Sputum Culture Health Article

Definition

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

Purpose

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.

Precautions

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.