I. Collection and Culture of Blood Specimens

(1) Timing of Blood Culture Collection

Requirements: For patients suspected of bacteremia or fungemia, blood culture specimens should be collected immediately before initiating antimicrobial therapy. At least two sets of blood cultures (each set comprising one aerobic bottle and one anaerobic bottle) must be obtained either simultaneously or at short intervals.

Note: Experimental data indicate that approximately one hour after bacteria enter the bloodstream, chills and fever typically begin within two hours. This is the optimal time to collect blood cultures for pathogen isolation. Blood sampling at other time points is warranted only in cases of suspected infective endocarditis or other intravascular infections—such as catheter‑related infections—where continuous bacteremia is anticipated.

(2) Number of Blood Culture Specimen Collections

Recommendation: For adult patients with suspected bacteremia or fungemia, it is recommended to collect 2–3 sets of blood cultures (from different sites) simultaneously (i.e., using two bottles and drawing from both arms). For infants and young children, it is recommended to obtain two separate blood culture specimens (from different sites) at the same time.

Note: A single blood culture result in adults is unreliable due to insufficient blood volume, making the findings difficult to interpret. Blood cultures should not be repeated within 2 to 5 days, as bacteria in the bloodstream are not eliminated immediately after the initiation of antibiotic therapy. Cockerill’s study demonstrated that, among three blood cultures, the pathogen isolation rate was 65% for the first sample, 80% for two samples, and 96% for three samples.

Promoting the use of fully automated blood culture systems can facilitate earlier detection of sepsis in transplant patients and patients with indwelling catheters. In most cases, the clinical course of bacteremia or fungemia allows clinicians to assess treatment response without the need for repeat blood cultures or serial monitoring. However, there are two exceptions: first, in patients with infective bacterial endocarditis, the clearance of bacteremia or fungemia can be used to evaluate and guide therapy; second, in staphylococcal bacteremia unrelated to infective endocarditis, follow-up of positive blood cultures over 48 to 96 hours can reliably predict the development of complicated staphylococcal bacteremia.

(3) Blood Collection Volume

Requirements: For adult patients, the recommended blood collection volume is at least 10 mL per set and at least 5 mL per tube. For pediatric patients, the recommended blood collection volume is at least 2 mL per tube (less than 1% of the patient’s total blood volume).

Note: The volume of blood collected is the most critical factor influencing the detection rate. In adults, the volume of blood drawn for culture ranges from 2 to 30 mL, and the pathogen detection rate increases proportionally with the volume collected—each additional milliliter of blood corresponds to a corresponding increase in the detection rate. Similar data in pediatric patients also demonstrate a positive correlation between detection rate and blood volume; however, some studies indicate that pediatric patients often have higher pathogen concentrations in their blood, suggesting that the required blood volume for culture need not be equivalent to that of adults. Laboratories must adhere to the manufacturer’s recommendations for blood culture bottles and collect an adequate volume of blood. For patients with hematologic disorders or those with relatively low blood volumes, it is advisable to use pediatric‑specific bottles to reduce the amount of blood drawn while maintaining or improving pathogen detection rates.

(4) Allocation of blood specimens between aerobic and anaerobic bottles

Requirement: One aerobic bottle and one anaerobic bottle constitute a blood culture set, to be used as the standard combination for routine blood cultures.

Note: Studies have shown that, compared with using paired aerobic blood culture bottles, the use of paired aerobic–anaerobic blood culture bottles yields higher isolation rates of staphylococci, enterococci, and anaerobic bacteria. When the volume of blood collected for culture is less than the recommended amount, the specimen should first be inoculated into the aerobic bottle; the remaining blood should then be transferred to the anaerobic bottle. Since most cases of bacteremia are caused by aerobic and facultatively anaerobic bacteria, these organisms are more effectively isolated from the aerobic bottle. Furthermore, pathogenic fungi, like strictly aerobic bacteria such as Pseudomonas and Stenotrophomonas maltophilia, are almost exclusively recovered from aerobic culture bottles.

II. Collection of Blood Culture Specimens

(1) Skin Disinfection

Recommendation: Use iodine tincture, chlorhexidine, or povidone‑iodine as skin disinfectants.

Note: Disinfectants must be allowed sufficient contact time to ensure effective disinfection (tincture of iodine: 1 minute; povidone‑iodine: 1.5–2 minutes). Chlorhexidine has a similar contact time to tincture of iodine but does not cause allergic reactions; therefore, it does not need to be wiped off after venipuncture. However, it should not be used for skin disinfection in infants younger than 2 months.

