Several modified methods of the niacin test have been made with a reagent impregnated paper strip. As it was thought that various methods have to be improved in their stability, sensitivity and feasibility, the authors prepared a new niacin test paper and compared with the routine niacin test.
I) Method to prepare the test paper
Following reagents were used; 50% acqueous chloramine T, 60% KSCN in 8% citricacid and 10% para-aminosalicylic acid (PAS) in 95% ethanol. Filter paper was cut into 50mm by 3mm strips. Acryl sheet was cut into the same size. One set of our test paper needs two paper strips and one acryl sheet. Using a capillary pipette, one drop of 60% KSCN in 8% citric acid and 3 drops of 50% acqueous chloramin T were added to the strip (a). Two reagents were not allowed to diffuse into one another on the paper strip. One drop of 10% PAS in 95% ethanol was added to the strip (b). Acryl sheet was placed between the two paper strips. Strip (b) is the front and strip (a) is the back of the test paper. They are covered with the vinyl sheets. The front of the cover is colorless transparent vinyl, and the back is the white one. The vinyl cover was sealed except its foot (Fig. 2). This test paper is kept in a brown, transparent plastic bottle with silicagel and left at room temperature.
II) Method of experiments
1.5ml of sterile distilled water was added into the 1% Ogawa's egg media with well grown colony, and laid down on the slope for 5 minites to gain the extract. 0.2ml of the extract was divided into a test tube for the routine niacin test. The test paper was placed in a tube of 1% Ogawa's egg media for 15 minites. The foot of the test paper is set in the extract. The development of a orange color at the part of the PAS is positive, and the absence of this color is negative. (Fig. 1)
III) Results
1) The test paper method and the routine niacin test showed nearly the same results, and the test paper presented positive reaction to 2mg/ml niacin. (Table 1)
2) The both methods presented nearly equal reactions in 158 strains isolated from sputa of pulmonary tuberculous patients. (Table 2)
3) The effect of the quantity of the sterile distilled water added into 1% Ogawa's egg media to gain the niacin extract was examined with 11 strains isolated from sputa of pulmonary tuberculous patients. 10^-2mg of each strain was inoculated and cultured. The less the quantity of the water, the stronger the reaction. (Table 3)
In conclusion, the merits of our test paper were summarized as follows;
(a) There is no fear of gas poisoning.
(b) This method does not need a procedure to divide the extract containing numerous bacilli.
(c) A paper is easily preserved.

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From our experiences in overseas technical cooperations in South-East Asia, we keenly felt the necessity of development for easier and more reliable technique on the isolation culture of tubercle bacilli applicable in any rural area.
This paper presents the process of examinations on the dissolving for this problem.
I. Comparative studies between Ogawa original and WHO swab culture method
Ogawa's sputum culture method which is the standard method in Japan is very simple and unique technique that the specimen is directly inoculated by pipett into medium after alkali treatment without neutralization. On the other hand, the sputum swab culture method which is appeared in WHO/Tbc/Techn. Guide is the simplified technique that the specimen stucked on cotton swab is treated by acid and then neutralized by weak alkali. This swab culture method is widely employed in many countries of South-East Asia.
The comparative studies between both methods using the same sputum specimens were performed by two leading laboratories in Thailand.
The results are shown in tables 1, 2 and fig. 2. It is concluded that the Ogawa's method is fairly superior to WHO swab method, especially in contamination rate, though there are no significant differences statistically in the positivity.
The swab inoculation procedure was applied, furthermore, to Ogawa's method for convenience in rural areas, and this method was compared with both Ogawa original and WHO swab culture method. The results are presented in tables 3, 4 and figs. 3, 4.
There are no differences between Ogawa original and Ogawa swab method in both positivity and contamination rate, while Ogawa swab method is a little superior to WHO swab method.
The advantage of Ogawa swab method is its extreme simplicity and stability.
II. Studies on the medium for swab method without neutralization
For the purpose of investigating more adequate medium for the swab method without neutralization, several examinations were performed.
A provisional formula of medium was theoretically set up as follows; KH₂PO₄2.0gr., Mg citrate 0.1gr., Na gultamate 0.5gr., distilled water 100ml dissolve by heating, after cooling, glycerol 4ml, 2% malachite green 4ml, egg homoginate 200ml mix and stire, dispense each 5 to 7ml into McCartney bottle or test tube and coaggulate in slant by heating at 90°C for 1 hour. (2% modified medium)
1. H37Rv strain was suspended in both distilled water and 4% NaOH solution. These suspensions were inoculated onto Ogawa, Loewenstein-Jensen and 2% modified medium. The numbers of colony in each reading week are shown in fig. 5.
2. Table 5 indicates the most suitable combination of the dose of KH₂PO₄contained in the modified medium and the concentration of NaOH in pretreatment agent. The best result is obtained from the combination of KH₂PO₄ 2gr. and 1.0mlof 3% NaOH in sputum culture.
3. The comparison among several salts with Mg or citrate was made as a component of medium. The results suggest that Mg citrate in this medium can be substituted for Na citrate which dissolve more easily and more economical than Mg citrate.

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Factors influencing the minimal inhibitory concentration (M.I.C.) of rifampicin for Myc. tuberculosis were examined.
Used strains were H37Rv and a strain isolated from the sputa of a previously untreated pulmonary tuberculous patient. 1% Ogawa's egg media and Kirchner's semiliquid agar media with 10% albumin were used.
Following results were obtained.
1) Influence of pH; on alkaline side, the M.I.C. was low with I% Ogawa's egg media and high with Kirchner's semi-liquid agar media.
2) On both media, the more the dose of inoculated bacilli, the higher the M.I.C.
3) The longer the inoculation period, the higher the M.I.C., on both media.
4) When the media are preserved in high temperature, the M.I.C. of rifampicin became higher in eary stage. The longer the preservation period, the higher the M.I.C. And the influence of the preservation temperature was more remarkable in I% Ogawa's egg media than in Kirchiner's semi-liquid agar media.

