Grain counting

Three years later, Lajtha, Oliver, and Ellis performed similar studies with human bone-marrow cultures exposed to 32P, or 14C-adenine. Control smears were treated with M HC1 at 60 °C for 6.5 min to remove 32P not incorporated into DNA. Grain counts were made over individual nuclei so that the rate of uptake into DNA could be estimated. The cycle time for the dividing cells in the culture was 40-48 h. DNA synthesis took 12-15 h in the second half of the cycle and was divided from mitosis by a 3-4 h non-synthesizing period (G2). 

Results and Discussion Till samples (about 8 kg of material) were collected up- and down-ice from the deposit, over a total distance of about 20 km (Fig. 2). The samples were put through a shaking table (<2 mm) and gold grain counts were reported. Sieving and heavy liquid (S.G. 2.8-3.2 and >3.2) separation followed. The PCIMs were identified in the 0.25-0.5 mm fraction. 

In cultured mammalian cells, acrylonitrile induced DNA strand breakage, gene mutation, sister chromatid exchanges and chromosomal aberrations, but not aneuploidy or unscheduled DNA synthesis in rat hepatocytes, at least if the silver grain counting method was used. [Studies using the less reliable scintillation counting method have not been summarized.] Cell transformation was induced in several test systems and gap-junctional intercellular communication was inhibited in one study with Chinese hamster V79 cells. 

Process for autoradiography and record the fraction of labelled mitoses, the fraction of labelled cells, and the grain count, i.e. the average number of autoradiographic grains over the labelled cells (Fig. 10.8.). 

The grain count will plateau when the labelled cells have progressed through the whole of S-phase in the presence of [3H]thymi-dine. However, when these cells divide the grains are now shared between the two cells and the grain count will fall. This introduces errors into the estimated /S. Errors also are introduced by the presence of those cells already in S-phase when the thymidine is added or when the cells are harvested. This method therefore overestimates the duration of fS. 

The endogenously synthesised dTTP dilutes the specific activity of the [3H]dTTP formed from the added [3H]thymidine. Thus on adding tritiated thymidine at 3 X 10 8M most of the DNA thymine is synthesised by the endogenous or de novo pathway, but when the [3H]thymidine concentration in the medium is raised to 0.3 mM it contributes 90% or more of the DNA thymine (Cleaver and Holford, 1965 Cooper et al., 1966 Cleaver, 1967). As the specific activity of [3H]dTTP is one of the factors which determine the amount of radioactivity incorporated into DNA (either total counts/min or grain counts) and as this varies (a) with external thymidine concentration and (b) with the state of the cells, the quantitative estimation of rates of DNA synthesis is full of pitfalls. 

Fig. 12.5. Effect of length of autoradiographic exposure on grain count. BHK21/C13 cells labelled with [3H]uridine were covered with Ilford L4 emulsion (diluted with an equal volume of water) dried in a horizontal position and exposed in air at room temperature for the indicated times. (Courtesy of K. Shaw and Dr. J.D. Pitts.)...

Fig. 12.6. Grain count distribution. L cells labelled for 10 min with [3H]thymidine (2.5/1 Ci/ml 0.36 Ci/mmol) and processed for autoradiography using NTB3 emulsion. Those cells (62% of the total) with one or more grains are recorded. A Poisson distribution with a mean of 30 is included for comparison. (Reproduced from Cleaver, 1967, with kind permission of the author.)...

Incubation of a population of cells with tritiated uridine or hypo-xanthine leads to incorporation of radioactivity into the RNA of all but the mitotic cells. Analysis of the grain counts shows these to fall into a Poisson distribution, the median point of which corresponds... 

A cell is considered undergoing DNA repair if the value of the net nuclear grain count is greater than five. 

The biological significance has to be taken into consideration for positive evaluation (i.e. cytotoxicity can artefactually lead to an increase in the value of net nuclear grain count). The positive control should induce a clear increase in the mean net nuclear grain count higher than the threshold value of five. 

Figure 8. a) Number of soil grains that reached the sea floor by the end of the period (0400 JST on October 25, 2011) calculated by the particle tracking analysis described in section 2.5 and coverage of (b) tabular coral and (c) branching coral at Stations 1-26. The sizes of grains counted in (a) were 1, 3, and 5 pm in diameter. The number of soil grains at Stations 17 and 18 in (a) were over 1000 (after [18]). 

UDS in cultured human liver slices was quantified as the net grain count in centrilobular hepaiocyies and as the percentage of ccntrilobular hepalocyte nuclei with >5 and >10 net grains. Control human liver slices were also cultured with dimethyl sulfoxide (DMSO) alone, in order to act as a negative control. 

Net nuclear grain count (nuclear grain count minus... 

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