Specimen Collection and Handling PDF

141 Specimen Collection and Handling GENERAL 1. Material from infection site CoHecti.on and Handling of...

141 Specimen Collection and Handling GENERAL 1. Material from infection site CoHecti.on and Handling of 2. Optimal time (ex.: Salmonella typhi- Blood Culture Specimem1 culture blood ffrst week, culture stool second and third weeks) Diagnostic Methods 3. Appropriate collection devices MICROSCOPY 4. NEVER refrigerate spinal fluids, 1. Light microscopy anaerobic or GC specimens a. Resolving power - 0.2 µm b. Ocular lens= l0X; oil immersion 5. Collect prior to antibiotic therapy lens= IO0X 6. Set up within 2 hours of collection 2. Darkfield - for spirochetes; reflected CRITERIA FOR REJECTION light 1. Preservatives used 3. Fluorescence - near UV range; auramine rhodamine, acridine orange 2. Insufficient quantity and calco-fluor white stains 3. Dry swab 4. Electron microscopy - can resolve 4. Leaky containers - contaminated particles 0.001 µm apart; u seful in viral specimen as well as biohazard I.D. BLOOD CULTURE COLLECTION CULTURE CONDITIONS 1. Must prep skin properly with alcohol 1. Most plates incubated at 35-37°C (70%) and iodine a. Campylobacter - 42°C b. Yersinia - 25-30°C 2. Best time to draw is just prior to fever c. 5-10% CO2 (Campy- spike mi.croaeropl1ilic) d. 50-70% humidity 3. Draw at least 2 cultures, but no more than 3 in a 24 hr period 2. Anaerobic Conditions a. Broths with thioglyco.llate or 4. May use antibiotic removal device (ARD) cysteine or resin bottles if patient on antil1iotics b. P re-reduced media ❖ Gas pak jars or anaerobic 5. Isolator® best for fungi and acid fast chamber organisms ❖ Environment: 10% H2, 5% CO2, 6. Need 1: 10 dilution of blood to broth; 85% N2, palladium crystals on adults draw at least 10 ml (5ml for MEDIA pediatric bottles) 1. Approximately 1.5% agar 2. Non-selective a. Supports most organism s b. Blood agar, chocolate agar, trypticase soy agar 3. Selective agar a. Contains chemicals, dyes, antibiotics to inhibit certain organisms (EMB, MAC, CNA, Campy-blood) b. May also be differential (HE, SS, XLD, EMB, MAC) 142 INOCULATION 3. Number of colonies x 100 (.OJ loop) or 1. Streak for isolation wit h nichrome or number of colonies x 1000 (.001 loop) pl atinum, or disposable loops 4. Read and report after 18-24 hrs 2. Calibrated 0.01 ml or 0.001 ml (. 001 ml for urine colony count plates) Stains Commonly Used in Microbio/ogy/Mycobacteriology PRIMARY DECOLORIZER COUNTERSTAIN RESULTS PRINCIPLE STAIN POS NEG Gram Stain Crystal Violet Alcohol/Acetone Safranin Purple Pink Iodine mordant Methanol or heat tix Violet dye & iodine form complex in cell; washes out of gram neg cells Kinyoun & Carbol Fuchsin Acid Alcohol Methylene Blue Pink Blue Acid tast Ziehl-Nielson For Mycobacteria Auramine- Auramine and Acid Alcohol Potassium Orange No For Mycobacteria Rhodamine Rhodamine Permanganate Fluoresc. Fluoresc. (Fluorescent Stain) Calcofluor Calcofluor White Bluish- No For yeast and fungi White + 10%KOH white Fluoresc. KOH to break down Fluoresc. debris and mucous Gram Positive Cocci STAPHYLOCOCCUS if methicillin-resistant S. aureus 1. "Grape-like" clusters (MRSA), vancomycin is drug of choice 2. S. a:ureus g. Laboratory diagnosis a. Coagulase positive ❖ BAP - soft, opaque, regular b. Most common pathogen of genus colonies 2-3 mm in diam; some are c. Common infections beta hemolytic and some have pale ❖ Furnncles (boils) and carbuncles golden color ❖ Biillous impetigo (blisters) ❖ Growth in 7.5% NaCl and ferment ❖ Paronycbia (nails) mannitol ❖ Post surgical wounds and ❖ Catalase positive and coagulase bacteremia positive d. Intoxications ❖ Pulse Field Gel Electrophoresis ❖ Scalded skin syndrome (PFGE) and susceptibility (exfoliatin - neonates) proflle for epidemiologi.c studies ❖ Toxic shock syndrome (TSST-1) - women ages 12-52 3. Coagulase negative Staphylococcus ❖ Food poisoning (en terotox.in) - a. Opportunist in symptoms in 1-5 hrs after immunocompromised hosts and ingestion (potato salad , cream patients with prosthetic valves and dislies) devices e. Exotoxins - h emolysins, leukocidins, coagulase and hyaluronidase (spreading factor), nuclease, protease and lipase f. Resistance/sensitivity ❖ Most resistant to penicillin due to Susceptibility Teating of plasmid mediated B-lactamase Staphylococcw ❖ Some sensitive to penicillinase- resistant penicillins (PRP's) (methicillin , oxacillin, etc);