Microbiology Specimens: Bacteriology and Mycobacteriology

Collection of Specimens for Culture: General Information

1. Labeling. Appropriate information is critical to proper processing of test requests. Although pertinent clinical information is highly desirable, if it is not available, please provide at least the following information.

a. Patient's name

b. Source of specimen or collection site

c. Date

d. Specimen and lesion status

e. Test desired

2. Obtain specimen correctly.

a. Explain completely to the patient.

b. Use a sterile container.

c. Label correctly and send the specimen to the laboratory promptly.

d. Avoid contamination of the container.

Note: Please examine specimen collection and transportation supplies to be sure they do not include expired containers.

3. Timing of collection.

a. Sputum, urine, stool, etc. are best collected in early morning and sent to the laboratory the same day.

b. Blood

  • A blood culture requires two bottles of blood—one for aerobic and one for anaerobic culture. Each blood culture should be collected from a separate venipuncture.
  • Collect blood specimens before antimicrobial treatment is initiated, if possible.
  • Collect two or three sets early in the illness; repeat if they are negative after 48 hours of growth.
  • Organisms are continuously shed during intravascular infections, such as endocarditis, but they are intermittently shed during occult infections. In some instances of occult infection, there is a predictable fever pattern. If this is the case, the blood for culture is best collected 30 minutes prior to the fever spike.
  • The yield beyond three or four cultures is minimal in most circumstances, and collection of more than this is discouraged.
  • Virtually any organism, including normal flora, can cause bacteremia. A negative culture result does not necessarily rule out bacteremia; false-negative results occur when pathogens fail to grow. A positive culture result does not necessarily indicate bacteremia; false-positive results occur when contaminants grow. Gram-negative bacilli, anaerobes, and fungi should be considered pathogens until proven otherwise. The most difficult interpretation problem is to determine whether an organism that is usually considered normal skin flora is a true pathogen.
Blood Culture Collection
Clinical Disease SuspectedCulture RecommendationRationale
*Mandell Gl, Bennett JE, Dolin R, eds. Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases. 5th ed. Philadelphia, Pa: Churchill Livingstone; 2000: 867-868.
Sepsis, meningitis osteomyelitis, septic arthritis, bacterial pneumoniaTwo sets of cultures − one from each of two prepared sites. Draw for the second culture after a brief time interval (30 minutes) and then begin therapy.Assures sufficient sampling in cases of intermittent or low level bacteremia. Minimize the confusion caused by a positive culture resulting from transient bacteremia or skin contamination.
Fever of unknown origin (eg, occult abscess, empyema, typhoid fever etc)Two sets of cultures − one from each of two prepared sites. Draw for the second culture after a brief time interval (30 minutes). If cultures are negative after 24 to 48 hours obtain two more sets, preferably prior to an anticipated temperature rise.The yield after four sets of cultures is minimal.
AcuteObtain three blood culture sets within two hours, then begin therapy.95% to 99% of acute endocarditis patients (untreated) will yield a positive in one of the first three cultures.*
SubacuteObtain three blood culture sets on day one, repeat if negative after 24 hours. If still negative or if the patient had prior antibiotic therapy, repeat again.Adequate sample volume despite low level bacteremia or previous therapy should result in a positive yield.
Immunocompromised host aids:
Septicemia, fungemia, mycobacteremiaObtain two sets of cultures from each of two prepared sites.Low levels of fungemia and mycobacteremia frequently encountered.
Guidance for Routine Blood Culture Collection
AgeBottle(s)Total Blood VolumeBlood Culture Set
≥15 years old

1 Aerobic (8 to 10 mL)

1 Anaerobic (8 to 10 mL)

16 to 20 mL

1 Set = 1 Aerobic Bottle and

1 Anaerobic Bottle

<15 years old2 Pediatric (1 to 4 mL)2 to 8 mL1 Set = 2 Pediatric Bottles
Neonates1 Pediatric (0.1 to 1 mL)0.1 to 1 mL1 Set = 1 Pediatric Bottle

