Synchronous Cultures of Micro-organisms

Introduction

 Synchronous culture technique enables separation of the smallest cells from an exponentially growing culture. This separation is achieved by passing the culture through a continuous flow centrifuge rotor under controlled conditions. The controlled conditions represent the seed of the rotor and flow rate. The separated smallest-sized class of organisms, which is suspended in their growth medium, remains in the effluent.  The growth of these cells is not disturbed throughout the procedure. This culture exhibit synchronous cell division.

This technique has been successfully applied to budding and fission yeasts, amoeboid, ciliated protozoa. This application of this procedure can be extended to any non-filamentous, non-aggregating, unicellular organism or to cells of higher plants or animals growing in liquid tissue-cultures.

Preparation of synchronous cultures can be broadly classified into two types: The first type induces all the cells of the culture to divide synchronously by some physical or chemical treatment.  This type is known as induction synchrony The other type selects cells at a particular stage of the cycle. The selected cycles are allowed to grow through their natural cycle. It is termed as selection synchrony. If a study needs to study of them in normal cell cycle selection synchrony is preferred as induction synchrony involves metabolic stress or interference.

Differential sedimentation of the exponentially growing cell population through a sucrose density gradient is the widely used method for selection of the smallest cells of a culture. This method is seriously limited by yield. Scale up of this process with better yield is achieved using zonal rotors which generate greater gradient capacity. The time taken for this separation is usefully high around 1 hour. During this  The organism is exposed different growing conditions such as sub-optimal temperature and anaerobic conditions.

The high osmotic pressure became a limiting factor in this modification. Separation of osmotically fragile organisms cannot be separated by this method. Even the substitution of high-molecular-weight gradient media to eliminate high osmotic pressures does not prevent distortion of the first subsequent cell-cycle. Rapid motile organisms cannot be separated or size selected using velocity sedimentation. Maintaining aseptic conditions are almost impossible. Even the alternative approach of cell-cycle fractionation also had similar problems but the cell yield is high during the use of large-volume zonal rotors. The difficulties discussed above are eliminated in the below-described method. The applied preparation has been successfully carried on for a period of two years.

 Methods & Organisms

Maintenance, growth, and harvesting of the organism

S. pombe 972h- was maintained and grown on a defined medium containing 1 % (w/v) glucose. Batch cultures (4-6 litres) were grown in a 6-liter capacity. Forced aeration was at 1 liter of air/min per liter of culture, stirring rate 400 rev./min and the growth temperature was 30°C. The exponentially growing culture was harvested during the phase of glucose repression when the population reached 2.0×107– 3.0 x 107 cells/ml, by centrifugation at 2000rev./min in the 4xl-liter rotor of an International centrifuge at 4°C. All subsequent operations were performed at this temperature.

 Candida utilis

 Stock cultures of T. utilis were maintained on slopes of Saboraud’s agar at 20°C. At about 2-monthly intervals subcultures were made, incubated at 30°C for 48 h and then stored at 20°C. To prepare an inoculum for initiating a continuous culture, a recent slope culture was recultured on a plate of Saboraud’s agar at 30°C for 48h. A suitable colony was then removed with a sterile platinum loop and used to inoculate 100 ml of glycerol medium with or without added FeCl3, as appropriate.

Tetrahymena pyriformis strain ST

Tetrahymena pyriformis strain ST was used throughout because, unlike strains T, w and GL, it stores little glycogen and isolated mitochondria did not show the instability of oxidative and phosphorylating activities previous reported for other strains. The organism was maintained in 75 ml. growth medium (250 ml. flasks) at room temperature and was transferred every 10 days to fresh medium. The growth medium contained 2 % (w/v) protease peptone , 0.1% (w/v) liver digest and 0.05 %(w/v) silicone MS antifoam RD.  The first two components were dissolved in a quarter of the final volume of distilled water, heated to 1200 for 15 min., cooled and centrifuged at 200g for 20 min. to remove suspended solids. The supernatant fluid was decanted, diluted by adding three volumes of distilled water, the antifoam added and the pH adjusted to 7.2 with KOH. The medium was then sterilized by autoclaving at 15 lb/in2 for 20 min. Growth of the organism. Cultures were grown at 29° under forced aeration through a glass tube without a sparger. There was approximately a 5 h. lag phase on inoculation of fresh growth medium with exponentially growing cells (1 day old), and this was followed by a phase of logarithmic growth to give a population of 70,000 to 80,000 cells/ml. at a rate of 0.44 h/l: the growth rate then became slower and the population finally reached 3 to 5 x 105 cells/ml.

