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Sputum Culture

Sputum culture


A sputum culture is a microbiology test performed to isolate and identify microorganisms causing an infection of the lower respiratory tract.


Infections of the lungs and bronchial tubes are caused by several pathogenic microorganisms, including bacteria , fungi , viruses , and parasites which are responsible for a variety of diseases including pulmonary tuberculosis , bacterial pneumonia , viral and mycoplasmal (atypical) pneumonia, chronic bronchitis and bronchiectasis. A chest x-ray provides visual evidence suggestive of a respiratory infection; a culture identifies the microorganism causing the infection.


For best results, the specimen should be obtained prior to initiating any therapy. The health care worker should wear a mask to avoid inhalation of airborne pathogens that may be introduced into the air during the collection process. The specimen should be taken to the laboratory within 30 minutes of collection.


Sputum collection

Based on the clinical condition of the patient, the physician determines what group of microorganism is likely to be causing the infection, and then orders one or more bacterial, viral, or fungal cultures. For all culture types, the sputum must be collected into a sterile container and care must be taken to minimize specimen contamination by the normal flora of the mouth and throat. Once in the laboratory, each culture type is handled differently.

Sputum must be expectorated from the bronchi by deep forceful coughing. The recovery of sputum is best in the early morning. The patient should rinse his or her mouth by gargling with water prior to coughing. Taking deep breaths and lowering the head helps bring up the sputum. Sputum must not be held in the mouth but immediately spat into a sterile container. For tuberculosis, the physician may request sputum samples from the patient on three consecutive mornings. In some cases the patient will be unable to produce the sputum, and an aerosol of saline will be needed to loosen the congestion. In such cases the sputum can be aspirated using a suction device.

In some cases sputum will be collected during a bronchoscopy or endotrachial procedure. These specimens, like coughed-up sputum, will be contaminated with normal flora from the mouth or throat and are not suitable for anaerobic culture. When anaerobic infection is suspected, the physician will collect the sample by transtrachial aspiration. These specimens and those collected by thoracentesis (removal of pleural fluid via chest wall puncture) are not contaminated by upper respiratory flora and are suitable for both aerobic and anaerobic culture.

Gram stain

The Gram stain is always performed when sputum is submitted for culture. Additional stains such as the acid-fast stain for tuberculosis are performed only upon request. The Gram stain is used to determine the acceptability of the specimen for culture, and aids the technologist in selecting special growth media that might be needed. Almost all bacteria are described by their Gram stain characteristics: color (purple or pink), shape (cocci or bacilli), and size; arrangement, presence, or absence of spores.

The Gram stain is performed by the following method:

  • A portion of the sputum is smeared onto a glass microscope slide, air dried, and heat-fixed.
  • The slide is flooded with crystal violet stain, which is allowed to set for 30-60 seconds.
  • The crystal violet is rinsed off with a gentle stream of water.
  • The slide is flooded with Gram's iodine, which is allowed to set for 60 seconds.
  • The iodine is removed with a gentle stream of water.
  • The slide is decolorized by rinsing with 95% ethanol, drop by drop, until the alcohol rinses clear.
  • The slide is flooded with safranin, which is allowed to set for 30 seconds.
  • The safranin is rinsed off with a stream of water.
  • The excess water is removed by blotting with bibulous paper.
  • The slide is allowed to air dry.
  • The slide is observed under the microscope using both low power and oil immersion lenses.

Gram-positive cells retain the crystal violet stain and appear dark purple, while gram-negative cells do not. Gram-negative bacteria are counterstained by the safranin and appear pink. Gram staining also helps determine the integrity of the sputum specimen. The presence of many epithelial cells and few white blood cells indicates a contaminated sample, one not adequate for culture. The presence of many white blood cells and bacteria in the specimen signifies an acceptable sample for culture, and provides a preliminary indication of infection.

Bacterial culture

Streptococcus pneumoniae is the most common pathogen causing bacterial pneumonia, but almost any organism can be implicated. Other bacterial isolates include Staphylococcus aureus, Haemophilus influenzae, E. coli, Enterobacter spp., Klebsiella pneumoniae, Pseudomonas aeruginosa, Legionella pneumophilia, Mycoplasma pneumoniae, and Mycobacterium tuberculosis.

