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►Standard Operating Procedure◄ Section: Laboratory Version No. # 1.0 Initials: ______ Title: 2.1B Processing of Blood Revision Date: 20 April 2011 Cultures_BacT/ALERT 1. Definitions 1.1. CRF- Case Report Form 2. Purpose / Background 2.1. The blood of healthy individuals is usually sterile. Blood cultures are a standard laboratory tool for detecting invasive bacterial/fungal disease. The BacT/ALERT is an automated blood culture system which incubates and continuously monitors blood cultures. A positive result is signaled immediately upon detection of carbon dioxide production in the blood culture bottle. 2.2. The quality of blood collection greatly affects the sensitivity and reliability of any blood culture system. Critical factors are: Timing of blood collection. Before administration of antibiotics. Volume of blood collected. Sensitivity increases with volume; bacteremias can be missed by relying upon small blood volumes, which is a particular issue in children. Skin disinfection. Contamination of blood cultures with bacteria that commonly reside on the skin can cause false positive results for bacteremia, may prevent identification of a true pathogen, and complicate the interpretation of our laboratory results. Thorough disinfection of the venipuncture site can significantly reduce the number of contaminated blood cultures. 2.2. The purpose of this SOP is to give guidance on isolation of organisms found in blood specimens. 3. Scope / Applicability 3.1. This SOP is applicable to all trained laboratory technicians/technologists/scientists working in the microbiology laboratory. 4. Roles / Responsibilities [Site specific] 5. Specimen 5.1. Blood received in the laboratory in BacT/ALERT Pediatric FAN blood culture bottles (see appendix 1, collection of blood for blood culture) 6. Prerequisites / Supplies Needed 6.1. Equipment 6.1.1 CO2 Incubators or Candle jar 6.1.2 Aerobic incubator 6.1.3 BD BacT/Alert machine 6.2 Media 6.2.1 5% Sheep Blood agar 6.2.2 Chocolate agar 6.2.3 MacConkey agar 6.3 Materials 6.3.1 Alcohol 6.3.2 Antimicrobial susceptibility discs. 6.3.3 Biochemical reagents 6.3.4 Paediatric blood culture bottles BacT/ALERT Pediatric FAN 6.3.5 Cotton wool/gauze 6.3.6 Gram stain reagents 6.3.7 Syringes and needles 6.3.8 Sterile gloves 6.3.9 Tourniquet 6.3.10 Microscope Slides 6.3.11 Sharps container 7. Safety/Risk Assessment 7.1 Wear Personal Protective Equipment at ALL times when processing blood culture samples. Process all blood cultures in a biosafety cabinet. 7.2 Waste disposal: Discard all sharps in sharp boxes. Autoclave all culture plates and other clinical wastes before taking them for incineration. Reusable material must be autoclaved before washing. 8. Procedural Steps 8.1. Pre-processing specimen handling Blood culture bottles should be placed in the BacT/ALERT blood culture instrument as soon as possible after arrival in the laboratory. Store at air-conditioned room temperature if unable to process immediately. [This will need to be modified based on specific manufacturers’ instructions, as some systems allow incubation in a standard incubator before placement on automated instrument] 8.2. Initial processing of blood culture bottles Each blood culture bottle should be weighed and the value subtracted from the un-inoculated weight of the bottle. The result is the weight of the inoculated blood and is recorded on the CRF. [specific to each automated system] SOP ID 2.1B Processing of Blood Cultures_BacT/ALERT v1.0 Page 2 of 8 8.3. Protocol for positive blood cultures (BacT/ALERT) Day 1 8.3.1. Unload positive blood culture bottle(s) according to BacT/ALERT instrument operation protocol (See instrument operation protocol below).