General Preanalytical Principles
To investigate the causes of respiratory infections, clinicians use sputum, nasal and throat swabs, bronchial washings (obtained via bronchoscopy), or tracheal aspirates.
The primary requirement for all types of biological samples is strict aseptic technique during collection and the use of sterile containers. The time interval between sample collection and laboratory inoculation must not exceed 1–2 hours. Exceeding this timeframe allows normal flora, which inevitably contaminate the sample, to actively multiply. Furthermore, putrefactive and fermentative processes may begin, rendering the test results invalid.
Sputum Collection (Lower Respiratory Tract)
Sputum should be collected in the morning during a coughing fit into a sterile, tightly sealed container. It is essential to ensure that saliva does not enter the container. If sputum production is poor, expectorants may be prescribed the day before the test.
Before collecting the sample, the patient must:
- Brush their teeth, but strictly without toothpaste, as it contains antibacterial components that can destroy the pathogen.
- Rinse the mouth with plain boiled water. This mechanically removes food particles, desquamated epithelium, and some oral microbes, reducing the risk of contamination.
Throat Swabs (Upper Respiratory Tract)
Unlike sputum collection, the patient must not rinse their mouth before a throat swab is taken. The procedure is performed either in a fasting state or 2–3 hours after eating or drinking.
The clinician depresses the tongue with a spatula and passes a sterile swab between the tonsillar pillars, across the uvula, and along the posterior pharyngeal wall. It is critical not to touch the lips, cheeks, or tongue to prevent the introduction of commensal flora from these areas. If purulent patches are present in the throat, the swab should be rubbed along the border between healthy and inflamed tissue—this is where bacteria accumulate most densely. After collection, the swab is immediately placed into a tube containing a specialized transport medium.
Alternative Collection Methods
If sputum is absent or insufficient, bronchial washings are used. During endoscopy, the bronchi are flushed with sterile normal saline, which is then aspirated and collected. However, this method has drawbacks: the secretions are heavily diluted, reducing bacterial concentration by a factor of 10 to 1,000 compared to standard sputum, and the saline itself may be detrimental to certain microbes. The procedure is also invasive and labor-intensive.
In cases of pulmonary infiltrates or abscesses, transthoracic needle aspiration is performed. This method is highly informative if the abscess has not yet ruptured into a draining bronchus. The obtained aspirate is inoculated onto nutrient media (blood agar, mannitol salt agar, Endo agar)—first rubbed onto a small area with a swab, and then streaked across the plate.
Interpretation of Results
Because the upper respiratory tract is heavily colonized by microorganisms under normal conditions, any sample passing through it inevitably becomes contaminated. Therefore, simply detecting a bacterium is insufficient; its quantity must be determined.
A semiquantitative method is used for evaluation by counting colonies on solid nutrient media:
- Grade I (up to 10 colonies) and Grade II (10–25 colonies) are considered normal carriage or accidental contamination.
- Grade III (from 50 colonies) and Grade IV (confluent growth) indicate that the isolated microbe is very likely the causative agent of the disease.
A definitive diagnosis is established by considering the degree of colonization, the predominance of a single microbial species (monoculture), smear microscopy findings, and the clinical picture of the disease.