LINE TEST 



182 



LIPASE 



phosphatase activity during amphibian 

 development. 



Other recent work that has come from 

 the Carlsberg Laboratory includes a 

 micro gasometric method for sulfur 

 compounds such as cystine, for which it 

 is accurate to 1 X 10~' g. within 2.5%, 

 Holter and L0vtrup {Ibid, 1949, 27, 

 72-78). Further development of the 

 Cartesian diver technique was effected 

 by using silicone coated divers, 

 Schwartz {Ihid, 1949, 27, 79-92), and 

 L0vtrup {Ibid, 1950, 27, 125-136) modi- 

 fied the diver balance of Zeuthen. In 

 the course of the latter study L0vtrup 

 designed a microbalance with a sensi- 

 tivity of 1 X 10~* g. To conduct studies 

 on the density of microorganisms such 

 as amoebae, a starch density gradient 

 tube was developed by L0vtrup {Ibid, 

 1950, 27, 137-144). From a combina- 

 tion of density and reduced weight 

 measurements the volume of amoebae 

 could be determined with an accuracy of 



1 X 10-9 ml. 



Aside from the classical applications 

 of the quantitative techniques and 

 methods that Linderstr0m-Lang and 

 Holter instituted, their procedures are 

 being employed ever more extensively 

 by an increasing number of scientists 

 in the diverse fields to which quantita- 

 tive histo- and cytochemistry is of 

 great importance. In fact, the develop- 

 ment of the fundamental aspects of all 

 of the life sciences can be expected to be 

 significantly enhanced by their con- 

 tributions. 

 Line Test for vitamin D. This is the basis 

 for calculating the U.S. P. unit of vita- 

 min D potency. The line test was 

 apparently first introduced by McCol- 

 lum, E. v., et al., J. Biol. Chem., 1922, 

 51, 41-49. A critique of the test_ is 

 given by Bills, C. E., et al., J. Biol. 

 Chem., 1931, 90, 619-636. See also 

 Sherman, H. C., The Chemistry of Food 

 and Nutrition, New York: MacMillan, 

 1941, 611 pp. A slightly modified tech- 

 nique is proposed and given in detail 

 by Martin, G. J., J. Lab. & Clin. Med., 

 1940, 26, 714-719. Inject rats intra- 

 peritoneally with 1 cc. 1% aq. sodium 

 alizarin sulfonate at pH 8.0 and give 

 supplements of measured amounts of 

 vitamin D orally. Animals similarly 

 stained but not given the vitamin serve 

 as controls. After test periods of 1 or 



2 days, kill the animals, remove radii 

 and ulnae and examine grossly and mi- 

 croscopically for alizarin stained^ lines 

 at epiphysis. See also use of Alizarin 

 Red S. Both this and the sulfonate are 

 better than Madder because they pro- 

 vide quicker and more intense colora- 

 tion of bony calcium laid down during 



the period that they are available in the 

 circulation as accelerated by vitamin D. 



Linguatulidae, see Parasites. 



Linin (L. linum, flax). The acidophilic, 

 thread-like framework of nucleoplasm 

 seen in sections but not in the living 

 nucleus. 



Lipase. Frozen sections 30ju thick and 4.5 

 mm. in diameter of beef adrenals are 

 extracted in 30% glycerol -f equal 

 volume 1% methyl butyrate in glycine 

 — NaOH buffer at pH 8.7; digested at 

 40°C. ; enzyme action arrested by addi- 

 tion of 2% phenol (10 parts) and 0.04% 

 brom-thymol blue (1.5 parts) to 3.5 

 times total volume; and end point ti- 

 trated at pH 6.5 with 0.05 N HCl. 

 This point is determined by comparing 

 color with standard color of brom-thy- 

 mol blue in phosphate buffer pH 6.5. 

 Nearby sections, some stained with 

 hematoxylin and eosin, and others, with 

 Sudan III, are examined histologically. 

 The medulla, which exhibits most 

 lipolytic activity, contains least lipid. 

 Estimations of esterase are also de- 

 scribed by Glick and Biskind (D.and 

 G. R., J. Biol. Chem., 1935, 110, 575- 

 582). See Barnes, J. M., Brit. J. Exp. 

 Path., 1940, 21, 264-275 for analysis of 

 lipase in lymphocytes and polymor- 

 phonuclear leucocytes and Hoagland, 

 C. L., et al., J. Exper. Med., 1942, 76, 

 163-173 for lipase determinations in 

 elementary bodies of vaccine virus. 



An important new technique is de- 

 scribed and well illustrated by Gomori, 

 G., Arch. Path., 1946, 41, 121-129: 



1. Fix thin slices of fresh tissue in 

 chilled acetone 12-24 hrs. in ice box. 



2. Dehydrate in 2 changes absolute 

 acetone, 12-24 hrs. each, room tempera- 

 ture. 



3. Impregnate in 5% acetylcellulose 

 (Eastman's cellulose acetate "high 

 acetyl, low viscosity, no. 4644") for 

 24 hrs. 



4. Drain off fluid, transfer to 2 

 changes benzene, 1 hr. each. 



5. Embed in paraffin (56-62°C), 2 

 changes, 1 to \\ hrs. each. Cut 4-8m 

 sections, float on water (db 35°C) and 

 mount on slides. Pass down through 

 xylol and alcohols to aq. dest. 



6. Incubate at 37°C 6-12 hrs. in 50 cc. 

 Solution I + 2 cc. Solution II. 



Solution I: Glycerin 150 cc, 10% aq. 

 calcium chloride, 50 cc; half-molar 

 maleate buffer pH 7 to 7.4 (maleic acid, 

 5.8 gm. ; 4% aq. sodium hydroxide 94 cc. 

 + aq. dest. 6 cc). If maleate buffer 

 is omitted mixture should be adjusted 

 to pH indicated. 



Solution II: 5% aq. Tween 40, or 

 Tween 60 (Atlas Powder Co., Wilming- 

 ton, Del.) or Product 81 with about 



