9. Holleman's Experiments, 1987 - 1989. (Experiment VI)

9.1. Materials and Methods

As stated in section 6.3.3 this final phase of Holleman's research led him to examine his algae in greater depth. Nevertheless developments were made in other areas as well.

9.1.1 Overview

Experiment VI, which was the only attempt at a replication of experiment II during this period, was incomplete and very few details were recorded. See section 6.3.3 for a somewhat speculative consideration of this experiment.

9.1.2. Apparatus and Materials

9.1.2.1. Lighting

The first lights used were fluorescent "double lamps"[?] of 9W and gave out 600 lumen, situated 7.5cm from the rack of culture tubes. A second lamp illuminated the other side of the rack.

On the basis of measurements by Sorokin and Myers (1950) the best lighting intensity for Chlorella vulgaris at 25 degrees was given as 2700lux (250f.c.). Accounting for losses due to the passage of light through the quartz wall, etc., the test tube rack was now 20[cm? from the lights??] so that the lighting was 2800Lux. No comparative lighting experiments were recorded.

9.1.3. Measurement of Cell Numbers

Cell numbers were counted directly under the microscope with the use of a Neubauer counting chamber [presumably such as is used in counting blood cells]. A sample of 0.1ml of the homogeneous culture was taken for this purpose. If necessary the culture was mixed using a rotor mixer. The counting chamber was delineated into a grid of squares. The number of cells present in each of sixteen of the grid squares was counted.

9.1.4. Procedure

As stated earlier, the information on experiment VI and other tests and experiments of this time were incomplete and details were limited or absent. See section 6.3.3 for details.

Any samples taken from cultures for chlorophyll measurement, etc. during growth were of 0.1ml in volume and were replaced by an equal amount of nutrient solution.

9.1.4.1. Light / Dark Regime

The regular implementation of a particular light/dark rhythm was proved able to induce a high degree of synchronicity of cell division. A number of light/dark rhythms were used, e.g. 8:12 hours light:dark (Ruppel 1962); 14:10 (Lorentzen 1963, Lorentzen and Schleif 1966); 9:15 (Sorokin [?]); 15:9 (Sorokin [?]); and 10:14 hours light:dark.

9.1.4.2. Ashing and Reconstitution of Nutrient Solution

The ashing procedure described in section 8.1.4.2 was further simplified. This prevented the potentially corrosive production of hot steam (section 6.3.2) and reduced the need for a second ashing. The culture was evaporated as normal in the aluminium block with filtered air blown over. It was then further dried in the oven at 120 degrees for one hour followed by one hour at 250-300 degrees and one hour at 480 degrees centigrade. After cooling the ashes were dissolved in distilled water and the above procedure repeated if necessary. Nitrate ions were added in the form of nitric acid. The possibility of adding Na2HPO4 [instead/as well?] was also considered (see section 10.1.5.2 for a further consideration).

9.1.4.3. Chemical Analysis

Holleman stated his intention to measure pH, nitrate, carbohydrate, protein, potassium and nitrogen. It is not clear whether any were actually measured. The element potassium was to be measured by the Kalignost method. This, as Holleman demonstrated both here and elsewhere, was not an easy test to perform.

Ash hydrolysis for analysis differed slightly from section 8.1.5.4 in as much as 0.2N HCl was used [the volume was not recorded]. The ash solution was then topped up to 10 [or 25?]ml with distilled water.

9.2. Results

No analysis was conducted of the results recorded in this section. The recorded results concerned dry weight of Chlorella after growth; optical density at the Walker dilution of 1:26 measured at 600nm; and cell counts, often of the same diluted optical density samples.

9.2.1. Synchronicity of Chlorella Cultures

The synchronicity of Chlorella cultures is at its most simplest, based on a measure of the relative proportion of cells undergoing cell division at any one time. This was easily measured [by myself from Holleman's cell count/size data]. A cell shortly before division is very much larger than cells immediately after division. Two cultures were sampled shortly after the majority of cells had undergone cell division; the numbers of large cells and the total numbers of cells within each of the grids were counted. The degrees of synchronicity were calculated to be 95% and 98%.

9.2.2. Health of the Cultures

In general Holleman considered that the cultures showed less clumping and sinking, as well as a greater uniformity between culture tubes than in previous experiments. A number of quantitative as well as qualitative observations were made.

The qualitative observations were as stated in sections 6.3.3 and 9.2. Under the microscope Holleman was able, not only to count, but also to observe the general behaviour of individual living cells. Cell densities were observed to vary from starting cultures of a few million cells per millilitre to fully grown cultures of up to a few thousand million cells/ml. Cell division was observed. For Chlorella this involves the production of so called autospores. A single reproducing cell can generally produce 2 or 4 or 8 or 16 daughter cells [and numbers in between] in a single division. Holleman often observed clusters of 3 or 4 small [post reproduction] cells. On one occasion he believed he witnessed the explosive production, from a large mother cell, of 4 small daughter cells.

A small but important experiment involved the sampling, after mixing/stirring, of algal culture from the top, middle and bottom of the culture suspension. The counts were 5.6x106, 17.7x106 and 29.6x106 cells/ml respectively.

Another small experiment examined the effects of an increase in nitrate concentration. Whilst a moderate increase led to a higher cell concentration, a large increase led to the development of a lower than expected cell count.