(2) Collection Site

Requirement: Blood samples should be collected from a peripheral vein.

Note: Collection is not recommended in the following situations: 1) Arterial blood, as it does not provide greater diagnostic value than venous blood; 2) Venous indwelling catheters, which often have a high contamination rate. If blood must be drawn from an indwelling catheter, a separate blood culture specimen should also be obtained from a peripheral vein to aid in interpreting and explaining any positive results.

(3) Collection Method

Requirements: 1. Prior to blood collection, the rubber stopper of the blood culture bottle must be disinfected with 70% isopropyl alcohol and allowed to dry, followed by skin disinfection at the puncture site. 2. Strictly adhere to the prescribed skin‑disinfection protocol (70% isopropyl alcohol, an iodine‑based antiseptic, and again 70% isopropyl alcohol), allowing sufficient contact time for each step. 3. Maintain strict aseptic technique; do not allow the skin to be touched by bare hands after disinfection unless sterile gloves are worn. 4. It is not recommended to change the syringe needle when drawing blood or inoculating blood culture bottles; using a vacuum‑collection system can reduce the rate of contamination.

Note: Regardless of the blood collection method used, the acceptable contamination rate for blood cultures is generally ≤3%.

III. Transport of Blood Culture Specimens

Requirements: 1. Blood culture bottles, once collected, must be transported to the laboratory within one hour. 2. Blood culture bottles must not be refrigerated or frozen either before or after inoculation.

Note: Delaying the placement of blood culture bottles in an automated incubation and detection system may delay or interfere with the detection of bacterial growth. It is not recommended to leave blood culture bottles at room temperature for more than a few hours. Refrigerating or freezing blood culture specimens can kill certain microorganisms, and freezing containers holding liquid may cause them to rupture. Centrifuged blood culture specimens should also not be left at room temperature for more than eight hours; exceeding this time may reduce the isolation rate of specific pathogens or delay their growth.

IV. Blood Cultures for Several Special Requirements

(1) Fungal Blood Culture

Requirement: For patients suspected of fungal bacteremia, specimens should be collected using aerobic blood culture bottles.

Note: Studies have shown that, compared with anaerobic bottles, yeasts are more readily isolated in aerobic bottles. Most yeasts and yeast‑like fungi can be detected within 2 to 5 days of incubation, whereas certain yeasts—such as Candida glabrata and Cryptococcus neoformans—require longer incubation periods. Although dimorphic fungi can grow on most commercially prepared aerobic blood culture media, detection may take 2 to 4 weeks. Consequently, automated blood culture systems are not suitable for the detection of dimorphic fungi. When a dimorphic or filamentous fungal infection is suspected, the blood sample should be subjected to lysis–centrifugation for concentration and then plated onto agar media.

(2) Mycobacterial Blood Culture

Requirements: Mycobacterial blood cultures must be performed using specialized commercial mycobacterial blood culture bottles. All cultures must be incubated for at least 4 weeks. The incubation temperature should be 25–30°C.

Note: Although mycobacteria are not microorganisms that require particularly complex nutrients, optimizing the isolation of mycobacteria from blood specimens necessitates supplementing the broth medium with fatty acids (e.g., oleic acid), albumin, and carbon dioxide. Certain mycobacterial species (e.g., Mycobacterium gilvum, Mycobacterium haemophilum) require additives such as mycolic acid, heme, hemoglobin, or ammonium ferric citrate.

(3) Catheter-Related Bloodstream Infection (CRBSI)

1. Short-term peripheral catheter

Requirements: Obtain two sets of peripheral blood cultures from the patient via venipuncture (two bottles, one from each arm). Perform aseptic catheter removal and culture using the Maki semi-quantitative method. Interpretation of culture results:

— If one or more blood cultures are positive, and the catheter tip culture is also positive (semiquantitative ≥15 colonies) with the same organism, this suggests a CRBSI.

— If one or more blood cultures are positive while the catheter tip culture is negative, no definitive conclusion can be drawn; however, if the organism is Staphylococcus aureus or a species of Candida, and all other potential sources of infection have been ruled out, this suggests a CRBSI.

— If the blood culture is negative but the catheter tip culture is positive, even with a colony count, this still suggests catheter colonization rather than CRBSI.

— If the blood culture is negative and the catheter tip culture is also negative, CRBSI can be ruled out.

