Chemical induction of growth 



up and forcibly ejected, is used to reduce the macerate as much as possible 

 to single cells. The total number of cells is calculated from counts made on 

 small aliquots placed in a haemocytometer. In principle this same method 

 is still being used but the following refinements have been added. 



The haemocytometer is placed upside down on the stage of a Zeiss Winkel 

 inverted microscope fitted with a camera attachment. A sufficient field 

 (approximately 12 mm-) on the haemocytometer is viewed with a low power 

 objective (4x). With a suitable eyepiece (6x), photographs are taken of 

 representative fields. The negatives are projected on a screen to count the 

 number of cells per unit area on the haemocytometer, and filed for a perma- 

 nent record. This method is very much more useful for routine application 

 because a large number of such macerates can be prepared and photographed 

 and the counting done subsequently at leisure, whereas actual microscopical 

 counting is arduous and severely limits the number of observations that are 

 possible on a given day. There is no reason to believe that the photographic 

 technique is any different in its accuracy from the visual technique, whose 

 accuracy is discussed below. 



The volume of liquid placed on the haemocytometer slide is a very small 

 part of the total macerate; therefore, the error is largely one of sampling. 

 To reduce this error, several aliquots are counted. By suitable precautions, 

 a relatively accurate estimate can be made of the total number of cells in a 

 tissue explant of this sort. 



The method also measures the average cell size. If one records the weight 

 of the tissue explant in milligrams and knows the number of cells per culture, 

 it is readily possible to determine cell size, either in the units micrograms per 

 cell, or cell number per microgram of tissue. Obviously this is a measure of 

 average cell size, but nevertheless it permits interesting conclusions to be 

 drawn when the growth passes predominantly from growth by cell division 

 into growth predominanth" by cell expansion. With these aids we now know 

 and may summarize much more about the growth of these tissue cultures 

 under controlled conditions than has previously been published. 



THE GROWTH CURVE OF TISSUE EXPLANTS UNDER CONTROLLED CONDITIONS 



The growth curve in terms of fresh weight follows the familiar sigmoid 

 pattern (Caplin and Steward, 1949). In terms of cell number, growth may be 

 somewhat similarly represented and changes in average cell size as the growth 

 proceeds may also be followed. 



In the initial phases of the growth there is first a lag period during which 

 little external growth occurs though preformed cells may enlarge. This 

 is followed by a more or less prolonged period in which cell numbers rise 

 exponentially with time and in which the growth is predominandy by cell 

 division. During this period the average cell size of the culture as a whole 

 steadily decreases so that the effect of any already mature cells is counteracted 

 by the large increment of cell number and of cells that do not conspicuously 

 enlarge. It is worth emphasizing how small are the cells typically produced 

 in coco-nut milk (see Table 1). Even so, however, as the culture enlarges, 

 some cells, particularly those internally, do enlarge and some areas occur in 

 the tissue in which relatively large air spaces may be formed. (See Steward, 



171 



