Ogawa Egg Medium Research Articles

A study on the frequency of ‘atypical’ mycobacteria in Japanese National Sanatoria

A co-operative study was carried out by thirteen Japanese National Sanatoria (chest hospitals) on the isolation of ‘atypical’ mycobacteria from patients admitted with the diagnosis of tuberculosis. For screening for ‘atypical’ mycobacteria, Ogawa egg medium containing 0·5 mg./ml. sodium salicylate (‘salicylate medium’) was employed. Strains growing on the salicylate medium (tubercle bacilli do not grow on it) were sent to a central laboratory for identification. As a result of one year's co-operative study, 666 strains of ‘atypical’ mycobacteria were isolated from 382 patients, among which 45 patients excreted the same ‘atypical’ mycobacteria more than three times. The frequency of isolation was significantly higher in two hospitals located in Tokyo and Osaka prefectures, which serve the largest and the second largest cities of Japan. The most frequently isolated ‘pathogenic’ ‘atypical’ mycobacteria were M. intracellulare followed by M. scrofulaceum. In comparison with data from the U.S.A., the relatively high frequency of isolation of M. scrofulaceum seemed to be noteworthy. The use of salicylate medium was shown to be useful for screening for ‘atypical’ mycobacteria.

Two Types of Slowly Growing, Nonphotochromogenic Mycobacteria Obtained from Soil by the Mouse Passage Method:Mycobacterium terraeandMycobacterium novum

Two types of slowly growing, nonphotochromogenic mycobacteria, Mycobacterium terrae and Mycobacterium novum, were isolated from soil by mouse body passage method. The former was presented previously by the present author as a new species. Its characteristics are better clarified in this paper based on the data of 93 strains. Mycobacterium novum is a slowly growing nonphotochromogen. It grows at 10 to 14 days on egg media and does not grow on Sauton agar. It grows on Ogawa egg medium containing either 0.2% (w/v) sodium p-aminosalicylate, 0.1% (w/v) sodium salicylate or 0.25 mg/ml NH2OH·HCl. It is differentiated from M. tuberculosis, M. bovis and M. microti by these characters. It grows at 28 C and 37 C, but does not grow at 45 C. Other characteristics are: nitrate not reduced; negative two week arylsulphatase; negative niacin test; and no amidase clemonstrated. None of the carbohydrates and nitrogen compounds tested could be utilized as the sole source of carbon or of nitrogen in synthetic agar medium. It survived in mouse organs for three to four weeks. It was noticed that these mycobacteria occur very commonly in soil.

マウス足蹠内へのらい菌接種

The authors observed a limited multiplication of acid-fast bacilli in mouse foot-pads inoculated with Mycobacterium leprae from an untreated patient with lepromatous leprosy in this country. There was no evidence of bacillary increase in any of the mice inoculated with a heat-killed suspension. Nor were any macroscopically visible changes noted. The acid-fast bacilli in the inoculum and the harvest were transferred to Ogawa's egg media, and incubated at 33°C and 37°C. The cultures were negative, eliminating the possibility of superinfection by culturable mycobacteria. Subcutaneous inoculations of mice in the abdomen did not produce any lesions, indicating that Mycobacterium lepraemurium, a known unculturable mycobacterium, did not exist in the inoculum and the harvest. There was an arrest in the multiplication of the bacilli at about 105 to 106 per foot-pad. These findings were compatible with those of Shepard and others, who recently reported degrees of successful transmission of leprosy to mice. It is likely that the authors isolated a strain of M. leprae in mouse foot-pads. The viability of M. leprae was not affected by freezing and storing at -30°C for 18 days.

STUDIES ON MEDIA FOR CULTIVATION OF TUBERCLE BACILLI 2. Effects of temperature of inspissation and storage Ogawa's egg media and different conditions of cultivation for growth of the tubercle bacilli.

Heating for coagulation of Ogawa's egg media was started from 55℃ for evaporation of air in inside of the media and the temperature was elevated gradually about 10℃ during fifteen minutes in speed by controlling ther-mostat of electric automatic inspissator. So that, for preparation of egg solid media coagulated at 85℃ it needs about forty-fuve minutes, starting from 55℃. The authors prepared Ogawa's egg media coagulated at different temperatures by using the above processes and compared effects of different temperature of coagulation and storage of Ogawa;s media and different conditions of cultivation for growth of tubercle bacilli. Inoculated media were sealed tightly with rubber caps. 1. Optimal coagulation temperature of Ogawa's egg media for growth of tubercle bacilli was 85℃ and this temperature was more optimal than 90℃ for preparation of Ogawa's media in number of colonies and also in size of colonies. The media coagulated one hour at 90℃ was worse than the above two kinds of media for growth of tubercle bacilli. 2. There were no significant differences for growth of tubercle bacilli between Ogawa's media stored at 10℃ during four months and freshly prepared Ogawa's media. And also Ogaawa's media stored at room temperature during four months from March the July were still useful for cultivation of tubercle bacilli. It seems important for long-time storage of Ogawa's egg media to protect dryness of media as much as low temperature. 3. Tight sealing of inoculated media was better for growth of small number of tubercle bacilli, i.e. from few to several hundred viable units, than incomplete sealing of inoculated media.

