cotton tipped applicator Sterile broth with SPS Broth Culture Transport Media With nutrients to maintain the growth of organism, buffer to maintain pH, small amount of agar to maintain moisture CARY BLAIR stool pathogens, Vibrio STUART’S bacteria and virus AMIES respiratory TRANSGROW & JEMBEC Neisseria TODD-HEWITT BROTH S. agalactiae (vaginal swab) LIM BROTH modified Todd-Hewitt BUFFERED GLYCEROL SALINE common STOOL medium (not preferred to ENTERIC BACTERIA like Vibrio and Campylobacter CHARCOAL added to transport media to absorb FATTY ACID SWAB For aerobes Swab is not recommended for fungi and anaerobes Dacron and Rayon Dacron cotton swab with charcoal good for bacteria and viruses Virus Urogenital specimen COTTON toxic to Neisseria, good for virus Calcium Alginate toxic to virus, good for Neisseria TRANSPORT TEMPERATURE CSF Boric Acid 4degC -20degC for 1 wk -70C ANAEROBE transport (RT), storage (35-37C) Urine preservative Accurate colony count Urine, stool, swab, viral specimens, sputum, foreign devices Serum for serology Tissue specimen for long term storage Should nor be refrigerated It inhibits the viability of certain organisms REFRIGERATOR ROOM TEMPERATURE stool, sputum, urine and swab except genital CSF, blood, body fluids, genital swab of N. gonorrhea Anticoagulants/Preservative To maintain accurate colony count, if there is delay in processing urine, this preservative may be added SPS 0.025% Sodium polyanethol sulfonate (SPS) Heparin EDTA EDTA & CITRATE to prevent clotting anti-complementary anti-phagocytic neutralizes aminoglycoside anticoagulant used in viral culture may inhibit gram (+) and yeast Viral PCR anticoagulant not to be used in Bacteriology Ways to facilitate ANAEROBIC CULTIVATION GAS PAK JAR Anaerobic atmosphere: 5% CO2, 10% H2, 85% N2 Boiling of culture medium to remove oxygen Use of GAS PAK JAR with palladium catalyst Indicators: Methylene Blue/Resazolin in the absence of air they become colorless Anaerobic cultures are incubated at 35-37 degC for at least 2 days ANAEROBIC CHAMBER ideal aerobic incubation system NITROGEN GAS is used as a filter for the remaining percentage of the ANAEROBIC ATMOSPHERE PALLADIUM PELLETS removes residual OXYGEN from the chamber by combining with H+ to form H20 It must be replaced or inactivated each time the jar is used heat the pellets in a dry oven for 2hrs (160-170degC) for rejuvenation SILICA GEL Dessicant used to absorb water formed when H+ combines free oxygen and catalyst CLINICAL SPECIMEN BLOOD Iodophor (betadine) and opposite arms Collect before height of fever 0.025% SPS: Most common anticoagulant inhibits Neisseria, G. vaginalis, P. anaerobius, S. monoliformis to counteract add 1% GELATIN TSB, BHIB with 0.025% SPS, 1% gelatin (+) cloudy, gas bubbles, hemolysis, pellicle Blood to broth ratio = 1:10-critical Children = 1:5 to 1:10 Adult = 1:10 to 1:20 No more than 3 sets should be collected in 24hr period CLINICAL SPECIMEN Subculture on BAP, CAP and MAC TAT: 7days 21 days: Brucellosis, Endocarditis, SBE 8 weeks: Leptospirosis Automation: Uses BACTEC 9120 TAT: 5 days Medium: BACTEC broth (1:10 ratio) with SPS 0.025% SPS (liquid) anticoagulant, anti-complementary, anti-phagocytic, neutralizes amingglycosides and bactericidal effect on serum Specimen Rejection for Blood Culture: Clotted specimen Specimen collected using only alcohol as the antiseptic Should be ALCOHOL + IODINE Reason: Contamination risk Specimen containing less than 20mL for adults are also questionable CLINICAL SPECIMEN CEREBROSPINAL FLUID (CSF) Usually collected using 3-4 tubes, tube #2 is for MICRO, gram stain and culture, if with 4th tube, use the for better exclusion of skin contaminants Examine immediately, PRIORITY/1st to process or hold either at 37degC storage temp transport temp: RT placed on bottle 2, not refrigerated only incubator Routine test: India ink and gram stain Used for the Isolation of: Neisseria meningitidis Streptococcus pneumonia and Haemophilus CLINICAL SPECIMEN CEREBROSPINAL FLUID (CSF) Centrifuge: sediment for culture and GS BAP, CAP (5-10% CO2) BHIB, MAC (incubator - NO CO2) India Ink method Detection of Cryptococcus neoformans capsule CSF Latex agglutination Detection od Capsular Antigen Storage: Bacteria: 37degC Virus: 4degC CLINICAL SPECIMEN RESPIRATORY TRACT SPECIMEN A. Throat Swab For