or darkness, the free movement comes to rest. The diatoms then excrete an EPS pad, which they use to adhere to the substrate. At light intensities, as utilized in cultivation, the effect of colony reduction is less than demonstrated in Figure 1.36, but still clearly recognizable. For Figure 1.37 a culture in light and dark phase was photographed with a daytime cycle of about 12.5 hours of light per day. To take pictures in the dark phase, the culture was illuminated with low intensity from below through a diffusing screen with a white LED. The light intensity in the bright phase was about 200 lx in the dark phase 15 lx. Probably because of this remaining brightness the movement never comes to rest completely. It is striking that all diatoms moving during the day have reconnected to a colony with a few exceptions.
Figure 1.35 Movement activity of diatoms between colonies.
Figure 1.36 Colonies at the beginning of intensive light irradiation (a) and after about two hours (b).
Figure 1.37 Cymbella culture in the light phase (a) and dark phase (b).
To qualitatively and quantitatively study the formation of colonies and the daytime variations of diatoms in and outside the colonies, a culture was observed over 24 days from the time of inoculation, with lighting conditions corresponding to those just mentioned. Another cell line with a diatom size slightly larger than 100 μm was used. Pictures were taken every 10 seconds. The visible area amounted to 8.27 mm × 6.21 mm, corresponding to 2.6% of the cultivated area.
A new colony can develop when a single diatom attaches itself to the substrate. It creates an adhesive area where other diatoms can get stuck. Many locations where single or a few diatoms adhere during the night are left by all diatoms in daylight. Due to the low water solubility of the excreted jelly, these areas are often an “anchorage” in the following dark phases. After a few days without settlement, the adhesion of such areas seems to decrease. Larger colonies do not disintegrate completely in the light phase. To quantify the growth of culture and colonies, 60 temporally equidistant images per day were converted to binary images after setting a brightness threshold. With the help of Fiji open-source software [1.32] particle analysis, the sizes of all connected image parts were determined for each image. The result is a list of areas ranging from individual diatoms to the largest colony. To estimate the number of freely moving diatoms and the diatoms bound in colonies, a classification was made:
• Areas smaller than a lower threshold of a few pixels (about 1 to 4 pixels) are caused by small particles and unclean boundaries and are sorted out.
• All objects larger than an upper threshold (in this evaluation, 70 pixels) are considered as colonies.
• All objects in between are interpreted as individual diatoms.
This classification may be incorrect, for example, if several diatoms overlap in the image and, due to their projection, occupy such a small area that they are categorized as one cell. An exemplary validation shows that this does not lead to significant distortions of the result. The total area occupied by diatoms shows notable daytime fluctuations. Diatoms bound in colonies occupy on average a different area than the diatoms between the colonies, because diatoms in colonies can overlap in the image and often do not lie horizontally. The area occupied by diatoms in colonies also does not grow linearly with the size of the colonies. In rough approximation a linear conversion factor of the areas has been determined. The criterion used was the minimization of the daytime fluctuations of the total number of diatoms. Apparently, the areas of the individual diatoms outside the colonies appear surprisingly small, as can be seen when explicitly comparing images in darkness and subsequent brightness (Figure 1.38). With the beginning of the bright phases, the number of individual diatoms increases rapidly. It falls off again in the course of the bright phase. Conversely, the area of the colonies is reduced. Figure 1.39 shows the total number of diatoms resulting from the sum of the two curves discussed. Compared to Figure 1.38, the daytime fluctuations are smaller, but still considerable.
On the one hand, this is due to the assumed and only roughly fulfilled proportionality of the area of the colonies and the number of diatoms contained therein, and on the other hand due to fluctuations of the number of moving diatoms caused by the leaving and entering of the region of interest. The second effect dominates in the first days, in which the formation of the colonies just begins. The culture is in good approximation in exponential growth.
The number of diatoms between the colonies can be determined by the described classification of the sizes of the connected clusters of an image. This is not identical to the number of diatoms that move, as some diatoms remain in place, especially at low brightness. When one superimposes two images whose recording times differ so much that moving diatoms have covered a distance of at least their own length, then they can be seen twice in this picture. The respective number of individual diatoms is obtained by particle analysis of one of the initial images and the superimposed image. The number of moving diatoms results from the difference. To improve the numerical quality, not only two, but six images were superimposed at one-minute intervals and the difference in the number of individual diatoms compared to the first image was divided by five, as moving diatoms appear five times in addition.
Figure 1.38 Number of free diatoms (blue) and the number of diatoms bound in colonies (red) over 24 days. A yellow bar indicates the phases of bright light.
Figure 1.39 Total number of diatoms (red) with exponential fitting (blue).
Figure 1.40 Number of diatoms in motion over the last 10 observation days.
Errors in this analysis mainly occur when diatoms do not continually move during the analyzed period (for example, by connecting to a colony) or when they cross the boundaries of the region of interest. Figure 1.40 shows the number of moving diatoms over the last 10 days of the observation time. Apparently, the movement activity drops off quickly after the beginning of the light phase. One can observe Cymbella species, which show high movement activity