250 H. A. KREBS VOL. 4 (1950) 



These procedures affect different tissues in different ways. In the case of kidney cortex, 

 Hver, striated, and cardiac muscle, mince and homogenates show higher initial rates of 

 respiration than sliced material when phosphate saline without a combustible substrate 

 is used as the medium. If the medium contains substrates which stimulate respiration 

 of slices, such as lactate, pyruvate, fumarate, and glutamate, homogenates, mince and 

 slices give approximately the same rates of respiration^' ^' '. In these tissues minced or 

 homogenized materials give the maximum rate of respiration. In other tissues, e.g., 

 spleen^, and lung^, minced and homogenized material gives consistently lower rates 

 of respiration than sliced material. The low values have been attributed to the hydrolysis 

 of coenzymes by nucleosidases released on the destruction of the tissue^. 



It is reasonable to assume that slicing leaves the tissue nearer to the 'natural' state 

 than mincing or homogenizing, because the number of physically damaged cells is 

 bound to be much smaller in slices than in mince or homogenates. Slicing is therefore 

 suggested as the procedure of choice for the measurement of the standard rate of 

 metabolism. 



2. Choice of medium 



In this section 5 different media are considered for the measurement of a standard 

 rate of respiration. They are: 



Serum 



Supplemented serum 



Saline serum substitute (later referred to as 'medium I') 



Phosphate saline without Ca, low in bicarbonate and CO2 (later referred to as 

 "medium IF) 



Saline low in phosphate, bicarbonate, and CO, (later referred to as 'medium IIP). 



Serum. Plasma or serum, being the natural environment of animal tissues, suggest 

 themselves as the most physiological standard media. Plasma requires the addition of an 

 anticoagulant and several of these, e.g., sodium fluoride and sodium oxalate, are unsuit- 

 able as they inhibit metabolic processes. Among the remaining substances heparine is 

 least likely to affect tissue metabolism, but relatively large amounts are required to 

 prevent coagulation in the presence of tissues. In general serum is preferable to plasma 

 because the absence of fibrinogen from the medium is less likely to affect the activities 

 of the tissue than the addition of an anticoagulant. 



Supplemented serum. Although serum resembles the physiological environment 

 more closely than any other medium it is by no means a perfect medium for in vitro 

 experiments. A tissue suspended in plasma or serum may, by its metabolism, soon cause 

 major changes in the concentration of important constituents, such as glucose, pyruvate, 

 lactate, and the acids of the tricarboxylic cycle, and also of bicarbonate. In the intact 

 body the balance of activities of all organs maintains a relative constancy of the con- 

 centration of serum constituents ; thus, glucose used up by some tissues, is replenished 

 from liver stores and by the absorption from the gut. But in vitro the metaboHc activity 

 of a single tissue can rapidly convert serum into an 'unphysiological' medium by 

 exhausting the available substrates. 



Another factor to be taken into consideration is the circumstance that in the intact 

 organ the path of diffusion is much shorter than in vitro, the average distance between 

 capillary wall and tissue cell being much shorter than the average distance between the 

 References p. 267-269. 