Acanthamoeba castellanii

 Cysts produced by the replacement technique were stored as suspensions in distilled water at 4°. The cysts were collected by centrifuging at 2000 g for 10 min. at laboratory temperature and were washed three times with sterile distilled water. Experiments were normally carried out in 50 ml. or 250 ml. conical flasks containing 10 ml. and 50 ml. of culture, respectively. The flasks were incubated in a reciprocal shaking water bath at a shaking rate of 100 cycles/min. at 30°. Oxygen uptake was measured on a Gilson differential respirometer. Respirometer flasks contained 5 mg. dry wt cysts in 2.8 ml. 4 % (w/v) mycological peptone. The gas phase was air, and CO2 was adsorbed by 0.2 ml. 20% (w/v) KOH. Flasks were shaken at 150 cycles/min. at 30°. Acid phosphatase activity was assayed. Turbidimetric measurements were carried out on a Unicam SP-600 spectrophotometer.

Crithidia fasciculata

The growth medium contained 2 % (w/v) proteose peptone, 0.1 % liver digest, 1 % (w/v) glycerol, 0.5 % Tween 80 and 0.6 %  triethanolamine. Half the triethanolamine and a11 the other components were dissolved in a sixth of the final volume of distilled water, heated to 100 °C, and centrifuged at 2000 g for 15 min to remove suspended solids. The supernatant was decanted and 25 mg haemin/l dissolved in the rest of the triethanolamine (made up as a 50 %, v/v, solution) were added. Folk acid dissolved in the minimum volume of  1.0 M-KOH was added to give 2-5 mg/l. The medium was adjusted to pH 8.0 to 8-2 with 2.0 M-HCI, made up to the final volume with distilled water and autoclaved (103 kNm-2 for 20 min) in 200 ml amounts in 1 L conical flasks or in 6 L amounts in a 14 L. The organism was maintained in tubes containing 10 ml of this medium or on slopes of medium containing 2 % agar. The growth of organisms was measured by counting in a haemacytometer slide. Cultures were inoculated with organisms from the late exponential phase of growth; the initial population was 2 x 106 organisms/ml. Cultures were grown at 29°C in a rotary orbital shaker for 48 h at 150 rev./min, or in the Fermentor with a stirring rate of 90 rev./min under forced aeration at 1 L- air/l medium/min. Sterile silicone MS antifoam RD (0.05 %) was added to prevent foaming. The population at the stationary phase of growth was 2 to 2.5 x 105 organisms/ml; the doubling time in the exponential phase of growth was 5 to 5.5 h. Except where otherwise stated, cultures were harvested in the late exponential phase of growth when the population was 105 organisms/ml. Harvesting was by centrifugation at 4 °C for 10 min at 1500 g in the 6 x 250 ml rotor of an MSE centrifuge or at 1500 g in the 6 x 11 rotor of an MSE Mistral centrifuge. Organisms were washed in 20 mM-potassium phosphate buffer (pH 8-0) and finally resuspended to a known density in this buffer.

The major carbon source in the yeast cultures was glucose. Cell numbers. Cells were counted in a Thoma hemocytometer slide or, for T. pyriformis in a Sedgewick-Rafter cell.

Centrifugation

The culture (mid- to a late-exponential phase of growth) was siphoned from the growth vessel through a ‘continuous action rotor’ fitted with a high-efficiency polypropylene insert running in a High Speed 18 centrifuge. This rotor achieves rate-separation of suspended particles during flow through the main compartment and through the vertical holes in the insert. The maximum flow rate is 2 l/min, and maximum rotor speed 18000 rev./min; suspended particles may be collected until a packed cell volume of 300 ml has accumulated in the rotor. Suitable flow rates (in the range 300 to 2000 ml/min) were provided by inclusion of one of a series of calibrated tapered glass tubes in the inlet gravity feed. Accurate control of rotor speed at less than 1800 rev./min was by means of a ‘low-speed zonal control’ ancillary circuit fitted to the centrifuge. Preliminary experiments established optimum conditions for the retention of about 90 % of the cells of a growing culture; those for a range of eukaryotic micro-organisms are given. Rotor effluent (containing the smallest cells of the culture) was aerated at the temperature of growth and provided the starting material for synchronous growth. The whole procedure could be carried out aseptically by autoclaving the rotor after plugging its ports with cotton wool and using a liquid seal in the rotor lid.

