WEBCLS Procedure Manual
BLOOD CULTURE: ADULT

AUTHOR: WEBCLS

ADOPTED: April 11, 2001

PURPOSE:

To provide a qualitative procedure for the culture and recovery of microorganisms (bacteria and yeasts) from blood.

PRINCIPLE:

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.
SPECIMEN:
    Patient Preparation:
    Whenever possible, blood cultures should be obtained prior to the initiation of antimicrobial therapy.
    Prepare the patient's arm for venipuncture:
    • Swab area with 70% alcohol
    • Swab area with PVP iodine in a concentric manner.
    Beginning in the center and swab toward outer ring.
    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.

    Type:
    Two vials should be drawn for each culture for anaerobic and aerobic atmospheres.
    Many bacteremias are non-continuous therefore timing of specimen collection may be critical. Theoretically, the best time to draw blood cultures is 30 minutes to one hour before a temperature spike, but this can be done only in patients with a set daily pattern of temperature elevation. Most physicians draw blood cultures when the temperature rises in hope of detecting organisms persisting in the blood stream. In this case, two or three separate cultures obtained within a 24-hour period are appropriate. If not specified, multiple blood cultures should be obtained in 30 minutes to 1-hour intervals.

    Amount Required:
    The recommended specimen volume is 8-10 ml.
    Sample volumes as low as 3 ml can be used, however recovery will not be as great as with larger volumes. Also, other considerations must be taken into account when sample volumes of less than 8 ml are used. The inoculated vial should be transported as quickly as possible to the laboratory. A yellow-top vacuum tube containing SPS may also be used to collect the blood sample from the patient. The tube should be transported to the laboratory as quickly as possible for transfer into the Blood culture vial.


    Collection:
    It is recommended that the specimen be inoculated into the Blood Culture vials in the drawing room.
    Most commonly, a 10 cc or 20 cc syringe with a Luer-Lok brand tip is used to draw the sample. If appropriate, a butterfly set may be used. If using a butterfly set (direct draw), carefully observe the direction of blood flow when starting sample collection. The vacuum in the vial will usually exceed 10 ml, so the user should monitor the volume collection by means of the 5 ml graduation marks on the vial label. When the desired 8-10 ml has been drawn, the flow should be stopped by crimping the tubing and removing the tubing set from the vial.

    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.

    Stability:
    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.

    Unacceptable Specimens:
    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.

WARNINGS:

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.

CALIBRATION:

    Preparation:
    START-UP PROCEDURE:
    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:
    Audible Alarm
    Temperature checks
    Backup disk procedure
    Quality Control Report

    Frequency / Use:
    Perform the start-up procedure at the beginning of each work shift.

    Acceptance Limits / Corrective Action:
    Refer to the operator's manual for the fluorescent instrument for acceptable limits and troubleshooting of the instrument.

    Recording:
    Document all calibration, quality control and corrective actions in the instrument maintenance and quality control manuals.

 

QUALITY CONTROL:
    Preparation:
    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.

    Frequency/Use:
    It is recommended that each shipment of media be tested for performance through the use of positive and negative vial tests.

    Acceptable Limits / Corrective Action:
    The inoculated vials should be detected as positive by the instrument within 24 hours. The negative control should remain negative. This verifies that the media were not subject to adverse storage or shipping conditions prior to receipt in your laboratory.

    Recording:
    Record the results of quality control testing in the quality control log.

PROCEDURE:

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:

    1. Using the current vial inventory list, the bottles are selected for negative reports to coincide with the actual collection date displayed on the original requisition.
    2. Print out status of all vials in the system.
    3. Report as "No Growth" those blood cultures that are listed by the fluorescent instrument as negative and are observed visually to be negative at the end of 5 days. Those bottles can be removed from the instrument and discarded.
    PROCEDURE for POSITIVE CULTURE:
    The following are the steps necessary in processing a positive blood culture:

    1. Remove Positive bottle(s) from the instrument.
    2. Aseptically remove an aliquot of blood from the bottle for subculture and gram stain.
      a. Wipe top of bottle with alcohol prep pad.
      b. Insert sterile needle of 3cc syringe, allow air to escape.
      c. Withdraw small volume of blood (1cc).
      d. Use 1 drop for gram stain.
      e. If gram stain is negative, return bottle to the instrument.
    3. If organisms are observed, determine type by gram stain. Determine total number of bottles drawn and number that are positive. Call the doctor or charge nurse with this preliminary report. Document report, date, time and person who receives the report in the LIS.
    4. Subculture the sample to aerobic sheep blood agar plate, chocolate agar plate, anaerobic blood agar plate. Incubate blood and chocolate agar plates in 5-10% CO2 at 35-37oC. Incubate anaerobic blood agar plate anaerobically at 35-37oC. Examine subcultures for growth at 24 and 48 hours.

CALCULATIONS:

No calculations are necessary.

REPORTING RESULTS:
Procedure:
A positive sample is determined by the fluorescent instrument and indicates the presumptive presence of viable microorganisms in the vial.

Reference Ranges:
No organisms should be found in the blood specimen.

Panic / Alert / Critical Values:
Notify positive results of gram stain and culture to the physician or charge nurse immediately. Document report, date, time and person who receives the report in the LIS.

LIMITATIONS:
Linearity:
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.

Dilution Protocol:
Not applicable.

Interferences:
Contamination:
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:
Because blood can neutralize the toxicity of SPS toward organisms sensitive to SPS, the presence of maximum volumes of blood (8-10 ml) can help to optimize recovery of these organisms. To enhance the growth of SPS sensitive organisms when less than 8 ml of blood is inoculated, additional whole human blood or defibrinated sheep blood may be added.

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.

Non-Viable Organisms:
A Gram-stained smear from a culture medium contains small numbers of non-viable organisms derived from medium constituents staining reagents, immersion oil, glass slides, and specimens used for inoculation. In addition, the patient specimen may contain organisms that will not grow in the culture medium or in media used for subculture. Such specimens should be sub cultured to special media as appropriate.

Antimicrobial Activity:
Neutralization of the antimicrobial activity by resins varies depending on dosage level and timing of specimen collection. The use of supplementary additives should be considered in appropriate situations; as an example, the addition of penicillinase when B-lactam therapy is employed.

For information on antimicrobial agents neutralized by resins, contact the Technical Services department.

Recovery of Streptococcus pneumoniae:
In aerobic media, S. pneumoniae will typically be visually and instrument positive, but in some cases no organism will be seen on Gram-stain or recovered on routine subculture. The organism can usually be recovered by performing an aerobic subculture of the anaerobic vial, since this organism has been reported to grow well under anaerobic conditions.

REFERENCES:
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.

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