Procedure for Specific Specimen Collection

Anaerobic Culture. Specimens are to be collected from a prepared site using a sterile technique. Contamination with normal flora must be avoided. Some anaerobes will be killed by contact with oxygen for only a few seconds. Ideally, pus obtained by needle aspiration through intact surface, which has been aseptically prepared, is put directly into anaerobic transport media. Sampling of open lesions is enhanced by deep aspiration using a sterile plastic catheter or needle. Curettings of the base of an open lesion may also provide a good yield. If irrigation is necessary, nonbacteriostatic sterile normal saline may be used. Pulmonary samples may be obtained by transtracheal percutaneous needle aspiration or by physicians trained in this procedure. Superficial collection (ie, a swab of the lesion) is not the best specimen for anaerobic culture. If swabs must be used, two should be collected; one for culture and one for Gram stain. Swabs of the throat or genital tract are not appropriate specimens for anaerobic culture.

The following are clinical symptoms suggestive of anaerobic infection:

  • Foul-smelling discharge
  • Location of infection in proximity to a mucosal surface
  • Necrotic tissue, gangrene, pseudomembrane formation
  • Gas in tissues or discharges
  • Endocarditis with negative routine blood cultures
  • Infection associated with malignancy or other process producing tissue destruction
  • Infection related to the use of aminoglycosides (oral, parenteral, or topical)
  • Septic thrombophlebitis
  • Bacteremic picture with jaundice
  • Infection resulting from human or other bites
  • Black discoloration of blood-containing exudates (may fluoresce red under ultraviolet light in B melaninogenicus infections)
  • Presence of “sulfur granules” in discharges (actinomycosis)
  • Classical clinical features of gas gangrene
  • Clinical setting suggestive for anaerobic infection (septic abortion, infection after gastrointestinal surgery, genitourinary surgery, etc)

Upper Respiratory Tract. This section describes procedures for obtaining culture specimens from the nasopharyngeal area and the throat.

1. A nasopharyngeal culture is obtained by inserting a thin sterile swab gently through the nose to touch the pharynx; gently rotate and remove.

2. A throat culture is obtained by introducing a sterile swab into the mouth. Use a tongue blade to avoid contaminating the specimen with oral secretions. Firmly swab both tonsillar fossae, posterior pharynx, and any inflamed or ulcerated areas.

Lower Respiratory Tract: Sputum. This section discusses sputum cultures, including such alternatives as induced sputum, tracheal aspiration, and bronchial washings.

1. Rinsing the mouth with saline or water (but not mouthwash) may reduce contamination with normal oropharyngeal flora.

2. Encourage deep cough with expectoration of the sputum into a sterile specimen collection cup that is labeled with the patient's name.

3. Do not send saliva (spit) for culture.

4. When the patient is unable to cough productively, notify the physician. An alternative method may be ordered, such as:

a. Induced sputum. This is done by a respiratory therapist on the orders of the physician. Involuntary deep coughing is induced by irritation.

b. Tracheal aspiration. The trachea is gently irritated with a small lumen suction catheter, which causes deep, productive coughing. Also, the specimen may be aspirated with a syringe.

c. Bronchial washings. These are done by the physician in the operating room at the time of bronchoscopic examination. Sputum following bronchoscopy can be very productive for the recovery of mycobacteria.

5. A small amount of sputum is all that is required, but it must be sputum and not oral secretions.

6. Three sputa collected on consecutive days is recommended for the recovery of mycobacteria.

Specimens of Wound Exudate. Follow these steps for using a sterile transport swab in collecting wound exudate specimens.

1. Gently cleanse the area, using dry, sterile gauze to remove any contaminants.

2. Using a sterile bacterial culture collection system, introduce deeply enough to obtain a moist specimen; replace the swab in the container. Do not break the container.

3. Store at room temperature.

Urine for Culture. When a urine culture is ordered, follow these steps for collecting a clean-catch specimen.

1. Explain carefully to patients the mechanics of midstream collection and the importance of collecting an uncontaminated specimen. Teach them how to handle the specimen container to keep it sterile.