Mycobacterium smegmatis. Its characteristics and identification

Physiological characters of twenty-four strains of Mycobacterium smegmatis, classified by a numerical classification, were investigated. The results are shown in tables 1 and 2.Colonial morphology: White or creamy, wet and smooth; non-photochromogenic. Non-motile. Acidfast rods or short rods. Growth at 3 days on egg media and on Sauton agar. Growth at 28°C, 37°C and 45°C; no growth at 52°C. Growth at 0.1% picric acid and 0.2% picric acid in Sauton agar. Growth at 0.2% Na-p-aminosalicylate and at 0.1% Na-salicylate in Ogawa egg medium. Growth at 0.25mg/ml. 8-azaguanine in Ogawa egg medium. No growth at 0.5mg/ml. NH2OH. HCl in Ogawa egg medium. Niacin: Negative.Nitrate reduction: Positive. Three-day-arylsulfatase: Negative. Two-week-arylsulfatase: Positive. PAS degradation: Negative. Salicylate degration: Negative. The following amidases are positive according to the meteod of Bönicke: Acetamidase, benzamidase, urease, isonicotinamidase, nicotinamidase, pyrazinamidase, succinamidase. The following amidase are negative in most strains: Salicylamidase, allantoinase and malonamidase.All of acetate, citrate, succinate, malate, pyruvate, benzoate, malonate and fumarate are utilized as sole carbon source for growth. The following carbohydrates are utilized as sole carbon source for growth: Glycerol, glucose, fructose, sucrose, mannose, galactose, arabinose, xylose, rhamnose, trehalose, inositol, mannitol, sorbitol, ethanol, propanol, propylene glycel, 1, 3-butylene glycol and 2, 3-butylene glycol. Raffinose and 1, 4-butylene glycol not utilized as sole carbon source for growth.Acid formed from glucose, mannose, galactose, arabinose, xylose, rhamnose, trehalose, inositol, mannitol and sorbitol, but no acid from raffinose.All of the following compounds are utilized as sole nitrogen source for growth: L-Glutamate, L-serine, L-methionine, acetamide, benzamide, urea, pyrazinamide, nicotinamide, isonicotinamide, succinamide, nitrate and nitrite.All of the following compounds are utilized as sole, simultaneous nitrogen and carbon source for growth: L-Glutamate, L-serine, glucosamine-HCl, acetamide, benzamide, monoethanolamine and trimethylene diamine.Identification. The following characters are almost specific for M. smegmatis and are considered to be “distinguishing characters” (Table 3). (1) Benzamidase; (2) isonicotinamidase; (3) succinamidase; (4) acid from galactose: (5) acid from rhamnose; (6) acid from sorbitol; (7) utilization of benzoate as sole carbon source for growth; (8) utilization of benzamide as sole nitrogen source for growth; (9) utilization of benzamide as simultaneous nitrogen and carbon source for growth.

Slowly Growing Mycobacteriaの分類と同定 (第2報)

Biological and biochemical characters of ten strains of Mycobacterium kansasii have been described.Colonial morphology: Rough. Photochromogenic. No pigmentation in dark. Growth at 7 days on Lowenstein-Jensen medium (LJ medium); at 2 weeks on Sauton agar. Growth at 28°C and 37°C; but no growth at 45°C. Growth on Ogawa egg medium containing 0.5mg/ml sodium salicylate. Niacin: Negative. Growth on Ogawa egg medium containing 50μglml thiophen-2-carbonic acid hydrazide. No tolerance to 0.1% picric acid in Sauton agar. No tolerance to 0.25mg/ml hydroxylamine in Ogawa egg medium. Positive urease and positive nicotinamidase, but negative reactions for other eight amidases among Bonicke's ten amidases. Nitrate reduced. Two-week-arylsulfatase: Positive. None of organic acids utilized as sole carbon source (organic acids tested as sole carbon source for growth: acetate, citrate, succinate, malate, pyruvate, benzoate, malonate and fumarate). No acid from glucose, mannose, galactose, arabinose, xylose, rhamnose, raffinose, inositol, mannitol and sorbitol. Glucose utilized as sole carbon source for growth; sucrose utilized by four of ten strains; fructose, ethanol and propanol not utilized. L-Glutamate not utilized or, if any, slightly utilized as sole (simultaneous) carbon and nitrogen source. L-Glutamate, succinamide and nitrate utilized as sole nitrogen source for growth; L-serine utilized by four of ten strains; urea utilized by two of ten; acetamide utilized by two of ten strains tested; benzamide, pyrazinamide, isonicotinamide, nicotinamide and nitrite not utilized.Albino type of M. kansasii was produced in the laboratory by Tsukamura (S) (1963) and Runyon (1964). Scotochromogenic type of this organism also was reported by Tacquet et al. (1965) and Hauduroy et al. (1965). These findings arised necessity for an identification method of this organism without use of photochromogeneity as a marker character. Over-all similarity of any test strains to the above characters would indicate that the test strains belong to the species M. kansasii, even if the test strains were nonphoto-chromogenic or scotochromogenic. Differential diagnosis of this organism from other mycobacteria also was discussed.