Using a sterile swab, a portion of the sputum sample is transferred to each plate and then streaked for isolation using a sterilized inoculating loop. Cultures should be performed using sheep blood agar, chocolate (heated blood) agar, and MacConkey agar. All plates are incubated in 5-10% carbon dioxide at 36°C for 24 hours. Plates are examined for growth and colonies are Gram stained and subcultured (transferred) to appropriate media for biochemical identification. Plates showing normal flora are incubated for an additional day. If L. pneumophilia is suspected, the sputum should be plated on BCYE-alpha agar (buffered charcoal-yeast extract with alpha-ketogluterate). Plates are cultured in air or 2.5% carbon dioxide for three to five days at 36°C. Small convex gray colonies are stained with fluorescent-labeled antibody specific for L. pneumophilia. If atypical pneumonia is suspected, the sputum is plated on a medium that supports the growth of Mycoplasma pneumoniae such as SP4. The plates are incubated in air at 36°C. Colonies grow slowly and are almost microscopic. They are identified by cutting a block of agar, and staining it with Dienes stain. M. pneumoniae demonstrate their typical fried egg appearance. Alternatively, the colonies can be identified using a fluorescent-labeled antibody specific for M. pneumoniae. Plates showing no growth are held up to four weeks before reporting as negative. Because of the long culture time, Mycoplasma pneumoniae is not usually cultured. Infections with this organism are diagnosed by enzyme immunoassay for IgM antibodies produced against the organism. A high concentration of these antibodies indicates acute infection.

The results of the initial Gram stain are available the same day, or in less than an hour if requested by the physician. A preliminary report on the status of the culture is usually available after one day. This report notes the presence or absence of bacterial growth, the Gram stain of any organism isolated, and presumptive identification (if possible). The final report, usually available in one to three days, includes organism identification, an estimate of the quantity of the bacteria, and the results of the antibiotic sensitivity testing.

Fungal culture

Fungal cultures of sputum are used primarily to identify the presence or Histoplasma capsulatum, Coccidioides immitis, Cryptococcus neoformans, Blastomyces dermatitidis, and Paracoccidioides brasiliensis. However, opportunistic yeast or fungi such as Candida spp., Aspergillus spp., and Fusarium spp. can cause pneumonia in immunocompromised patients. Definitive diagnosis depends upon the presence of clinical signs of pulmonary infection, a positive chest x-ray, and laboratory identification of the organism. In addition, histological results of open lung biopsy may reveal the organisms by microscopic examination of stained tissue. For the laboratory identification, the sputum is first examined microscopically via a direct smear using one or more of the following methods:

  • 10% KOH preparation
  • India ink preparation
  • calcofluor white stain
  • Gram stain
  • Kinyoun's acid fast stain
  • lactophenol cotton/aniline blue wet mount

In many cases, direct smears are sufficient to establish a preliminary diagnosis based upon the appearance of the yeast seen or the characteristic spores and hyphae produced by the fungus. If a fungal culture is ordered or suspected from direct smear, a portion of the sputum is transferred to an appropriate growth medium such as Sabauroad-dextrose agar or brain-heart infusion-blood agar. When infection with a dimorphic fungus is suspected (i.e., infection by Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Paracoccidioides brasiliensis), the cultures are incubated at both 25°C and at 36°C in order to demonstrate conversion of the mold form at 25°C to the yeast form at 36°C. Rapidly growing fungi such as Coccidioides immitis may appear in three to four days, while slow growing fungi such as Histoplasma capsulatum may require several weeks. For this reason cultures are held for four weeks before signing out as negative. Identification is made on the basis of growth rate, appearance and color of colonies, and microscopic appearance demonstrating characteristic hyphae and/or spores. Microscopic evaluation is performed by transferring a loop or drop of the fungal culture onto a glass slide, adding one to two drops of lactophenol cotton blue stain, placing a coverglass over the material, and examining under the microscope. In some cases, a culture filtrate is prepared and tested using antibodies to the exoantigens produced by the dimorphic fungi. This is done by the double immunodiffusion technique that permits direct comparison of precipitation reactions for the cultures and known fungal antigens controls.

