The cultural amœbæ mentioned on p. [42] were grown on such media or modifications thereof. One modified medium actually used was brain tissue, to which blood was added from day to day, and an easily assimilable bacterium (one of the influenza group of bacilli) was present, which did not overgrow the medium at a temperature of 38° C. Different conditions of food and of temperature produced morphological variations in the cultural amœbæ.
Couret and J. Walker[1275] (1913) state that they have cultivated five varieties of intestinal amœbæ, the associated bacteria having been previously separated. They used a medium consisting of agar to which sterile autolysed tissue had been added. The sterile tissue, such as brain or liver, was kept in a sterile thermostat at a temperature of 40° C. for ten to twenty days. The surface of the agar should be broken up before use, and the medium must not be too acid (not over 1·5 per cent.). They consider that autolysed tissue is necessary for the growth of Entamœbæ, and that naturally associated bacteria aid growth by autolysing the tissues.
(2) Culture Media for the growth of Protozoa parasitic in the Blood.—MacNeal and Novy,[1276] in 1903, used a mixture of blood and agar for the cultivation of trypanosomes such as T. lewisi and T. brucei. They employed varying proportions of the blood and agar, a medium consisting of two parts of defibrinated rabbit’s blood mixed with one part of agar being useful. The trypanosomes grew in the water of condensation. Some of the authors’ earlier formulæ contained different proportions of blood and agar with a little peptone, while one of these media contained meat extract, agar, peptone, salt and sodium carbonate. The temperature, like the proportion of blood and agar, varied with the trypanosome investigated, but the optimum was 25° C.
Mathis[1277] (1906) somewhat simplified the technique of Novy and MacNeal. He collected the blood of a suitable animal, such as rabbit, cow or dog, strict asepsis not being essential. The blood was defibrinated in the ordinary way. One part of blood was added to two parts of agar at 50° C. The mixture was sterilized several times by heating to 75° C. or 100 ° C. Slopes were made and the water of condensation was inoculated with a little blood containing the trypanosomes. Blood may be obtained from a superficial vein or from the heart.
Novy-MacNeal-Nicolle or N. N. N. Medium.—In 1908 C. Nicolle[1278] brought forward a modification of the Novy-MacNeal (N.N.) medium. The formula is as follows:—
| Agar | 14 | grm. |
| Sea salt | 6 | " |
| Water | 900 | " |
Apparently pure sodium chloride can be substituted equally well for sea salt. The mixture is placed in tubes and sterilized in an autoclave. To each tube one-third of its volume of rabbit blood, taken by aseptic puncture of the heart, is added. The salt agar is kept liquid at 45° C. to 50° C. and the blood is added to the mixture. The culture medium so prepared is maintained for five days at 37° C., and then for a few days at room temperature. This medium was devised for the cultivation of Leishmania (see p. [106]), but trypanosomes may also be grown thereon. Subsequently, Nicolle recommended the use of citrated rat’s blood heated to 45° C. for half an hour, instead of defibrinated rabbit’s blood. On such a medium, J. G. Thomson and Sinton[1279] (1912) succeeded in growing Trypanosoma gambiense and T. rhodesiense (see pp. [76], 83).
Noguchi’s media for the cultivation of Spirochætes and Treponemata are described on pp. [123], 125. Hata’s modification is discussed on p. [126].
Bass’s glucose-blood medium for the cultivation of malarial parasites is described on pp. [170]–172. It has also been used successfully for the cultivation of Piroplasma or Babesia (see p. [172]).