BLOOD CULTURE: ADULT
ADOPTED: April 11, 2001
To provide a qualitative procedure for the culture and recovery of microorganisms
(bacteria and yeasts) from blood.
Two blood culture vials (enriched Soybean-Casein Digest broth with CO2) are used for aerobic and anaerobic cultures. The principal use is with fluorescent methodology instruments. Media have been formulated to allow the addition of up to 10 ml of blood. The addition of these larger sample volumes results in overall higher detection ranges and earlier times to detection.
The sample to be tested is inoculated into the vials, which are inserted into the fluorescent instrument for incubation and periodic reading. Each vial contains a chemical sensor, which can detect increases in CO2 produced by the growth of microorganisms. The instrument monitors the sensor every ten minutes for an increase in its fluorescence, which is proportional to the amount of CO2 present. A positive reading indicates the presumptive presence of viable microorganisms in the vial. Detection is limited to microorganisms that will grow in a particular type of medium.
Resins have been described for the treatment of blood specimens both prior to and after their inoculation into culture media. Resins have been incorporated into the culture media to enhance recovery of organisms without a need for special processing.
If microorganisms are present in the test sample, CO2 will
be produced when the organisms metabolize the substrates present in
the vial. The fluorescent instrument monitors increases in the fluorescence
of the vial sensor caused by the higher amount of CO2. Analysis
of the rate and amount of CO2 increase enables the fluorescent
instrument to determine if the vial is positive, i.e., that the test
sample contains viable organisms.
CLINICAL SIGNIFICANCE:The isolation of any organism from a blood culture must be considered significant and correlated with the clinical picture.
Whenever possible, blood cultures should be obtained prior to the initiation of antimicrobial therapy.
Prepare the patient's arm for venipuncture:
Allow iodine to remain on skin one minute before performing venipuncture
The specimen must be collected using sterile techniques to reduce the chance of contamination.
Remove the flip-off cap from the vial top and inspect the vial for cracks, contamination, excessive cloudiness, and bulging or indented stoppers. DO NOT USE if any defect is noted. Before inoculating, swab the septum with alcohol (iodine is not recommended). Aseptically inject or draw directly 8-10 ml of specimen per vial.
Label each bottle with patient's name, identifying number, date, time,
and your initials.
The uninoculated vials are ready for use as received and require no reconstitution or dilution. Store in a cool (2-25 degrees C), dry location out of direct sunlight.
Specimens should be transported to the laboratory and incubated as
soon as possible. If a delay in transport or processing is anticipated,
do not refrigerate the vials; leave them at room temperature.
Specimens may be rejected if they: Are unlabeled Are leaking or cracked Have evidence of insufficient quantity Have evidence of refrigeration Call the charge nurse or physician to request a repeat sample before discarding the sample.
REAGENTS / MATERIALS:
70% alcohol Swab PVP iodine Swab 10 cc or 20 cc syringe with a Luer-Lok brand tip or butterfly set Two culture vials containing Soybean-Casein Digest Broth, Polyanetholesulfonate (SPS) (0.05% w/v), Nonionic Adsorbing Resin, and Cationic Exchange Resin. Media is dispensed with added CO2. Anaerobic media are pre-reduced and dispensed with CO2 and N2.
The prepared culture vials are for in vitro diagnostic use. Pathogenic microorganisms including Hepatitis B Virus and Human Immunodeficiency Virus may be present in specimens. Standard Precautions should be followed in handling all items contaminated with blood or other body fluids.
Prior to use, each vial should be examined for evidence of contamination such as cloudiness, bulging or depressed stopper, or leakage. DO NOT USE any vial showing evidence of contamination. A contaminated vial could contain positive pressure. If a contaminated vial is used for direct draw, contaminated culture media could be refluxed into the patient's vein. Vial contamination may not be readily apparent. If a direct draw procedure is used, monitor the process closely to avoid refluxing materials into the patient.
Prior to use, the user should examine the vials for evidence of damage or deterioration. Vials displaying turbidity, contamination, or discoloration (darkening) should not be used. On rare occasions, the glass bottleneck may be cracked and the neck may break during removal of the flip-off cap or in handling. Also, on rare occasions, a vial may not be sealed sufficiently. In both cases, the contents of the vials may leak or spill, especially if the vial is inverted. If the vial has been inoculated, treat the leak or spill with caution, as pathogenic organisms/agents may be present. Before discarding, sterilize all inoculated vials by autoclaving.
Positive culture vials for sub culturing or staining, etc.: Before sampling, it is necessary to release gas, which often builds up due to microbial metabolism. Sampling should be performed in a biological safety cabinet if possible, and appropriate protective clothing, including gloves and masks, should be worn. See procedure section for more information on sub culturing.
To minimize the potential of leakage during inoculation of specimen into culture vials, use syringes with permanently attached needles or Luer-Lok brand tips.