Viral culture

Viruses that are a common cause of respiratory tract infection include adenoviruses, rhinoviruses, influenza , parainfluenza, enteroviruses, respiratory syncytial virus, retroviruses, herpes simplex, and cytomegalovirus. Because viruses need host DNA to replicate, they will not grow on artificial media. Therefore, viral cultures are inoculated onto cell cultures. These may be cancer cells grown in monolayers in the laboratory or cells taken from animal tissues and used to prepare a monolayer. Cell types commonly used for viral isolation are human diploid fibroblasts (HDF), HEp2 cells (epithelial cancer cells from the larynx), A549 cells (human lung cancer cells); primary monkey kidney cells (PMK), and rabbit kidney cells (RK). Cell cultures are inoculated and allowed to grow for one to three days at 36°C in 5-10% carbon dioxide. Within one to three days a characteristic cytopathic effect (CPE) can be seen by observing the cells under a microscope.

Mycobacterial culture

Mycobacterium tuberculosis is a respiratory infection commonly transmitted via the air to the lungs, where it thrives, causing fever , cough, and blood spitting. Pneumonia can also be caused by M. bovis, M. avium complex, and M. kansasii.

Most mycobacteria that cause pneumonia such as Mycobacterium tuberculosis grow very slowly requiring from two to six weeks for culture. Mycobacterium tuberculosis stains very poorly with Gram stain; therefore, acid-fast (light microscopy) and fluorescent staining methods are used to identify mycobacteria by direct microscopic examination of sputum. A smear can provide a presumptive diagnosis of mycobacterial disease; confirm that cultures growing on media are acid-fast; and demonstrate a patient's response to antibiotic therapy from post-treatment sputum cultures.

There are three staining methods commonly employed to test for mycobacteria. Two acid-fast stains, Ziehl-Neelsen and Kinyoun use light microscopy; the third method uses auramine or a combination of auramine and rhodamine and requires a fluorescent microscope. The advantage of fluorescent staining resides in the ability to examine much larger areas of the smear in a shorter period of time. Fluorescent staining is more sensitive and detects approximately 18% more cases. Its disadvantage is that the stain also detects organisms that are non-viable. While a positive finding provides a basis for initiating antibiotic treatment, the sensitivity of the direct smear is highly variable. Therefore, when acid-fast culture is requested, all sputum samples (meeting the laboratory's criteria for sputum) are cultured, even when the direct smear exam shows no evidence of acid-fast bacteria.

Sputum for culture of mycobacteria must be decontaminated. This is commonly done by adding a mucolytic agent such as N-acetyl-L-cysteine and a clearing agent, sodium hydroxide, to an equal amount of the sputum. The suspension is mixed and allowed to stand for 15 minutes, then phosphate buffered saline is added and the sample is centrifuged. The supernatant is decanted and the remaining sample is resuspended with bovine albumin and used for culture.

An acid-fast culture can detect as few as 10 to 100 CFU/mL of sputum. Culture media may be enriched with egg, albumin, or a mixture of salts, vitamins , cofactors, fatty acids, glucose, and other nutrients. Media also contain malachite green or antibiotics to retard the growth of other bacteria and yeast. The sputum should be inoculated on at least one solid medium such as American Thoracic Society or Lowenstein-Jensen and one liquid medium such as Middlebrook 7H9. Cultures are set up at several different temperatures and examined daily for several weeks to characterize the rate of growth. Colonies are subcultured and transferred to appropriate media for biochemical identification. This process can take several more weeks and therefore, other identification methods are often performed concurrently. These include analysis of cell wall fatty acids by either gas or high-performance liquid chromatography (HPLC) and DNA probe testing. High-performance liquid chromatography can rapidly identify the species of mycobacteria from cultures, but according to the CDC, laboratories that use HPLC report that the method requires a highly experienced technologist and usually takes a long time (about six months) for initial incorporation into their laboratories. DNA probe testing can be done on as little as a single colony and demonstrates a far more rapid turnaround time than biochemical testing. Probes are available for many species but not all mycobacterium, and may be falsely negative (i.e., a low hybridization rate) if contaminating organisms are present. For these reasons, this method is used in conjunction with biochemical testing.

Other microorganisms that cause various types of lower respiratory tract infections also require special culture or staining procedures to grow and identify. For example, Pneumocystis carinii in bronchial lavage, sputum, or lung biopsy samples is detected by observing the organisms with special stains such as methenamine silver stain or a fluorescent monoclonal antibody stain. Pneumocystis carnii causes pneumonia in people with weakened immune systems, such as people with AIDS , and does not grow in culture. The diagnosis is based on the results of these stains, the patient's symptoms, and medical history.

