The Direct ixperimental Observation of Cells in Phenomic Lag abstract. By employing colls mitating at a very high spontaneous rate and 4 grid plate containing 4 medium totally selective aysuinst mutant cells, direct microscopic observation was made of mutant cells of recent crigin passing through a limited mumber of phenomic lag divisions. The concept that cells immediately fcllowing mitation are phenotyrical- ly normal aml require several generations for mutant phenotype expression has becn invoked to interpret discrepancies in mutation rates estimated by vari- ous methods (1), and to interpret the effect of intermediate cultivation after expcesure to mutagens, on the number cf wutants expressed (¢,3). Ryan (ui) re- viewed the evidence for phenomic lag and concluded that it wus not compellin:. The livelihood of direct observation of a newborn mutant in phenomic lag is unredsonably small where the mutation rate is low. where the rute is high (@eG~e 102405 as in the cytoplasmic mutution to resriration deficiency in some yeast strains) the direct observation of phencmic lag vecomes rossible on a medium tetalily selective against the mutant. Under these conditions one pre~ dicts that normal cells should form colonies (complete develcpnant), old nu- tant cells or dead cells should fuil to divide (no development), but mutant ceils of recent crigin should exhibit only one or a few divisions (limited development), if phenomic Lag does in fact occur. intermittent observation cf tie development of many individual cells on an agar surface may be made if each cell can be relecatud on a pride . con~ venient prid olatc may Le constructed as fellows: Ten razcr blades are clam- ed toyether with the edges aligned on @ olene surface. Tie clamped blade edges are used to croSs stam: the dry agar surface of a wc day old plate lightly, yielding a grid uf squaree approximately 9.lim on edge, One drop of a cell suspension (ca 1 x 104 per ml) is »laced dircetly on the «rid. If tue apar is dry the drop is rapidly absorbed and microscopic observation may be vegun &lnost immediately. ‘The mechanical staye is modified to accomodate a petri dish, the corner of the grid is locuted under low und then high power and, as the grid is scanned, each cell or configuraticn (singlet, doublet, triplet etc.) is drawn on a paper faesimile of the grid. .t timed intervuls the grid is scanned and the number of cells in each configuraticn is asain recorded, In- termittent microscopic observation may be made of the cpen olate for 4 hours without visible interference from airborne contaminants, The tendency of growth to become confluent interferes with longer periods of observation, Two plating media have been emloyed. Cne, a plucose nutrient ayar al- lows both respiration deficient (aer) and resriration sufficient (ER) cells tc form colonies, Te other, a lactate nutriont agar allows only AER cells to form colonies (5). When a respiration deficient clonal isolate of strain W940 (6), growing in exponential phase in glucose nutrient broth was trans- ferred to glucose grid plates, essentially all cells or configurations de- veloped to form colonies, aA low frequency (less than 1%) failed to rroduce &@ single bud and were considered dead cells, when aliquots cf the same cul- ture were studied on lactate grid plates, not a single new bud was formed in more than 1000 configurations examined. To test whether the aer cells had died on the lactate agar a doughnut shaped ring cf filter paper impregnated with glucose was placed on the agar surface surrounding the grid. «after some lag essentially all configurations budded on the plates to which glucose had been adied, The aer cells were therefore still viable, Although previous observations (5) had indicated that aer cells were incapable of forming macro- colonies on lactute agar, the observation cf the com: lete failure to form new buds on the lactate grid plate is more critical evidence for the totally se- lective character of the medium ayainst wer cells, It des not, however, yield any information concerning the ability of aer cells recently arisen from aER cells to produce buds in the selective medium, Strain 14940 exhibits a high spontaneous rmtation rate to respiration deficiency when growing exnonentially in lactate nutrient broth at 35°C(ca 40%). The population consists principally of doublets (cells with attached buds) with a lower frequency of singlets, triplets, and quadruplets. when samples of this culture were transferred to glucose grid plates, essentially all con- figurations developed tc microcolonies within 2 hours and to macrocolonies by 48 hours. «a small number (less than 1%) failed to develop at all and war: considered dead cells, On the lactate grid plate, however, many of the con- figurations exhibited limited development. In a population consisting of 36.84% singlets, 54% doublets, 7.8% triplets, ls quadruplets and only 0.3% of any larger configurations, 61% of the singlets, 23% of the doublets, and 17% of the triplets showed limited development. To test whether all the cells in a configuration exhibiting Limited development had died, glucose was added to some of the lactate grid plates after 2h hours via the filter naper ring de- scribed above, By lf hours the cells which had showr limited development had resuned their growth. (n contre] clates the cells which had shown limited . development for 2, hours produced few additional buds between 24 and 48 hours, A strain (14716 x 8256) characterized by a low spontaneous mtation rate exhibited limited development in cnly a small proportion of its cells, The prediction of limited development for considerable number of cells is thus fulfilled only with a strain showing a high mutation rute on a med- dum totally selective apainst the mutant. Further development is produced by delayed supplementation of the medium with a nutrilite which sustains mutant