1. Print Vial inventory
2. Print Current Positives if applicable
3. Perform QC checks and maintenance, reading and recording the following on the maintenance form:
Backup disk procedure
Quality Control Report
Frequency / Use:
Acceptance Limits / Corrective Action:
The user should examine the culture vials for evidence of deterioration prior to use. Vials displaying turbidity, contamination, or discoloration (darkening) should not be used. Do not use vials past their expiration date.
The performance of the media may be checked by inoculation with pure cultures of appropriate organisms. The positive vials should be inoculated with Escherichia coli and Staphylococcus aureus to a level below visual turbidity. These vials and an uninoculated control vial should be logged into the instrument and tested.
Quality Control Certificates are provided with each carton of media. Quality control certificates show test organisms, including ATCC cultures specified in the NCCLS standard, Quality Assurance for Commercially Prepared Culture Media.
Acceptable Limits / Corrective Action:
Inoculated aerobic and anaerobic vials should be placed in the fluorescent instrument as soon as possible for incubation and monitoring. If placement of an inoculated vial into the instrument has been delayed and visible growth is apparent, it should not be tested in the fluorescent instrument, but rather it should be sub cultured, Gram-stained and treated as presumptively positive bottle.
Vials entered into the instrument will be automatically tested every ten minutes for the duration of the testing protocol period. Positive vials will be determined by the fluorescent series instrument and identified as such. The sensor inside the bottle will not appear visibly different in positive and negative vials, however the fluorescent series instrument can determine a difference in fluorescence.
If at the end of the testing period, a negative vial appears visually positive (i.e., chocolatized blood, bulging septum, lysed and/or very darkened blood), it should be sub cultured and Gram-stained and treated as a presumptive positive.
Positive vials should be sub cultured and a Gram-stained slide prepared.
In a great majority of cases, organisms will be seen and a preliminary
report can be made to the physician.
PROCEDURE for NEGATIVE CULTURE:
The following are the steps necessary in processing a positive blood culture:
No calculations are necessary.
A positive sample is determined by the fluorescent instrument and indicates the presumptive presence of viable microorganisms in the vial.
Panic / Alert / Critical Values:
Optimum recovery of isolates will be achieved by adding maximum amounts of blood. Use of lower volumes may adversely affect recovery and/or detection times of organisms such as Peptostreptococcus, Peptococcus, Bacteroides asaccharolyticus. Blood may contain antimicrobials or other inhibitors, which may slow or prevent the growth of microorganisms. False negative readings may result when certain organisms are present which do not produce enough CO2 to be detected by the system or significant growth has occurred before placing the vial into the system. False positive may occur when the white blood cell count is high or the bottle is overfilled.
Due to the nature of biological materials in media products and inherent organism variability, the user should be cognizant of potential variable results in the recovery of certain microorganisms.
Care must be taken to prevent contamination of the sample during collection and inoculation into the Blood Culture vial. A contaminated sample will give a positive reading, but will not indicate a relevant clinical sample. Such a determination must be made by the user based on such factors as type or organism recovered, occurrence of the same organism in multiple cultures, patient history, etc.
Recovery of SPS Sensitive and Fastidious Organisms from Blood Samples:
Some fastidious organisms, such as certain Haemophilus species, require growth factors, such as NAD, or factor V, which are provided by the blood specimen. If the blood specimen volume is 3.0 ml or less, an appropriate supplement may be required for recovery of these organisms. whole human blood, or defibrinated sheep blood may be used as nutritional supplements.
For information on antimicrobial agents neutralized by resins, contact the Technical Services department.
Recovery of Streptococcus pneumoniae:
1. Wallis, C. et al., Rapid Isolation of Bacteria from Septicemic Patients by Use of an Antimicrobial Removal Device, J. Clinical Microbiology, 1980, 11:462-464.
2. Applebaum, P.C. et al, Enhanced Detection of Bacteremia with a New Bactec Resin Blood Culture Medium, J. Clinical Microbiology, 1983, 17:48-51
3. Jungkind, D.L., et al, Evidence for a second mechanism of action of resin in Bactec NR16A aerobic blood culture medium. Abstracts of the Annual Meeting of American Society for Microbiologists, 1989.
4. Recommendations for Preventing Transmission of Human Immunodeficiency Virus and Hepatitis B Virus to Patients during Exposure-Prone Invasive Procedures, MMWR, 1991, Vol. 40, No. RR-8.
5. Bloodborne Pathogens, Code of Federal Regulations, Title 29, Part 1910.1030, Federal Register, 1991, 56:64175-64182.
6. Data available from Becton Dickinson Diagnostic Instrument Systems.
7. Balows, A., et al, Manual of Clinical Microbiology, 5th Edition, American Society for Microbiologists, Washington, DC, 1991.k.
8. Howden, R.J., J. Clinical Pathology, 1976, 29:50-53.
9. Bactec Peds Plus/F Package Insert.