The preparation of clinical specimens for mycobacterial culture followed the recommended guidelines (9 ). They were cultured on 3% Ogawa medium (Eiken Chemical, Tokyo, Japan) for 8 weeks and inoculated into liquid medium for 6 weeks. For liquid medium culture, Mycobacteria Growth Indicator Tube (MGIT) medium (Becton Dickinson, Franklin Lakes, NJ, USA) was used at PNUYH. At PNUH, the BacT/ALERT 3D system (Organon Teknika, Boxtel, the Netherlands) was used. All cultures that were positive for mycobacteria were routinely subjected to the polymerase chain reaction (PCR) assay (LG Life Science, Daejon, Korea) and MPT 64 test (Standard Diagnostics, Seoul, Korea) for differentiation between M. tuberculosis and NTM. Further species identification was performed at the Korean Institute of Tuberculosis (KIT, Osong, Korea) at the request of the charge physician. A multiplex PCR restriction fragment-length polymorphism assay was performed for the rpoB gene until 2009, after which the test method was changed to a PCR-based reverse line blot hybridization assay for the ITS gene (AdvanSure Mycobacteria GenoBlot Assay; LG Life Sciences, Seoul, Korea) at the KIT.
Ogawa medium
Manufactured by Eiken Chemical
Sourced in Japan
Ogawa medium is a laboratory culture medium used for the isolation and cultivation of mycobacteria, particularly for the detection of tuberculosis (TB) infection. It is a solid, egg-based medium that provides essential nutrients and growth factors required by mycobacteria. The medium is commonly used in mycobacteriology laboratories for the diagnosis and research of TB and other mycobacterial diseases.
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Lab products found in correlation
8 protocols using ogawa medium
1
Mycobacterial Culture and Identification
2
Radiographic Evaluation of Pulmonary Tuberculosis
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Chest radiographs and computerized tomography (CT) results of the patients were reviewed for the presence of cavities, and to determine the extent of the disease. If patients with PTB had a definite cavity on chest radiography, then a chest CT was not performed. In contrast, if it was unclear whether the patient with PTB had a cavity, then a chest CT was performed for confirmation. The extent of each lesion was categorized based on lobar involvement. Unilobar and multilobar PTB were defined as the involvement of ≤1 and ≥2 lobes, respectively. Acid-fast bacilli (AFB) smears were examined after auramine-rhodamine fluorescent staining, and they were graded on a scale from 0 to 4+. Mycobacterium tuberculosis culture was simultaneously performed using solid media, 3% Ogawa medium (Eiken Chemical, Tokyo, Japan), and liquid media in the mycobacteria growth indicator tube 960 system (BD Biosciences, Franklin Lakes, NJ, USA).
3
Bronchoscopic Diagnosis of Pulmonary Tuberculosis
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Bronchoscopy was conducted by the faculty staff of the pulmonology division in each hospital. The choice between bronchial washing (BW) and bronchoalveolar lavage (BAL) via bronchoscopy was based on the judgment of individual bronchoscopists. When the multiple lesions of suggested PTB were observed on CT, BW or BAL was conducted on the most severe lesion.
All the bronchoscopic specimens for the Acid-Fast Bacilli (AFB) smears and cultures were processed and pretreated as recommended [20 (link)]. The AFB smears were examined after auramine–rhodamine fluorescent staining, and graded on a scale from 0 to 4+.[21 (link)] All the specimens were simultaneously cultured on both solid and liquid media using 3% Ogawa medium (Eiken Chemical, Tokyo, Japan) and the mycobacterial growth indicator tube 960 system (Becton Dickinson, Mountain view, CA, USA). TB-PCR was conducted using either the Xpert MTB/RIF assay (Cepheid Inc., Sunnyvale, CA) or AdvanSure TB/NTM RT-PCR kit (LG Life Sciences, Seoul, Korea) [22 (link), 23 (link)].
All the bronchoscopic specimens for the Acid-Fast Bacilli (AFB) smears and cultures were processed and pretreated as recommended [20 (link)]. The AFB smears were examined after auramine–rhodamine fluorescent staining, and graded on a scale from 0 to 4+.[21 (link)] All the specimens were simultaneously cultured on both solid and liquid media using 3% Ogawa medium (Eiken Chemical, Tokyo, Japan) and the mycobacterial growth indicator tube 960 system (Becton Dickinson, Mountain view, CA, USA). TB-PCR was conducted using either the Xpert MTB/RIF assay (Cepheid Inc., Sunnyvale, CA) or AdvanSure TB/NTM RT-PCR kit (LG Life Sciences, Seoul, Korea) [22 (link), 23 (link)].
