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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 likely to be causing the infection, and then orders one or more specific types of cultures: bacterial, viral, or fungal (for yeast and molds). For all culture types, the sputum must be collected into a sterile container. The sputum specimen must be collected carefully, so that bacteria that normally live in the mouth and saliva don't contaminate the sputum and complicate the process of identifying the cause of the infectious agent. Once in the laboratory, each culture type is handled differently.
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 carinii 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.
It is possible that sputum cultures will eventually be replaced in the diagnosis of tuberculosis by newer molecular techniques. These advanced methods speed the diagnostic process as well as improve its accuracy. As of late 2002, four molecular techniques are increasingly used in laboratories around the world to diagnose TB. They include polymerase chain reaction to detect mycobacterial DNA in patient specimens; nucleic acid probes to identify mycobacteria in culture; restriction fragment length polymorphism analysis to compare different strains of TB for epidemiological studies; and genetic-based susceptibility testing to identify drug-resistant strains of mycobacteria.
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. Bronchoscopy, however, is not regarded as a cost-effective way of obtaining a useful sample.
If tuberculosis is suspected, collection of sputum should be carried out in an isolation room, with all attending healthcare workers wearing masks.
In addition to special precautions in collecting sputum when tuberculosis is suspected, workers in hospital laboratories must take extra care to inactivate unstained smear preparations that may contain M. tuberculosis. As of 2002, the most effective deactivation technique is the use of a solution of 5% phenol in ethanol.
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 micro-organisms 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.
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.
Sensitivity test — A test that determines which antibiotics will kill the bacteria that has been isolated from a culture.
Sputum — Material coughed up from the lower respiratory tract and expectorated through the mouth.
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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American Lung Association. 432 Park Avenue South, New York, NY 10016. (800) LUNG-USA. www.lungusa.org.
National Heart, Lung, and Blood Institute (NHLBI). P. O. Box 30105, Bethesda, MD 20824-0105. (301) 592-8573. www.nhlbi.nih.gov.