Recent Advances in the Bacteriological Examinations of Tuberculosis

　

Chiyoji Abe

Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association,

Kiyose, Tokyo 204-0022, Japan

　

　

1.　Isolation of mycobacteria from clinical specimens

　

Individual investigators have different reasons for cultivating Mycobacterium tuberculosis from clinical specimens in their laboratories.　The most common reasons for cultivating tubercle bacilli is to confirm a clinical diagnosis of tuberculosis.　Isolation of tuberculosis is time-consuming.　Therefore, laboratories today look toward more efficient systems to provide physicians and patients with faster results and to free laboratory personnel from unnecessary hours of tedious labor.　In the last ten years, new detection systems of mycobacteria in clinical specimens (which were based on liquid media) were developed.

　

1-1.　Septi-Chek AFB¹⁾

The biphasic culture system consists of a bottle containing the basic broth, a lyophilized supplement in a vial, and a slide enclosed in a plastic tube for subculturing.　The liquid phase consists of 20 ml of modified Middlebrook 7H9 broth in a screw-cap glass bottle.　The slide consists of Middlebrook 7H11 agar, egg-based 7H11, and chocolate agar.　An aliquot of any size (0.1-1.0 ml) filled with the pretreated sample is inoculated into the bottle.　The slide for the subculture is immediately screwed on the bottles, and it is incubated in a vertical position at 37℃, inverting and rotating it 2 to 3 times during the first week, and weekly for up to eight weeks in the event of a negative finding.

　

1-2.　BACTEC system²⁾

In the radiometric culture technique, the growth medium for culturing mycobacteria is supplemented with a substrate (palmitic acid) labeled with radioactive carbon (¹⁴C).　This substrate is utilized by mycobacteria and during metabolism, ¹⁴CO₂ is produced from the substrate.　The ¹⁴CO₂ in the head space gas is measured quantitatively on a scale from 0 to 999 by an automated instrument (BACTEC TB460).　The actual number is designated the growth index, or GI reading.　The radiometric technique can detect very small quantities of ¹⁴CO₂ which allows detection of mycobacterial growth at a very early stage.　BACTEC vials are read two or three times a week for the first 2 to 3 weeks thereafter for a total of 6 weeks.

　

1-3.　Mycobacteria Growth Indicator Tube (MGIT)³⁾

A fluorescent compound (ruthenium metal-complex) is embedded in silicone on the bottom of 16 x 100 mm round-bottom tubes.　The fluorescent compound is sensitive to the presence of oxygen which is dissolved in the broth. Initially, the large amount of dissolved oxygen quenches emissions from the compound and little fluorescence can be detected.　Later, actively respiring mycobacteria consume the dissolved oxygen and allow the fluorescence to be observed using a 365 mm UV transilluminator.　Growth can also be detected by the presence of non-homogenious turbidity or small grains or flakes in the culture medium.　Up to 0.5 ml of the pretreated sample can be inoculated into the tube.　Then they are mixed by inverting several times, and incubated at 37℃.　The MGIT tubes are read 2 to 3 times a week for 8 weeks or longer depending on the type of specimen.　Fluorescence is detected as a bright orange color in the bottom of the tube and also an orange reflection on the meniscus.

　

1-4.　BacT/Alert⁴⁾

BacT/Alert is an automated microbial detection system based on the colorimetric detection of CO₂ produced by growing microorganisms.　A CO₂ sensor is bonded to the bottom of each bottle and is separated from the broth medium by a semipermeable membrane.　The membrane is impermeable to most ions, including hydrogen ions, and to components of media and whole as well as degraded blood.　It is nearly impermeable to water but is freely permeable to CO₂.　Carbon dioxide produced by growing organisms diffuses the membrane into the sensor and dissolves in the water, thereby generating hydrogen ions according to the following equation:

CO₂ + H₂O ⇔ H₂CO₃ ⇔ H⁺ + HCO₃

Free hydrogen ions can interact with the sensor, which is blue to dark green in the alkaline state.　As CO₂ is produced and dissolves in the water, the concentration of hydrogen ions increases and the pH decreases.　This causes the sensor to become a lighter green and eventually yellow, which results in an increase of red light reflected by the sensor.　The system is a self-contained incubator, shaker, and detector.

　

1-5.　MB-REDOX⁵⁾

MB-REDOX is a modified, serum-supplemented Kirchner medium containing a colorless tetrazolium salt used for self-indicating microbial growth, because this tetrazolium salt is reduced by the redox system of the mycobacteria to a pink-, red- and

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