LINDERSTRPM-LANG ET AL 



181 



LINDERSTRPIM-LAXG ET AL 



prevent their curling etc., and Holter 

 and Lindahl (Ibid, 1940, 23, 249-256) had 

 already studied the distribution of pep- 

 tidase in the Paracentrotus embryo in 

 continuation of the line of cytochemical 

 work on marine invertebrates that had 

 been instituted earlier. 



Considering the disruptive influence 

 of the occupation of Denmark by the 

 Nazis, it is remarkable that the Carls- 

 berg Laboratory workers continued to 

 be productive, but perhaps the very 

 need to have a refuge from the wild 

 events outside served to hold them to 

 their laboratory benches for periods of 

 sweet reasonableness. The record 

 shows that during these years the Car- 

 tesian diver technique was subjected to 

 intensive theoretical analysis, Linder- 

 str0m-Lang and Holter {Ibid, 1942, 24, 

 105-138) and Linderstr0m-Lang {Ibid, 

 1942, 24, 249-280; Ibid, 1943, 24, 333- 

 398), and laboratory refinement, Holter 

 {Ibid, 1943, 24, 399-478). Zeuthen, E. 

 {Ibid, 479-518) devised a diver with a 

 gas volume a hundred times smaller 

 than the first ones used, and this opened 

 many new possibilities. 



Applications of Cartesian diver gas- 

 ometry at this time included studies on 

 the respiration of the egg and enibryo 

 of the ascidian, Ciona intestinalis L., 

 Holter and Zeuthen {Ibid, 1944, 25, 

 33-65), the respiration of syncytia 

 formed by abnormal development of 

 Ciona eggs, Andresen, Holter, and 

 Zeuthen {Ibid, 1944, 25, 67-85), and ox- 

 ygen uptake during mitosis of frog eggs, 

 Zeuthen {Ibid, 1946, 25, 191-228). 

 Holter {Ibid, 1944, 25, 156-167) also de- 

 veloped a colorimetric method for 

 measuring the volume of large amoebae. 



Parallel with these developments 

 during the war years were others. 

 Schmidt-Nielsen {Ibid, 1942, 24, 233- 

 247) worked out a titrimetric method 

 for analysis of fat in quantities of 10"* 

 g. and later he extended this investi- 

 gation to methods for the determination 

 of the iodine number in amounts of fat 

 of this order {Ibid, 1944, 25, 87-96), and 

 he described the extraction and frac- 

 tionation of fats in 1 mg. of tissue 

 {Ibid, 97-105). Previous work on the 

 stomach was also continued by Rask- 

 Nielsen {Ibid, 1944, 25, 1-32) who 

 studied the peptidase content of the 

 pyloric portion of the hog stomach, and 

 Bottelier, H. P., Holter, H., and Linder- 

 str0m-Lang, K. {Ibid, 1943, 24, 289-314) 

 investigated the peptidase in the roots 

 of barley. 



Further extension to new uses of the 

 microtechniques that had been de- 

 veloped marked the post-war years. 

 Bruel, D., Holter, H., Linder8tr0m-Lang, 



K. and Rozits. K. {Ibid, 1946, 25, 289- 

 324) elaborated a titrimetric method for 

 the determination of total nitrogen 

 with an accuracy of 5 X 10~* g. nitrogen, 

 Levi and Zeuthen {Ibid, 273-288) 

 adapted the density gradient tube to 

 micro weighing, and later Zeuthen, E. 

 {Ibid, 1948, 26, 243-266) constructed a 

 Cartesian diver balance that gave re- 

 duced weights to 1 X 10~* g. The re- 

 duced weight is the weight of an object 

 minus the weight of an equal volume of 

 water. This quantity is convenient for 

 expressing the amount of a small bio- 

 logical sample since it is a measure of 

 the quantity of the substance independ- 

 ent of its water content. Zeuthen 

 {Ibid, 1948, 26, 267-276) followed the 

 reduced weight and volume of amoebae 

 during starvation, and Holter and 

 Zeuthen {Ibid, 277-296) studied metabo- 

 lism and reduced weight of amoebae. 

 A theoretical treatment of the use of the 

 Cartesian diver for respiration measure- 

 ments on single cell organisms was 

 given by Linderstr0m-Lang {Ibid, 1946, 

 25, 229-272). 



The main program of the cytochemi- 

 cal department in recent years has been 

 the continuation of Holter's previous 

 work on the biochemistry of unicellular 

 organisms and the developing inverte- 

 brate and amphibian embryo. The 

 demand for microanalysis of different 

 constituents during the course of these 

 studies has led to further expansion of 

 method development. 



Apparatus that will permit colori- 

 metric analysis of substances at the level 

 required for work on microtome sec- 

 tions, single early embryos, or proto- 

 zoa was developed more recently by 

 Holter and his coworkers. The vol- 

 umes of liquid needed for the color- 

 imetry are of the order of 0.010 ml. The 

 cuvettes employed to hold these small 

 volumes are pieces of capillary tubing 

 having a lumen of about 1 mm. in 

 diameter and about 10 mm. long. The 

 cuvette is sealed to a microscopic slide 

 with stopcock grease, filled with the 

 colored liquid, and a cover slip is placed 

 on top. This unit is set on the stage of a 

 microscope and a fine beam of filtered 

 light is passed through it. The trans- 

 mitted light is collected by a low-power 

 objective and sent on to a photocell 

 fixed over the ocular. Galvanometer 

 readings are taken as for any other 

 photoelectric colorimeter. With this 

 equipment Holter and L0vtrup {Ibid, 

 1949, 27, 27) studied the proteolytic 

 enzymes in amoebae and Krugelis 

 {Ibid, 1950, 27, 273-290) investigated the 

 properties and changes of alkaline 