2. A clean-catch specimen is necessary to confirm the presence or absence of infecting organisms in urine. The specimen must be free of any contaminating matter that might be present on the genital organs; therefore, patients should be urged to follow the steps outlined below.

a. Instructions for the Female Patient.

  • If you are menstruating, first insert a fresh tampon or use cotton to stop the flow.
  • Separate the skin folds around the urinary opening.
  • Wash the urinary opening and its surroundings from front to back with a sterile antiseptic pad.
  • Begin urinating into the toilet, making sure you keep the skin fold apart with the fingers of one hand.
  • Wait until the urine stream is well established before moving the container into the path of the stream to catch the rest of the urine. Do not touch the container to the genital area.

b. Instructions for the Male Patient.

  • Wash the end of the penis well with soapy water. Let it dry.
  • Begin urinating into the toilet. Wait until the urine stream is well-established before moving the container into the path of the stream to catch the rest of the urine. Do not touch the container to the genital area.

3. Cleansing agents, such as soap or detergent, must be rinsed away from the urethral area before the specimen is collected.

4. A urine specimen from a catheterized patient is obtained by using a sterile 21- to 23-gauge needle and a 3-mL syringe. Prepare an area on the distal end of the rubber catheter with an antiseptic sponge. Insert the needle at a 45° angle, pointed toward the drainage tubing. If urine is not obtained, try lifting the catheter tubing carefully. If necessary, kink the tubing three inches from the catheter and hold in place with a rubber band until urine is visible.

5. Urine for culture must be transferred to a urine transport tube that contains preservative immediately after collection.

Note: Do not collect urine specimens from a drainage bag.

Stool for Culture. When collecting stool specimens, follow these guidelines.

1. A small amount is all that is required, about the size of a walnut. If several different types of cultures are requested, submit a walnut-sized sample for each. Place the specimen in stool culture transport medium (C&S vial).

2. When stool specimens are not readily obtainable, rectal swabs are acceptable; however, it must be indicated whether the specimen is a stool or a rectal swab. Place the swab in stool culture transport medium (C&S vial).

Use of Sterile Swab Bacterial Collection Kit

The swab system is guaranteed sterile until the seal is broken. Directions for use:

1. Peel open and remove the swab from the package.

2. Remove the cap/swab stick from the tube.

3. Collect the appropriate specimen and put the cap/swab into the tube. Push the cap to bring the swab into contact with the transport medium.

4. Print the patient's name and the culture site on the specimen tube.

5. Place the specimen in a specimen bag and put the completed test request form in the side pouch.

6. Store it at room temperature.

7. Send specimen to the laboratory.

Normal Flora. The common practice in microbiology is to identify “significant” organisms from cultures. Significance is determined in part by the quantitation of an organism relative to other organisms present, the “pathogenicity” of isolates, and the site from which the specimen was obtained. When the organisms present are known to be part of the expected flora from a particular body site, the result reported is often “routine (site) flora”. The following list representative flora from various body sites.

Skin Flora

  • α-Hemolytic (Alpha-hemolytic) Streptococcus species
  • Bacillus species
  • Coagulase-negative Staphylococcus species
  • Corynebacterium species

Respiratory Flora

  • α-Hemolytic (Alpha-hemolytic) Streptococcus species not Enterococcus
  • Corynebacterium species
  • Neisseria species
  • Nonhemolytic Streptococcus species

The following potential pathogens may be part of the routine flora if not predominating:

  • Coagulase-negative Staphylococcus species
  • Haemophilus influenzae
  • Haemophilus parainfluenzae
  • Moraxella catarrhalis
  • Neisseria meningitidis
  • Streptococcus pneumoniae

Genitourinary Tract Flora

  • α-Hemolytic (Alpha-hemolytic) Streptococcus species not Enterococcus
  • Coagulase-negative Staphylococcus species (if not predominating)
  • Corynebacterium species
  • Lactobacillus
  • Nonhemolytic Streptococcus species


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