2. Central Venous Catheters and Venous Access Ports (VAP)

(1) Method One

Requirements: For patients suspected of having a CRBSI, collect at least two sets of blood cultures. At least one set should be obtained via peripheral venipuncture and clearly labeled; the other set should be collected aseptically from the catheter hub or the VAP septum, with a collection time closely aligned with that of the peripheral blood culture, and both sets must be properly labeled.

Interpretation of culture results:

If two sets of blood cultures are positive and yield the same bacterial species, and infections from other sources have been ruled out, this suggests a CRBSI.

If both sets of blood cultures are positive and yield the same bacterial species, and the catheter‑derived blood culture turns positive 120 minutes earlier than the peripheral venous blood culture, this suggests a CRBSI. If infections from other sources have been ruled out, and the time difference between the two positive blood cultures is less than 120 minutes with identical antimicrobial resistance profiles, this also indicates a possible CRBSI.

If both sets of blood cultures are positive, and the bacterial count from the catheter‑derived blood culture is at least five times that of the peripheral venous blood culture (CFUs/mL), and if infection from any other source has been ruled out, this suggests a possible CRBSI. (This applies to manual quantitative blood culture systems, such as the dissolution–centrifugation concentration method.)

If only the blood culture drawn from the catheter is positive, a diagnosis of CRBSI cannot be made. This suggests either catheter colonization or contamination during the specimen collection process.

If only a peripheral venous blood culture is positive, it cannot be classified as a CRBSI; however, if the culture yields Staphylococcus aureus or Candida species and other potential sources of infection have been ruled out, this suggests a possible CRBSI.

If both sets of blood cultures are negative, it is not a CRBSI.

(2) Method Two

Requirement: Obtain two sets of blood cultures via aseptic collection from separate peripheral venous access sites.

Remove the suspected catheter, aseptically excise a 125‑px segment from its distal end, and submit it to the laboratory for either Maki’s semi‑quantitative roll‑plate assay or quantitative culture following agitation or sonication.

Interpretation of culture results:

If one or more blood cultures are positive, and the catheter tip culture is also positive, and if both cultures yield the same bacterial species according to identification and susceptibility testing, this suggests a possible CRBSI.

If one or more blood cultures are positive while the catheter tip culture is negative, and if the culture results identify Staphylococcus aureus or a Candida species with no other apparent source of infection, this suggests a possible CRBSI. Confirmation may require additional positive blood cultures yielding the same organism as supporting evidence.

If the blood culture is negative but the catheter tip culture is positive, this suggests catheter colonization rather than a CRBSI.

If both blood cultures are negative and the catheter tip culture is also negative, it is not a CRBSI.

(4) Infective Bacterial Endocarditis

1. Acute Infective Endocarditis:

Requirement: Blood cultures must be drawn within 30 minutes prior to the initiation of empirical antibiotic therapy. Initially, three sets of blood cultures should be collected. If these cultures remain negative after 24 hours, an additional two sets of blood cultures should be obtained, for a total of five sets.

2. Subacute Infective Endocarditis:

Requirement: Several sets of blood cultures must be drawn consecutively within 30 minutes to 1 hour to confirm persistent bacteremia and provide clinicians with additional valuable information. Initially, three sets of blood cultures should be collected. If these cultures remain negative after 24 hours, an additional two sets should be obtained, for a total of five sets.

Note: When obtaining blood cultures from patients suspected of having infective endocarditis, skin disinfection is critically important. In patients with subacute infective endocarditis or prosthetic heart valves, the predominant pathogens are skin flora, such as coagulase-negative staphylococci; therefore, it is especially crucial that blood cultures be collected via venipuncture rather than through indwelling vascular access devices. Blood cultures obtained from catheters increase the risk of contamination, potentially leading to false‑positive diagnoses of infective endocarditis.

Studies have shown that another possible cause of “culture-negative” infective endocarditis is the presence of streptococci exhibiting nutritional variants. Therefore, it is particularly important to collect both aerobic and anaerobic blood cultures simultaneously.

It is recommended to perform proper skin disinfection and to obtain blood cultures from separate peripheral venous sites; blood should not be drawn from an indwelling catheter.

(5) Fungal Infective Endocarditis

Requirement: Refer to bacterial infective endocarditis.

Note: Many of the techniques used to diagnose bacterial endocarditis can also be applied to the diagnosis of fungal endocarditis. The culture media employed in many standard manual or automated blood‑culture systems are capable of supporting the growth of yeasts, such as Candida. If these routine blood cultures remain negative, specialized “fungal blood cultures” should be utilized.