Sputum is material coughed up from the lungs and expectorated (spit out) through the mouth. A sputum culture is done to find and identify the microorganism causing an infection of the lower respiratory tract such as pneumonia (an infection of the lung). If a microorganism is found, more testing is done to determine which antibiotics will be effective in treating the infection.
A person with a fever and a continuing cough that produces pus-like material and/or blood may have an infection of the lower respiratory tract. Infections of the lungs and bronchial tubes are caused by several types of microorganisms, including bacteria, fungi (molds and yeast), and viruses. A chest x ray provides visual evidence of an infection; a culture can grow the microorganism causing the infection. The microorganism is grown in the laboratory so it can be identified, and tested for its response to medications, such as antifungals and antibiotics.
Based on the clinical condition of the patient, the physician determines what group of microorganism is
A portion of the sputum is smeared on a microscope slide for a Gram stain. Another portion is spread over the surface of several different types of culture plates, and placed in an incubator at body temperature for one to two days.
A Gram stain is done by staining the slide with purple and red stains, then examining it under a microscope. Gram staining checks that the specimen does not contain saliva or material from the mouth. If many epithelial (skin) cells and few white blood cells are seen, the specimen is not pure sputum and is not adequate for culture. Depending on laboratory policy, the specimen may be rejected and a new specimen requested. If many white blood cells and bacteria of one type are seen, this is an early confirmation of infection. The color of stain picked up by the bacteria (purple or red), their shape (such as round or rectangular), and their size provide valuable clues as to their identity and helps the physician predict what antibiotics might work best before the entire test is completed. Bacteria that stain purple are called gram-positive; those that stain red are called gram-negative.
During incubation, bacteria present in the sputum sample multiply and will appear on the plates as visible colonies. The bacteria are identified by the appearance of their colonies, by the results of biochemical tests, and through a Gram stain of part of a colony.
A sensitivity test, also called antibiotic susceptibility test, is also done. The bacteria are tested against different antibiotics to determine which will treat the infection by killing the bacteria.
The initial result of the Gram stain is available the same day, or in less than an hour if requested by the physician. An early report, known as a preliminary report, is usually available after one day. This report will tell if any bacteria have been found yet, and if so, their Gram stain appearance—for example, a gram-negative rod, or a gram-positive cocci. The final report, usually available in one to three days, includes complete identification and an estimate of the quantity of the bacteria and a list of the antibiotics to which they are sensitive.
To look for mold or yeast, a fungal culture is done. The sputum sample is spread on special culture plates that will encourage the growth of mold and yeast. Different biochemical tests and stains are used to identify molds and yeast. Cultures for fungi may take several weeks.
Viruses are a common cause of pneumonia. For a viral culture, sputum is mixed with commercially-prepared animal cells in a test tube. Characteristic changes to the cells caused by the growing virus help identify the virus. The time to complete a viral culture varies with the type of virus. It may take from several days to several weeks.
Tuberculosis is caused by a slow-growing bacteria called Mycobacterium tuberculosis. Because it does not easily grow using routine culture methods, special procedures are used to grow and identify this bacteria. When a sputum sample for tuberculosis first comes into the laboratory, a small portion of the sputum is smeared on a microscope slide and stained with a special stain, called an acid-fast stain. The stained sputum is examined under a microscope for tuberculosis organisms, which pick-up the stain, making them visible. This smear is a rapid screen for the organism, and allows the physician to receive a preliminary report within 24 hours.
To culture for tuberculosis, portions of the sputum are spread on and placed into special culture plates and tubes of broth that promote the growth of the organism. Growth in broth is faster than growth on culture plates. Instruments are available that can detect growth in broth, speeding the process even further. Growth and identification may take two to four weeks.
Other microorganisms that cause various types of lower respiratory tract infections also require special culture procedures to grow and identify. Mycoplasma pneumonia causes a mild to moderate form of pneumonia, commonly called walking pneumonia; Bordetella pertussis causes whooping cough; Legionella pneumophila, Legionnaire's disease; Chlamydia pneumoniae, an atypical pneumonia; and Chlamydia psittaci, parrot fever.
Pneumocystis carnii causes pneumonia in people with weakened immune systems, such as people with AIDS. This organism does not grow in culture. Special stains are done on sputum when pneumonia caused by this organism is suspected. The diagnosis is based on the results of these stains, the patient's symptoms, and medical history.
Sputum culture is also called sputum culture and sensitivity.
The specimen for culture should be collected before antibiotics are begun. Antibiotics in the person's system 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 having anything to eat or drink. The patient should first rinse his or her mouth with water to decrease mouth bacteria and dilute saliva. Through a deep cough, the patient must cough up sputum from within the chest. 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 want the patient to collect sputum samples on three consecutive mornings.
If coughing up sputum is difficult, a health care worker 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. Sputum may also be collected by a physician during a bronchoscopy procedure.
If tuberculosis is suspected, collection of sputum should be carried out in an isolation room, with all attending healthcare workers wearing masks.
Sputum from a healthy person would have no growth on culture. A mixture of microorganisms, however, normally found in a person's mouth and saliva often contaminate the culture. If these microorganisms grow in the culture, they may be reported as normal flora contamination.
The presence of bacteria and white blood cells on the Gram stain and the isolation of a microorganism from culture, other than normal flora contamination, is evidence of a lower respiratory tract infection.
Microorganisms commonly isolated from sputum include: Streptococcus pneumoniae Haemophilus influenzae Staphylococcus aureus Legionella pneumophila Mycoplasma pneumonia Klebsiella pneumoniae Pseudomonas aeruginosa Bordetella pertussis, and Escherichia coli.
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Koneman, Elmer W., et al. Color Atlas and Textbook of Diagnostic Microbiology. 4th ed. Philadelphia: J. B. Lippincott Co., 1992.
Pagana, Kathleen Deska. Mosby's Manual of Diagnostic and Laboratory Tests. St. Louis: Mosby, Inc., 1998.
Shulman, Stanford T., et al., eds. The Biologic and Clinical Basis of Infectious Diseases. 5th ed. Philadelphia: W. B. Saunders Co., 1997.
Buono, Nancy J., et al. "The Fight Against TB: A New Laboratory Arsenal Fights Back." Medical Laboratory Observer (Aug. 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 (Dec. 1997): 308-321.
Nancy J. Nordenson
Acid-fast stain—A special stain done to microscopically identify the bacteria that cause tuberculosis.
Culture—A laboratory test done to grow and identify microorganisms causing infection.
Gram stain—Microscopic examination of a portion of a bacterial colony or sample from an infection site after it has been stained by special stains. Certain bacteria pick-up and retain the purple stain; these bacteria are called gram-positive. Other bacteria loose the purple stain and retain the red stain; these bacteria are called gram-negative. The color of the bacteria, in addition to their size and shape, provide clues as to the identity of the bacteria.
Normal flora—The mixture of bacteria normally found at specific body sites.
Pneumonia—An infection of the lungs.
Sputum—Material coughed up from the lower respiratory tract and expectorated through the mouth.
Sensitivity test—A test that determines which antibiotics will kill the bacteria that has been isolated from a culture.