S. pyogenes, C. albicans (Oral thrush) and for H. influenzae (epiglottis); Medium of choice: SBA Swab in the posterior pharynx, tonsils and inflamed areas MOST ABUNDANT THROAT FLORA Viridans Streptococci Alpha hemolytic Streptococci MOST COMMON THROAT PATHOGEN: S. pyogenes / Group-A Strep / Beta-hemolytic Strep B. Sputum May often be contaminated with normal flora so it is important evaluate the quality of the specimen Ideally collected in the morning; rinse mouth or gargle with WATER then instruct the patient to deep cough and expectorate into sterile container. THREE SPUTUM FOR ACID FAST SMEARS morning specimens are preferred since they are more concentrated DOH: 2spx Should be Gram stained and checked for acceptability Gentamicin BAP S. pneumoniae Bacitracin CAP H.influenza B. Sputum BARLETT’S CLASSIFICATION assess the quality and for scoring of sputum Note the number of squamous epithelial cell (SEC)/LPF and PMNs to evaluate acceptability of the specimen in Bartlett's classification, the number of neutrophils and epithelial cells per lowpower field (LPF) is enumerated. Based on these findings, the sputum is given a score. Scores of 0 or less indicate a lack of inflammation or the presence of saliva while scores greater than 1 indicate inflammation or infection Number of Neutrophil per LPF 28 +2 Number of EC of per LPF 6 0 Total Score 2 Purulent Secretions Respiratory Secretions Oral Secretions more than 25 PMNS and fewer than 10 SECS/LPF whereas a predominance of AMs or CCs (greater than 25/LPF) more than 25 SECs/LPF and fewer than 10 PMNS/LPF. C. NASAL SWAB Moistened swab in the nares/nose detects carrier state of S. aureus D. NASOPHARYNGEAL SWAB Flexible swab inserted through the nose and into posterior nasopharynx and then rotated for 5 seconds Detect carrier state of N. meningitidis and for detection of B. pertussis CLINICAL SPECIMEN URINE (RANDOM, CATHETERIZED, MIDSTREAM, SUPRAPUBIC) Specimen of choice for bacterial culture is Clean-catch Catheterized for those unable to produce. Foley Catheter - urine IV catheter - blood Ideally, First-morning specimen is used since it is more concentrated. Must be preserved or refrigerated if not processed, suitable if preservative BORIC ACID to maintain accurate colony count Major cause of UTI is E. coli and the most common cause of UTI among young females S. saphrophyticus Suprapubic collected via needle aspiration above the symphysis pubis ANAEROBIC PROCESS MAC and BAP are suitable combination Urine specimen MUST BE CULTURED WITHIN ONE HOUR OR REFRIGERATE however, NEVER REFRIGERATE FOR LONGER THAN 24 hours. Colony count should be performed on all urine sample when performing colony count use CALIBRATED LOOP (1uL or 10uL) Formula to compute for colony count per mL of urine: No. of colonies counted X DF X 1000 (if 1 uL loop was used) = colony count/mL of urine No. of colonies counted X DF X 100 (if 10 uL loop was used) = colony count/mL of urine CONSIDERED SIGNIFICANT: Greater than or Equal to 100,000 colony count/mL or 10^5 GASTROINTESTINAL TRACT SPECIMEN A. Stool 1-2grams stool on sterile vial for detection of ENTERIC PATHOGEN Routinely screen for Salmonella Shigella, Campylobacter May be directly plated: Differential Media: EMB or MAC Selective media: HEA and SSA Enrichment media: Tetrathionate Test/s: Presumptive: oxidase test, biochemical test Confirmatory: Serotyping B. Rectal Rectal swab on Cary Blair Collected by inserting a swab 2.5cm past anal sphincter. Feces should be visible on the swab GENITAL TRACT SPECIMENS To detect the presence of N. gonorrhea, G. vaginalis, C. trachomatis Transport medium such as JEMBEC or Gono-Pak Mostly fastidious organisms require the use of (CAP) Chocolate Agar Plate WOUNDS AND ABSCESSES Preferred specimen for wound/abscess culture is needle and syringe aspirate TB CULTURE 1 sputum for GS; 2 sputa for AFS NALC-NaOH GOLD STANDARD NALC (n-acetyl-L-cystine): digestant/mucolytic 2.4% NaOH: decontamination Oxalic acid Pseudomonas aeruginosa contamination (cystic fibrosis) Critical Values Positive blood cultures, CSF Gram Stain or culture and positive acid-fast stain S. pyogenes in surgical wound Gram stain suggestive of gas gangrene Blood smear for patient with malaria Positive cryptococcal antigen test Detect of select agents or other significant pathogens antibiotic-resistant strain)