Specimens such as sputum, body fluids and feces are commonly used in microbiological tests, which contain diverse microorganisms and complex endogenous components. Mycobacterium tuberculosis has high nutritional requirements and slow growth. During culture, contaminants in specimens grow rapidly and consume nutrients, interfering with the detection of Mycobacterium tuberculosis. Therefore, specimens containing contaminants (e.g., sputum specimens, gastrointestinal specimens, bronchoalveolar lavage fluid, transbronchial aspirates) require pre-treatment prior to culture for specimen liquefaction and decontamination. Urine, autopsy tissues and other contaminated liquid specimens also need decontamination before culture. Specimens collected from sterile sites of the body without contamination, such as blood, cerebrospinal fluid and synovial fluid, can be directly centrifuged, and the sediment is used for inoculation.
Common digestion reagents for specimen pre-treatment include sulfuric acid, sodium hydroxide, N-acetyl-L-cysteine (NALC), benzalkonium bromide, oxalic acid, trypsin, dithiothreitol (DTT), etc. Sputasol (sputum digestion solution) mainly consists of DTT and phosphate salts, sterile-processed, and can be directly applied for homogenization of sputum specimens. For research use only. Not for clinical diagnosis or other applications.
Required Materials:
1. Sputum, body fluid, stool and other samples
2. Normal saline, PBS, culture medium
3. Inoculating loop, centrifuge tubes, centrifuge, scissors, mortar
Operating Procedures (For Reference Only):
(I) Sputum Specimens
1. Transfer approximately 5 mL of thick sputum into a labeled 50 mL centrifuge tube.
2. Add an equal volume of sputum digestion solution for homogenization. Vortex for 20 seconds, then incubate at 30–37 °C for 15–20 minutes.
3. Add sterile PBS (pH~6.8) up to 50 mL, centrifuge at 3000 r/min for 15 minutes.
4. Discard the supernatant, then resuspend the pellet in 1–3 mL of PBS.
(II) Body Fluid Specimens
1. For sterile body fluids: directly take 10 mL fluid into a centrifuge tube for centrifugation, and inoculate the sediment.
2. For contaminated body fluids: process following the protocol for sputum specimens.
(III) Tissue Specimens
1. Take 1 g tissue, cut into small pieces with scissors, add 5 mL culture medium, homogenize using a mortar, then transfer the homogenate into a 50 mL centrifuge tube.
2. Follow the subsequent steps of the sputum specimen protocol.
(IV) Fecal Specimens
1. Add 1 g feces into a 50 mL centrifuge tube, mix with 5 mL culture medium thoroughly.
2. Follow the subsequent steps of the sputum specimen protocol.
Precautions:
1. Sputum samples are preferably collected in the early morning. The subject should rinse the mouth thoroughly with cool boiled water before sampling to avoid contamination by normal oral flora.
2. For urine samples, the midstream portion of the first morning urine is recommended.
3. Samples should be delivered to the laboratory immediately after collection.
4. If the sample is used for the detection of Mycobacterium tuberculosis, an adequate volume of sputum is required.
5. Patients with pulmonary infection may be complicated with bacteremia; blood culture should be performed simultaneously.
6. Use the reagent promptly after opening the package to avoid compromised experimental results.
