Journal of Applied Microscopy. 



35 



sample is diluted with one hundred cubic 

 centimeters of the culture medium. One 

 drop of this mixture is added to ninety- 

 nine cubic centimeters more of a liquid 

 medium. The last mixture is divided 

 into ten Freudenreich culture flasks. 

 The dilution may be carried as far as 

 necessary until a single organism occurs 

 in the flask. 



For culture two formulae are used as 

 fellows: 



Formula "A." 



Magnesium sulphate, 10 gr. 

 Calcium chlorate, 10 gr. 

 Sodium sulphate, 5 gr. 

 Ammonium nitrate, 1 gr. 

 Potassium nitrate, 2 gr. 

 Sodium nitrate, 2 gr. 

 Potassum bromide, 0.2 gr. 

 Potassium iodide, 0.1 gr. 

 Water, 100 gr. 



Formula "B." 



Sodium phosphate, 4 gr. 

 Calcium chlorate, slcc, 4 gr. 

 Hydrochloric acid, 20 degrees, B. 2 ccm. 

 Perchloride of iron, 2 ccm. 

 Water, 80 ccm. 



The culture media to be kept separated. 

 To one liter of water add forty drops of 

 " A " and twenty drops of "B," five centi- 

 grams of straw, and as much moss. This 

 should be sterilized. Water lost by 

 evaporation can be replaced by sterilized 

 water. According to Miquel and H. Gill, 

 direct sunlight is injurious. Pleurosigma 

 angulatum, Cymatopleura solea, and 

 other species have been cultivated. 

 (Zeitschrift f. Angewandte Mikroskopie 



3; 225). L. H. P. 



Culture of Bacterial Spores. 

 Mayer recommends staining with pot- 

 assium iodine iodide (3-t-3-f-20), and 

 chloriodide in an hour colors the spores 

 of Astasia as follows: Extine with its 

 spines and rod in spore yellow. Alcoholic 

 ruthuim red colors extine first, later 

 the rod. Alcoholic safranin (0.1 alcohol 

 and v/ater 50) colors the spores intensely 

 when added while alive. Concentrated 

 Delafleld's haematoxylin when allowed 

 to act for two hours, colors the mem- 

 brane deep blue. Short treatment with 

 carbolic fuchsin, and wash rapidly with 

 hydrochloric acid alcohol (20 gr. Hcl., 

 100 ccm. alcohol, 200 ccm. of water) colors 

 the spores as well as the fiagella. 



(Flora 84: 191-192) L. H. P. 



Staining of Cytoplasm and Vacuoles. 

 Mayer gives the following directions: 

 To a drop of living culture of Astasia 

 add a trace of methyl blue solution (one 

 pint concentrated alcohol methyl blue 

 solution -\- 10 pints of water) the 

 motile rods color without losing 

 their motion. The membrane colors 

 first, a delicate blue line surrounds 

 the protoplast. Preparations are treated 



for five minu'tes in vapors of for- 

 maline to fix, then stained with safra- 

 nin. The structure of the cytoplasm 

 comes out nicely. The nucleus stains 

 nicely when treated with iodide potas- 

 sium iodide solution. It is only necessary 

 to add to a drop of the culture a little 

 iodine on the edge of a cover slip. The 

 nucleus of the Astasia asterospora 

 usually occurs near the wall. To show 

 vacuoles of rods, heat rather rapidly on 

 a cover slip with a little water and then 

 color with ruthium red. The vacuoles 

 will mostly color red and darker than 

 cytoplasm. (Flora 84: 206-207) L. H. P. 



Asparagin Liquid Culture Medium for Bac- 

 teria. 

 Mayer recommends the following 

 method of preparation: One gram 

 each of magnesium sulphate, sodium 

 chloride, potassium phosphate dissolved 

 in one hundred cubic centimeters of 

 water, heated and neutralized with 

 sodium carbonate, heat again and filter. 

 Increase to one hundred cubic centimet- 

 ers. Take five cubic centimeters of this 

 stock solution and add to it one gram 

 of asparagin, two grams cane sugar 

 ninetj'-two cubic centimeters of water. 

 Astasia asterospora grew rather slowly 

 in this medium at first, but later mucil- 

 age was produced rapidly. 



(Flora 84 186-187) L. H. P. 



Staining Haematozoa of Malaria. 



Dr. E. Marchoux recommends the fol- 

 lowing formula: Saturated solution cf 

 thionin in 507o alcohol, 20 cubic centi- 

 meters; 2% carbolic acid, 100 cubic centi- 

 meters. It is necessary to let the mixture 

 mature for a few days until the carbolic 

 acid has combined with the thionin. — 

 Jour. Royal Mic. Soc, 1897, p. 450. 



Proper Angle of Microtome Knife. 

 Dr. B. Rawitz adduces experimental 

 proof to show that the microtome knife 

 should be placed at an acute rather than 

 at a right angle. When placed at the 

 latter angle, the sections, according to 

 their thickness, are always more or less 

 crowded together, thus distorting the 

 finer structures of the tissues cut. The 

 experimental proof consists of the 

 measurement of the sections cut with 

 th»^ knife at a right angle and also at an 

 angle of forty-flve degrees. The sections 

 wez-e made from a block measuring 

 20.5X11.5 mm., and were 15.10 and 5 mu. 

 thick. With the knife at the acute angle 

 they all measured 11 mm. in breadth, 

 while with the knife at a right angle 

 they measured 9.5 mm. for 15 mu., 9 mm. 

 for the 10 mu., and 8 mm. for the 5 mu. 

 sections; thus showing a shrinkage of 

 2. 2.5, ?nd 3 mm. respectively. — Jour. 

 Royal Micros. Soc, 1897, p. 447. 



