B. Culture examination
Culture examination takes a long time to get results and requires much more materials, equipment, and skilled personnel. Hence, it is expensive, and the cases so found are less infectious than smear-positives. However, the sensitivity and specificity of the culture examination cannot be underestimated, permitting diagnosis of paucibacillary cases before they break down to highly infectious smear-positive cases. Sputum culture screens approximately 20-25% more cases from symptomatics in the highly prevalent countries than smear examination. In developed countries, however, perhaps no more than one half (or two thirds) of the cases are smear-positives at the time of diagnosis, the remainder being detected only by culture. For this reason, culture facility is necessary for case-finding in the developed countries, but it is less important in the developing countries. The main reason why most of the newly found cases in developing countries are smear-positive, is that they only go to the health institutes after their symptoms have been severely advanced.
Culture is essential for drug susceptibility tests, which are helpful for the selection of effective retreatment regimes.
There are a variety of culture methods, which have different advantages according to the circumstances. Each country should select a method better suited to the local situation taking into account of availability of financial resources, facility, laboratory personnel, etc.
1. Pretreatment of specimens
For decontamination, sputum specimens can be treated either with acids, such as 5% oxalic acid or 4-8% sulfuric acid or with alkali, such as 2-4% sodium hydroxide and, for homogenization, N-acetyl L-cysteine (or dithiothreitol) with NaOH or trisodium phosphate-benzalkonium chloride is most widely used. Special emphasis must be given to the selection of digestant-decontaminant and it's exposure time and temperature because viability of organisms and decontamination efficiency are seriously affected by these factors. As seen in Figure 2. decontaminating agents kill a considerable number of tubercle bacilli, thus care must be taken not to treat specimen too harshly.
2. Inoculation onto culture medium
There are basically two different inoculation methods of decontaminated specimen onto medium, one is a simple direct inoculation method and the other, a concentration method. A variety of medium are using for culture examination. Broth based or agar based medium is used in some countries, but the most commonly used medium is egg-based Lowenstein-Jensen (L-J) medium, which has been accepted as an international standard medium, and acid-buffered Ogawa medium, which is useful for SC. These egg-based media contain glycerin that promote growth of M. tuberculosis, but to isolate M. bovis, pyruvic acid should be enriched instead of glycerin.
(a) Simple culture methods in most common use are a modified Ogawa method and Nassau oxalic acid method. In Ogawa method, sputum is digested and decontaminated with an equal amount of 4% NaOH and then inoculated onto acid-buffered (2-3% KH2PO4) Ogawa medium without centrifugation and neutralization, so it is simple and economical. Nassau method is also simple technique in which sputum adhered on swab stick is carefully decontaminated in 5% oxalic acid and the neutralized in 5% sodium citrate prior to inoculate onto medium. This technique together with 4% sulfuric acid (5 volumes of sputum) treatment is useful for isolation of tubercle bacilli from specimens contaminated with alkali resistant contaminants (for example. Pseudomonas spp.). The case-yield rate of oxalic acid method seems to be inferior to Ogawa method.
(b) Concentration method is performed by centrifuging decontaminated and digested specimen at more than 3000g after diluting with six volumes of a sterile distilled water (DW). The sediment is either directly inoculated onto acid-buffered medium or washed with a sterile DW by repeated centrifugation instead of neutralization before inoculating onto L-J medium.
As mentioned before, two smears screen nearly same number of positives found by a single culture, one must not bestow undeserved favour on sensitivity of culture.
The crowth of positive cultures are mostly observed after 3-4 weeks incubation at 37 ℃. As seen in Figure 3, 91.6% of 4 + cultures have been reported after three weeks of incubation, while 90.9% of less than 1 + culture result have been issued after six weeks of incubation.
