General rules of the pre-analytical phase
Sputum, nasal and throat swabs, bronchial secretions (after bronchoscopy), or tracheal aspirates are used to study the causes of respiratory infections.
The main requirement for all types of biomaterial is strict adherence to asepsis during collection and the use of sterile containers. The time interval between sample collection and its inoculation in the laboratory must not exceed 1–2 hours. Violating this timeframe will cause the normal microflora, which inevitably enters the sample, to actively multiply. In addition, putrefactive and fermentation processes may begin, rendering the test results invalid.
Sputum collection (lower respiratory tract)
Sputum is collected in the morning, during a coughing fit, into a sterile container with a tightly fitting lid. It is important to ensure that saliva does not enter the container. If sputum is difficult to expectorate, the patient is prescribed expectorants the day before the test.
Before collecting the material, the patient must:
- Brush their teeth, but strictly without toothpaste, as it contains antibacterial components capable of destroying 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, mouth rinsing is prohibited before taking a throat swab. The procedure is performed either on an empty stomach or 2–3 hours after eating or drinking.
The physician depresses the tongue with a spatula and runs a sterile swab between the tonsillar pillars, along the uvula, and the posterior pharyngeal wall. It is extremely important not to touch the lips, cheeks, and tongue to avoid introducing microflora from these areas. If there is purulent plaque in the throat, the swab is run along the border of healthy and inflamed tissue—this is where the most bacteria accumulate. After collection, the swab is immediately placed into a tube with a special transport medium.
Alternative collection methods
If there is no sputum or too little of it, bronchial washings are used. During endoscopy, the bronchi are washed with a sterile saline solution, which is then collected. However, this method has drawbacks: the secretion is heavily diluted, the bacterial concentration drops by 10–1000 times compared to regular sputum, and the saline itself can be detrimental to some microbes. The procedure is invasive and labor-intensive.
In pulmonary infiltrates or abscesses, transthoracic puncture is performed. This method is the most informative if the abscess has not yet ruptured into a draining bronchus. The obtained punctate is inoculated onto nutrient media (blood agar, mannitol salt agar, Endo agar)—first rubbed with a swab onto a small area, and then streaked across the plate.
Interpretation of results
Since the upper respiratory tract is normally heavily colonized by microorganisms, any material inevitably becomes contaminated when passing through it. Therefore, simply finding a bacterium is not enough—its quantity must be determined.
A semi-quantitative method is used for evaluation by counting colonies on solid nutrient medium:
- Grade I (up to 10 colonies) and Grade II (10–25 colonies) are regarded as carriage or accidental contamination.
- Grade III (from 50 colonies) and Grade IV (confluent growth) indicate that the isolated microbe is most likely the causative agent of the disease.
The final diagnosis is made taking into account the degree of contamination, the predominance of a single microbial species (monoculture), smear microscopy data, and the clinical picture of the disease.
Briefly: answers to topical questions
What cytological criteria in smear microscopy are used to evaluate the suitability of sputum for bacteriological culture?
The suitability of sputum for microbiological examination is assessed by the number of leukocytes and epithelial cells in a Gram-stained smear. Criteria for a quality sample when viewed under a microscope (low magnification, at least 20 visual fields):
- Neutrophils (segmented neutrophils) — more than 25 cells per field of view.
- Epithelial cells (squamous epithelium from the oropharynx) — less than 10 cells per field of view.
- Microorganisms — bacteria of a single morphological type dominate the preparation.
If the smear contains many epithelial cells and few leukocytes, the study is impractical due to the high risk of contamination with oral cavity contents.
Which pathogenic bacteria are most frequently the true causative agents of lower respiratory tract infections?
Sources list the following among bacterial pathogens of pneumonia:
Gram-positive bacteria include:
- pneumococci (Streptococcus pneumoniae);
- streptococci (Streptococcus pyogenes, Streptococcus viridans);
- staphylococci (Staphylococcus aureus, Staphylococcus haemolyticus).
Gram-negative bacteria include:
- Haemophilus influenzae (Haemophilus influenzae);
- Klebsiella (Klebsiella pneumoniae);
- Pseudomonas aeruginosa (Pseudomonas aeruginosa);
- Escherichia coli (Escherichia coli);
- Legionella (Legionella pneumophila).
Atypical flora include mycoplasmas and chlamydia. For hospital-acquired pneumonia, Acinetobacter spp. are also indicated.
Class and exam questions
Why is it forbidden to brush teeth with toothpaste when collecting sputum?
Toothpastes contain bactericidal substances that can destroy pathogenic microorganisms in the sample and lead to a false-negative result.
What is the difference in patient preparation for sputum collection versus a throat swab?
Before sputum collection, you need to rinse your mouth with boiled water to remove oral cavity flora. Before a throat swab, rinsing the mouth is strictly prohibited so as not to wash away the pathogen.
How to distinguish the true pathogen from normal microflora during sputum culture?
The main criterion is the number of grown colonies. Abundant growth (grades III and IV, more than 50 colonies) indicates the causative agent, while scant growth (up to 25 colonies) is considered contamination with normal flora.
What else to study on this topic
- Nutrient media for culture (blood agar, mannitol salt agar, Endo agar)
- Serial dilution method for determining exact microbial concentration
- Primary smear microscopy
- Transport media for throat swabs
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