Antibiotic susceptibility testing

With the exception of Streptococcus pneumoniae or other strep which are sensitive to penicillin and related antibiotics, antibiotic susceptibility testing is preformed on all isolates. Susceptibility testing is performed for most other organisms by the microtube broth dilution or Kirby Bauer method. The selection of antibiotics for testing depends upon the organism isolated (i.e., gram-negative, gram-positive, aerobe, anaerobe, mycobacteria, or yeast).

The Kirby-Bauer antibiotic susceptibility test method is commonly used for gram-positive and gramnegative aerobic bacteria. Antibiotic disks are placed on a plate containing a clear medium such as Mueller-Hinton agar that has been swabbed uniformly with a standardized broth suspension of a pure culture of the bacteria to be tested. The plate is then incubated at 36°C for 18-24 hours. The zone of no growth (zone of inhibition) around each disk is measured, and compared to predetermined cutoffs for each antibiotic concentration used. If the zone size equals or exceeds the cutoff, the organism is susceptible. If not, the organism is resistant. The results are reported as sensitive (organism inhibited by antibiotic), intermediate (inconclusive effect of antibiotic on organism), or resistant (organism not inhibited by the antibiotic).


The specimen for culture should be collected before antibiotics are begun as the antibiotics may prevent microorganisms present in the sputum from growing in culture. The best time to collect a sputum sample is early in the morning, before the patient has had anything to eat or drink. The patient should first rinse his or her mouth with water to decrease mouth bacteria and dilute saliva.

If coughing up sputum is difficult, a nurse or respiratory therapist can have the patient breathe in sterile saline produced by a nebulizer. This nebulized saline coats the respiratory tract, loosening the sputum, and making it easier to cough up.

It is also useful to obtain information concerning travel to foreign countries, exposure to animals, and diagnosed or suspected immunosuppressive disease.


There are no specific requirements for care after obtaining the specimen. However, if the patient is found to have tuberculosis, several measures will be taken to prevent the spread of this airborne disease.


There are no complications associated with this test.


Sputum from a healthy person will have no growth on culture. However, a mixture of microorganisms, typically found in a person's mouth and throat often contaminates the culture. In such cases the report will indicate the presence of normal flora contamination.

The preliminary report will note the presence of bacteria and white blood cells on the Gram stain and describe the appearance of the bacteria and the number of cells seen. Preliminary culture results will identify the Gram stain or presumptive identification of any organisms recovered.

Health care team roles

Sputum culture is requested by a physician. A nurse or respiratory therapist will provide instructions to the patient for collecting a sputum sample. Bronchoscopy, transtracheal aspiration, bronchial lavage or brushing, and thoracentesis are preformed by a physician. Clinical laboratory scientists/medical technologists who specialize in microbiology will perform the culture and antibiotic sensitivity tests.


Cocci —Spherical shape bacterium.

Bacilli —Rod-shaped bacterium.

Bronchiectasis —A chronic dilation of one or more bronchi.

Epithelial cells —Skin cells.

Mycobacterium —A slender acid-fast organism resembling Mycobacterium tuberculosis.



Chernecky, Cynthia C., and Berger, Barbara J. Laboratory Tests and Diagnostic Procedures. 3rd ed. Philadelphia, PA: W. B. Saunders Company, 2001.

Isada, Carlos M., et al. Infectious Diseases Handbook. Hudson, Ohio: Lexi-Comp., 1995, pp 76-79.

Kee, Joyce LeFever. Handbook of Laboratory and Diagnostic Tests. 4th ed. Upper Saddle River, NJ: Prentice Hall, 2001.

Pagana, Kathleen D., and Timothy J. Pagana. Manual of Diagnostic and Laboratory Tests. St. Louis: Mosby, 1998, pp 681-83.

Shulman, Stanford T., et al., eds. The Biologic and Clinical Basis of Infectious Diseases. 5th ed. Philadelphia: W. B. Saunders Co., 1997, pp 123, 530.


Buono, Nancy J., et al. "The Fight Against TB: A New Laboratory Arsenal Fights Back." Medical Laboratory Observer (August, 1996): 38-40, 42, 44, 46, 48, 50, 52.

Skerritt, Shawn J. "Diagnostic Testing to Establish a Microbial Cause is Helpful in the Management of Community-Acquired Pneumonia." Seminars in Respiratory Infections (December, 1997): 308-321.


Centers for Disease Control. <>.

Laboratory Corporation of America. <>. 2001.

Victoria E. DeMoranville

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