Assessment of synchrony. The degree of synchrony was assessed by the synchrony index, F, calculated from the equation:

equation synchronous theory where F has a maximum value of 1.00 in a culture exhibiting theoretically perfect synchrony, N is the number of organisms at time t, No the number of organisms at zero time, and g the mean generation time.

In the presentation of results, vertical lines indicate the mid-points of doublings in cell numbers, and Fl and F2 denote the synchrony indices of the first and second doublings in cell numbers, respectively.

Discussion

For each of the first four organisms, cell counts doubled in numbers over a time interval which was short compared with the mean generation time. The achieved synchrony of cell division was satisfactory. The mean generation time of exponentially growing cultures and their duration of the cell cycle is similar for all cultures.   For C. fasciculata the degree of synchrony was not good; only 45 % of the organisms underwent division because many of the smallest cells were not viable.

Characteristics

  • Changes in temperature do not impact growth.
  • Fall in oxygen tension does not impact growth, as the culture was only in the rotor for a few seconds.
  • As organisms are not removed from the medium, nutrient status is preserved.
  • Establishment of synchronous cultures is achieved within a few minutes.
  • This method can be applied to large volumes; for large cultures.
  • Rapid flow rates (up to 2 l/min) are necessary to reduce the total time of collection to a minimum, especially for organisms with short cell-cycle times. The above-described method was successfully applied to a culture volume of 20 liters.
  • Also, this procedure can be carried out under strictly aseptic conditions. This technical characteristic can be used in the separation of slow-growing organisms in complex growth media.
  • The growth can be carried out uncontaminated for long periods having an upper limit of 24 h.
  • A Lowering the shear stress as low as 60 Nm-2 have been achieved to disrupt amoebae exposed to mechanical stress for periods of the order of 1 ms. This property makes this procedure useful for an easily disrupted organism as A . castellanii. This suggests the liquid shearing forces generated are not sufficient to damage even the most fragile cell-types.
  • This procedure can be used with any centrifuges given they are fitted with a variable speed-control rheostat.
  • Increasing the path length of a continuous flow zonal rotor does not improve the synchrony indices. Also, the problem of contamination increases with long path length.
  • Size variation of the cells of the same stage of the cycle limits the maximum useful size resolution.
  • The application of this method, to any non-filamentous, non-aggregating organism, is not restricted to size, shape or motility given smallest cells of the culture is viable.
  • This procedure is even applied to mammalian cells in tissue culture.
  • A maximum rotor speed (18000 rev./min) is adequate for the size-selection of bacteria.
  • Limitation of this method is, defined in terms of cell density,  sufficient limiting nutrient must remain to support 10 % of the population through one further cycle.
  • The election must be carried out before the original culture attains a population one generation before the stationary phase of growth.

Applications

Physical Methods For Obtaining Synchronous Culture

According to the organism employed methods used for synchronous culture techniques purpose differ. A rhythmic growth is achieved in an early phase of growth resulting from inoculation of aged cells of Protozoa into a fresh medium. The same characteristic is shown in case of bacteria and for yeast. Cycling temperature in the culture has also resulted in the synchronous growth of bacteria and of Protozoa. Also, synchronization of cell division is induced by proper regulation of the growth medium using wild-type and a deficient Escherichia coli strain B. The achievement of synchronous growth is obtained by intermittent illumination in case of an autotrophic alga, Chlorella.

The above-mentioned methods are based on the physiological conditioning of the microbial cells, This can lead to the possibility of synchronous cells obtained may have some abnormality in their physiological pattern. Fractional sedimentation and fractional filtration were tested for the separation of larger (mature) and smaller (immature) cells present in a logarithmic phase culture of E. coli are the methods tested to counter the physiological abnormalities formation. Methodology and inference of this procedure is discussed in this application part.

Materials & Methods

E.coli strain B was cultured in a medium containing NH4Cl, 1 g; Na2HPO4 12H20, 18 g; KH2PO4, 1 g; MgSO4.H20, 0.2 g; glucose, 1 g; and 0.01 g of “tween 80” in 1 L of distilled water adjusted to pH 7.4. Cells grown in this medium were inoculated into a fresh medium and when the number reached 108 cells per ml at 37 C, the culture was centrifuged at 4,000 rpm for 12 min. The sedimented cells were used for the experiments to be described. Viable counts were carried out by the capillary tube method. The standard error of this method was 11 per cent under the present experimental conditions.