LINDERSTR0M-LANG ET AL 



180 



LINDERSTR0M-LANG ET AL 



quiring 0.2 ml. of liquid, was used by 

 Lundsteen, E. and Vermehren, E. 

 (Ibid, 1936, 21, 147-166) at the Carlsberg 

 Laboratory for the determination of 

 phosphorus and phosphatase in 0.05 

 ml. blood samples. But for many histo- 

 chemical purposes the liquid volume 

 needed for the colorimetric measure- 

 ment was still too great. This problem 

 was largely solved subsequently as will 

 be mentioned later. 



In 1937 two new analytical techniques 

 were ingeniously developed by Linder- 

 str0m-Lang for micro-biochemical re- 

 search. One of these was based on the 

 dilatometric principle that in certain 

 reactions a volume change of the total 

 solution occurs which results in a 

 change in specific gravity great enough 

 for measurement. As applied by Lin- 

 derstr0m-Lang (Nature, 1937, 139, 713- 

 714), a density gradient is set up by 

 half -filling a glass tube with a kerosene- 

 bromobenzene mixture of appropriate 

 specific gravity, and then filling the re- 

 mainder of the tube with a similar mix- 

 ture having a greater proportion of 

 kerosene to give a lower specific gravity. 

 If the liquid in the middle of the tube 

 is stirred, and the tube mounted in a 

 thermostat, a stable vertical density 

 gradient is produced. If into this 

 gradient an aqueous drop of a reaction 

 mixture is placed, it will fall until it 

 reaches the level at which its specific 

 gravity equals that of the surrounding 

 medium. As the reaction within the 

 drop proceeds, the resultant volume 

 change of the drop will alter its specific 

 gravity and the drop will then move to 

 a new level. In some cases the distance 

 the drop will move has been found to 

 be directly proportional to the extent 

 that the chemical reaction has occurred. 

 By employing a cathetometer the move- 

 ment of the drop can be followed to 0.01 

 mm. and finer movements can be ob- 

 served with an ocular micrometer in the 

 telescope of the instrument. After cali- 

 bration of the gradient with drops of 

 salt solution of known specific gravity, 

 measurements of the excursion of the 

 drop can be used to calculate the 

 amount of reaction products formed in 

 certain instances. 



The dilatometric method was em- 

 ployed by Linderstr0m-Lang and H. 

 Lanz, Jr. (Ibid, 1938, 21, 315-338), for 

 peptidase measurements, and a study 

 was made by Holter, Lanz, and Linder- 

 str0m-Lang {Ibid, 1938, 23, 1^9) of this 

 enzyme during the first cell divisions in 

 the egg of Psammechinis miliaris. 

 Linderstr0m-Lang, Jacobsen, O. and 

 Johansen, G. {Ibid, 17-25) also em- 

 ployed the density gradient tube for 



the measurement of the deuterium con- 

 tent of heavy water mixtures. 



The other new technique that was 

 evolved at this time was based on the 

 principle of the Cartesian diver (Lind- 

 erstr0m-Lang, K. Nature, 1937, 140, 

 108-108). The diver is a small vessel, 

 open at one end, that will rise or fall, 

 while submerged in a salt solution, as a 

 result of a change in its gas volume. 

 The volume of the first divers used was 

 about 0.01 ml., and a reaction mixture 

 of about 0.001 ml. placed in the bottom 

 of the diver was employed for gaso- 

 metric measurements of enzyme activity 

 or respiration. The measurement was 

 carried out by applying a known posi- 

 tive or negative pressure to the air over 

 the tube containing the submerged 

 diver to bring it to a marked level in the 

 tube. At this level the gas volume 

 in the diver has a fi.xed value, and by 

 determining the external pressure 

 necessary to bring the gas volume back 

 to this value after expansion (in case of 

 a reaction evolving gas) or contraction 

 (in case of reaction using up gas), the 

 actual amount of gas evolved or con- 

 sumed can be calculated. In other 

 words the apparatus is a constant 

 volume, variable pressure, unit similar 

 in this respect to the ordinary Warburg 

 apparatus but capable of measuring gas 

 changes of a magnitude a thousand 

 times smaller. Further refinements 

 were developed later and will be 

 mentioned. 



The first applications of the Cartesian 

 diver method were to the gasometric 

 measurement of cholinesterase activity 

 in ganglia, Linderstr0m-Lang and Glick 

 (Compt. rend. trav. lab. Carlsberg, 

 1938, 22, 300-306), the determination of 

 thiamine and cocarboxylase in yeast, 

 Westenbrink, H. G. l<i.\lbid, 1940, 23, 

 195-212), and the respiration of sepa- 

 rated parts of the embryos of Para- 

 centrotus lividus, Lindahl, P. E. and 

 Holter {Ibid, 249-256), and of cells 

 grown in tissue culture, Zamecnik {Ibid, 

 1941,24,59-67). 



The following period in the work of 

 Linderstr0m-Lang and Holter, although 

 beclouded by the German occupation of 

 Denmark and the accompanying diffi- 

 culties and distractions, was devoted to 

 the consolidation of the advances 

 already made by further refinements of 

 technique and the extension of the 

 methods to new uses. Meanwhile, 

 Linderstr0m-Lang and Mogensen, K. R. 

 {Ibid, 1938, 23, 27-35) had already 

 described a cryostat for the accurate 

 sectioning of fresh frozen tissue at a 

 constant low temperature and the tech- 

 nique of handling the cut sections to 



