INTRODUCTION 



A B C D E F G 

 PREPARATION 



Fig. 3. Percentage distribution of various 

 types of tissue preparations employed in studies 

 on invertebrate tissue respiration from 1929 

 through 1959. Types of preparations indicated as 

 follows: A. Whole organ. B. Pieces (including) 

 fragments, strips, teased tissue, zones, parts). 



C. Slices (including sheets, thin sections). 



D. Suspension (including cell suspension, ground 

 tissue, mince). E. Homogenate. F. Particulate 

 fraction (including mitochondria and microsomes). 

 G. Other fractions (nuclear, supernatant). 



ical aids. For a more complete treatment of 

 work with tissue slices, see Field (1948) and 

 Robbie (1948). 



Soon a method was devised for fractionating 

 finely ground or homogenized tissues into their 

 cellular components by centrifugation at differ- 

 ent speeds for different lengths of time (see 

 Claude, 1946a, 1946b: Schneider, 1946). Thence- 

 forth, workers directed much of their attention 

 to characterizing these cell fractions. They 

 studied the effects of various metabolites, ions, 

 suspending media, and poisons on the respiratory 

 rates of the various fractions and also on the 

 ability of these fractions to form the high-energy 

 phosphate compounds found to be coupled to 

 their respiration. 



In figure 3 there appears the per cent of the 

 total number of studies on invertebrate respira- 

 tion for which each type of procedure was used. 

 Selection of the whole organ (A), pieces of organ 

 (B), and homogenate (E) took place with approx- 

 imately equal frequency. A more revealing 

 graph is that of figure 4. The diagonal line from 

 the lower left to the upper right corner of the 



graph emphasizes the trend through the years 

 towards the use of more and more finely divided 

 tissue preparations. The vertical arrow indi- 

 cates the approximate time when methods for 

 the preparation of suspensions and homogenates 

 first appeared. The use of homogenates and 

 particulate fractions derived from homogenates 

 in studies on invertebrate tissue respiration 

 followed quickly upon this development. 



At no time during the 30-year period have 

 whole organs or tissue slices fallen into dis- 

 favor as subjects for respiratory studies. Indeed, 

 in 1950 Krebs questioned the trend towards the 

 disruption of cell structure in attempts to ex- 

 plore certain aspects of cell physiology. He 

 pointed out that one can attribute much of the 

 conflicting data on the respiratory rates of 

 homologous tissues from different animals to 

 the type of tissue preparation and the type of 

 medium used. From his studies he concluded 

 that tissue slices suspended in particular syn- 

 thetic media comprise the type of preparation 

 most likely to yield meaningful results. 



For the future, therefore, a reverse trend has 

 something to recommend it. According to Green- 

 stein (1956, p. 651): "It is possible that studies 

 of cellular metabolism, which began with obser- 

 vation on the whole animal and then progressed 

 successively through studies of isolated organs, 

 tissue slices, homogenates, cell fractions, and 

 finally highly purified individual metabolic fac- 

 tors can with profit turn back to the whole ani- 

 mal. Studies of the effect of constitutional factors 

 on metabolic reactions . ... in vitro provide a 

 certain interest but, like all in vitro approaches, 

 are at the mercy of the experimental conditions 

 which the investigator chooses to select." 



Just as methods of preparing tissues for study 

 of their respiratory rates have undergone 

 marked changes over a period of years, so also 

 have the ways in which respiratory measure- 

 ments are made. First, let us examine the per- 

 centage distribution of the various methods. 

 More than one-half of the investigations cited in 

 Section 2 have involved the use of the Warburg 

 manometric method (see fig. 5A), while 16 per 

 cent have involved various forms of differential 

 manometer, including Fenn, Barcroft, and Thun- 

 berg (fig. 5B). Another 11 per cent of these stud- 

 ies were concerned with the spectrophotometer 

 (fig. 5E), 5 per cent with chemical methods 

 (Winkler and micro -Winkler, fig. 5D), 4 per 

 cent with micro volumetric techniques (fig. 5C), 