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Tuberactinomycin-N (TUM-N) is a peptide antibiotic which has been isolated from the culture filtrate of streptomyces griseoverticillatus var. tuberacticus N6-130. This antibiotic is principally effective against tubercle bacilli and the activity is proved to be almost same as that of viomycin. The cross-resistance is observed between TUM-N and viomycin or capreomycin, however, only one way cross resistance is shown with kanamycin, and TUM-N is effective against kanamycin-resistant bacilli.
In the present study, authors have observed the therapeutic effect and the side effects of TUM-N in 88 patients of severe pulmonary tuberculosis with cavities, who were sensitive to viomycin. The duration of TUM-N treatment has been fixed for six months and the antibiotic was administered by intramuscular injections at the dose of 1 gram once daily for the first three months and twice weekly thereafter combined with other antituberculous drugs which had been used orally just prior to TUM-N treatment.

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Infection of bacteria with deoxyribonucleic acid (DNA) extracted from bacterio phages, that is called “transfection”, was found in Mycobacterium smegmatis in 1963. Evidence for showing that DNA but not any phage particles contaminated in the DNA samples produces infective centers was presented as follows: (1) the in fectivity was destroyed completely by catalytic amounts of DNase, (2) phage antiserum did not reduce number of plaque-forming units, (3) Tween 80, which prevents, phage adsorption, did not prevent the infection, (4) a phage-resistant mutant of the host was infected by the sample, (5) host cells in a late log phase of the growth were competent for the infection (Fig. 2).
Since it was known in Bacillus subtilis, Pneumococcus and Haemophilus that cells, competent for transfection are also competent for genetic transformation, the phenomenon of transfection found in Mycobacteria has been widely studied by many investigators from the standpoint of genetic transformation. For instance, we found that glycine has capacity to sensitize mycobacterial cells not only for the induction of cell lysis by lysozyme but also for the transfection (Fig. 3) These observations were expanded from glycine to various d-type amino acids. Based on these knowledges, we devised a new, gentle method for isolating DNA from amino acid-sensitized mycobacteria with lysozyme and phenol (Fig. 4). Employing the amino acid-sensitized cells competent for transfection and DNA isolated gently from various strains of Mycobacteria, experiments aiming at genetic transformation. were carried out. However all attempts have been ended in negative results.
Apparently, mycobacterial cells have ability to uptake DNA and have dark repair system for UV-damaged DNA. As described later, they also have intracellular mechanisms of recombinating bacterial DNA with their chromosomes. The reason why mycobacterial cells competent for transfection can not be transformed with bacterial DNA has not yet been clear; we assume that they might lack ability to recover after possible DNA uptake and recombinative events.
Transduction with mycobacteriophage was reported by Ramakrishnan and his coworkers using phage 13 and M. smegmatis strain SN2. We confirmed their results and added some positive data using additional genetic markers (Table 2). Attempts for obtaining transducing phages other than phage 13, however, were all failed.
In 1970, genetic recombination was demonstrated in M. smegmatis. Crosses between nutritionally complementary auxotrophs derived from one strain were infer tile whereas those from different strains formed recombinants (Fig. 6, Table 4 and 5). Conjugation rather than transformat ion and transduction was suggested as the gene transfer mechanism. Backcrosses of recombinants by either parental strain indicated four different types of mating behavior, suggesting that the mycobacterial compatibilities are controlled by at least two different factors (Table 7).
Sexual conjugation rather than cell fusion was proposed as the zygote-formation mechanism, based on the following facts: (1) analysis of segregation of unselected markers in recombinant progeny obtained in various cross systems revealed that one particular parent contributes the majority of alleles in nearly all of the recombinants, (2) mating medium containing streptomycin (SM) prevented recombination when one particular parent was SM resistant and another was sensitive, but it did not prevent recombination when the former parent was SM-sensitive and the latter was resistant (Table 6), and (3) crosses were infertile when one particular parent was recombination deficient mutant (rec-) and another was not (rec+), but crosses were fertile when the former strain was rec+ and the latter was rec-(unpublished data).

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Cell-mediated immune reactions involve the interaction of sensitized. T-lymphocytes with their specific antigen and cell activation. The activated lymphocytes exert a direct cytotoxic action on cells with which they are in contact and release soluble mediators producing a variety of effects, both cytotoxic and stimulatory, on adjacent tissue. These include lymphotoxin, permeability factors, chemotactic factors for neutrophils, eosinophils, monocytes, and other lymphocytes, factors affecting DNA synthesis and the proliferation of adjacent cells, activation factors for macrophages, interferon, pyrogenic factor, and transfer factor conferring specific reactivity on other lymphocytes. T-Iymphocytes appear to be heterogenous with respect to the identity and amount of the mediators they release. The resulting inflammation may therefore vary between pure lymphocytic infiltration with tissue destruction (as in some tumor immunity), macrophage infiltration and activation (as in most “delayed” reactions), predominantly basophilic or eosinophilic infiltrates (as in cutaneous basophil hyper sensitivity and the “retest” reaction), and plasmacytic infiltration. Macrophage activation is accompanied by enhanced destruction of intracellular microorganisms and appears to be the principal effector mechanism of immunity in such infections as tuberculosis and leishmaniasis.

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