The survival of chloroplasts in vitro: Particle volume distribution patterns as a criterion for assessing the degree of integrity of isolated chloroplasts

Abstract

Volume distribution patterns of chloroplast suspensions obtained electronically with a Coulter counter have been compared with the corresponding diameter measurements and with the appearance of chloroplasts observed by phase-contrast microscopy. It was found that the Coulter counter pattern could be used to detect gross morphological changes of chloroplasts in isolation, but could not be used for the quantitative determination of the properties of chloroplasts in different morphological states. The pattern for a suspension containing both intact and damaged chloroplasts in approximately equal numbers has only one maximum. Two maxima are present if the proportion of intact chloroplasts is considerably greater than 50%. Chloroplasts which have lost both their outer limiting membrane and also their stroma (i.e. naked, but intact, lamellae systems) make only a very small contribution to the size distribution pattern in the region recording apparent volumes between 0 and 11.2 µ(3).Previous workers have described intact chloroplasts as "Class I", and damaged chloroplasts lacking limiting membrane and stroma as "Class II". We suggest that a third "Intermediate Class" should be recognized for chloroplasts devoid of their limiting membrane, but still retaining stroma. Such chloroplasts can be distinguished from Class II chloroplasts by their less clearly visible grana, and slightly lighter appearance under phase contrast, and also by their considerable contribution to Coulter counter volume distribution patterns with a peak maximum between 35 and 55 µ(3). Such Intermediate Class chloroplasts would also be expected to have biochemical properties differing considerably from those of both Class I and Class II chloroplasts.