4
Mycobacterium tuberculosis Isolation Protocol
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Biopsy samples were homogenized in a sterile mortar and pestle and digested and decontaminated using the N-acetyl-l -cysteine (NALC)-NaOH method. Ziehl–Neelsen staining was performed on tissue smears directly after homogenization and after concentration. Specimens were cultured for M. tuberculosis on liquid modified Middlebrook 7H9 medium (BacT/ALERT MP System; bioMérieux, Durham, NC, USA) and/or on 3% solid Ogawa medium (Eiken, Tokyo, Japan).
5
Rapid TB Diagnosis via Xpert and PCR
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The Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) was performed and interpreted following the manufacturer’s instructions. For the Xpert assay, 2–3 mL of bronchial washing fluid without decontamination or concentration was used. The bronchial washing fluid and the Xpert sample reagent were mixed at 1:2 ratio and incubated at room temperature for 15 minutes. Then 2 mL of the mixture was transferred into an Xpert cartridge.
Remaining bronchial washing fluid was pretreated with equal volumes of 4% sodium hydroxide and centrifuged at 3,000 ×g for 20 minutes. A TB-PCR assay was performed using an AdvanSure TB/NTM real-time PCR kit (AdvanSure, LG Life Science, Daejeon, Korea), following the manufacturer’s protocols. Acid fast Bacilli smears were performed using auramine-rhodamine fluorescent staining and confirmed using Ziehl-Neelsen staining. Sediments were inoculated for culture on 3% Ogawa medium (Eiken Chemical, Tokyo, Japan) and in Mycobacteria Growth Indicator Tube medium (Becton Dickinson, Franklin Lakes, NJ, USA).
Remaining bronchial washing fluid was pretreated with equal volumes of 4% sodium hydroxide and centrifuged at 3,000 ×g for 20 minutes. A TB-PCR assay was performed using an AdvanSure TB/NTM real-time PCR kit (AdvanSure, LG Life Science, Daejeon, Korea), following the manufacturer’s protocols. Acid fast Bacilli smears were performed using auramine-rhodamine fluorescent staining and confirmed using Ziehl-Neelsen staining. Sediments were inoculated for culture on 3% Ogawa medium (Eiken Chemical, Tokyo, Japan) and in Mycobacteria Growth Indicator Tube medium (Becton Dickinson, Franklin Lakes, NJ, USA).
6
Pleural Fluid TB Diagnosis Protocol
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Pleural fluid specimen were obtained during thoracentesis and transported to the laboratory. It was concentrated but not decontamination then inoculated for MTB culture in the laboratory as recommended21 (link). The acid-fast bacilli (AFB) smears were examined after auramine–rhodamine fluorescence staining. All specimens including pleural fluid were simultaneously cultured on both solid and liquid media, 3% Ogawa medium (Eiken Chemical, Tokyo, Japan) using the mycobacteria growth indicator tube 960 system (Becton Dickinson, Mountainview, CA, USA). TB polymerase chain reaction was performed using the AdvanSure TB/NTM RT-PCR kit (LG Life Sciences, Seoul, Korea) according to the manufacturer’s protocol.
7
Standardized Sputum Examination for TB
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All IS specimens were processed and pretreated in accordance with the recommended standard techniques for mycobacterial examination of sputum samples26 (link). Acid-fast bacilli (AFB) smears of IS were examined following auramine–rhodamine fluorescence staining and graded on a scale of 0 to 4+27 (link). All IS specimens were simultaneously cultured on both solid media (SM) and liquid media (LM) using 3% Ogawa medium (Eiken Chemical, Tokyo, Japan) and the mycobacterial growth indicator tube 960 system (Becton Dickinson, Mountain View, CA, USA). All MTB isolates from the baseline IS specimens were sent for drug sensitivity testing to the Korean Institute of Tuberculosis (KIT), a World Health Organization-designated supranational reference laboratory. At KIT, resistance to anti-TB agents was defined as mycobacterial growth above 1% in Löwenstein-Jensen medium, using the proportional method.
8
Identification of Nontuberculous Mycobacteria
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All specimens submitted for NTM identification were obtained as tissue or serous/purulent discharge samples. NTM infection was determined by performing AFB liquid or solid culture. AFB staining and PCR test were also performed for some of the patients. AFB cultures were observed during a 6- to 8-week incubation before being considered negative. In Korea, when NTM is identified in culture samples from any laboratory at a medical institution, the specimen must be sent to the Korean National Tuberculosis Association (KNTA). Species identification and drug susceptibility tests are conducted by the KNTA, and the results are forwarded after 2 to 3 weeks to the respective institution. In the case of solid culture with 3.0% Ogawa medium (Eiken, Tokyo, Japan) was used for 8 weeks; in the case of liquid culture with 7 mL of mycobacterium growth indicator tube (Becton Dickinson, Sparks, MD, USA) was used for 6 weeks.
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