Synchronous culture: Definition, Methods, Application
Synchronous culture is a culture in which all cells divide simultaneously with a constant division rate so that all the cells are in the same stage of the growth phase and all the cells are dividing at the same time, this pattern of growth is known as synchronous growth. Many problems concerning cell multiplication that cannot be studied on conventional cultures may be solved by using the cells dividing more or less in similar ways. There are many laboratory methods by which one can manipulate the growth of cells so that they are all in the same phase of their growth cycle.
Methods of obtaining synchrony
Several means of obtaining synchrony have been devised which are as follows —
|I) Induction method||II) Selection of cells|
|1) Low temperature||1) Low temperature|
|2) Effect of single temperature shift||2) Effect of single temperature shift|
|3) Cyclic temperature shift|
|4) Thymine starvation|
|5) Inhibition of RNA and protein synthesis|
|6) Glucose starvation, periodic supply of growth factors
I) Induction methods
1) Low temperature
i) A log culture of Pseudomonas which is taken from 37-25°C, held at low temperature for 155minutes has been found in phase with two respects-
a) During 5 minutes, following 25°C about 2/3 of the cells divide for two generations and thereafter cell division remain partially synchronized
b) after returning the cells to 37°C, their susceptibility to the transforming effect of Pneumococcal DNA changes cyclically.
ii) In the case of Bacillus megaterium it was further observed that freezing young cells produce a characteristics compact nuclear composition and as well as synchrony uniformly in all cells. Thus a culture of Bacillus megaterium growing at 34°C which is cooled to 15°C and held for 30 minutes at that temperature and then returned to 34°C will divide synchronously after a lag of about 40 minutes. This division may be followed by a new lag and then a second burst of cell division. The cells will continue to grow but for many hours the cells will not divide and DNA: RNA ratio slowly increases and growth is underbalanced for some time.
2) Effect of single temperature shift
In the case of Salmonella tphimurium when the temperature is rapidly increased from 25°C to 37°C the following dissociation pattern occurs. The rate of cell vision remains unchanged for about 30 minutes, after 30 minutes a burst of cell division occurs. The rate of RNA synthesis and mass increased almost immediately at that level. the DNA synthesis is greatly enhanced and the total DNA increase by about 50%.
3) Cyclic temperature shift (25°C to 37°C)
Cell division is particularly dependent upon temperature, so a lower temperature, growth of cells is arrested and gives a chance for more tardy members to catch with others i.e. to come to other’s level and when the temperature is suddenly changed or raised, the barrier of cell division is lifted and virtually entire population proceed to divide so by repeating several stages entire population can be maintained. The primary effect of the shift from 25°C to 37°C is specific stimulation of DNA synthesis.
Example – When a culture of Salmonella typhimurium is subjected at fixed intervals to alternate growth cycle at 25°C and 37°C. the entire population will divide synchronously. Cell mass increases at both temperatures but the rate at 25°C(20-30 minutes) is low as 37 °C (8 minutes) is closer to the optimal one where the synchrony can persist for several generations.
4) Thymine starvation
- This method is based on the observation that certain proteins are required before a round of replication can be initiated. However, once started the round runs to completion even if the cell is temporarily unable to synthesize proteins.
- The experiment can be done by starving the culture for a required amino acid (thymine) long enough for all cells to complete replication then the culture is switched over to the medium permitting protein but not DNA synthesis.
- The immediate effect of withdrawal thymine is the prevention of DNA synthesis without affecting RNA and protein synthesis.
- During the second starvation of 30 min in glucose salt medium, protein synthesis is progressed thus preparing the cell for replication and if thymine is added back at on right time.
- Replication synchrony ensues and after a short lag a burst of cell division is observed, thus when protein synthesis is blocked, replication terminates very soon in some and much later in other cells.
- The DNA: Mass ratio, therefore remain almost in the former, whereas if nearly doubled in cells that go through most of their replication cycle while starved of thymine.
- Thymine starvation has certain effects in common with cyclic pretreatments to obtain division synchrony, in both systems pre-treatment allows cells to grow an abnormal and causes than to under large rapid division.
5) Inhibition of RNA and protein synthesis
- In bacteria, DNA synthesis will continue for some time after RNA and protein synthesis has been initiated.
- This inhibition can be effected by transferring cells growing in rich media to minimal media by withdrawing the required was or by the addition of chloramphenicol to media.
a) DNA produced under protein and RNA inhibition represents the completion of already initiated replication.
b) It seems that protein and RNA synthesis is required to initiate a new replication cycle.
This theory predicts that the cells at the time of inhibition had almost finished replication and continued DNA synthesis for short time, whereas cells with had just begun replication should continue synthesis for a longer time and effectively double the DNA under conditions of inhibition of protein and RNA synthesis.
6) Glucose starvation
Division synchrony has been obtained in yeast culture which after the glucose has been exhausted, is diluted by a factor of two with fresh medium at regular intervals. Division synchrony can be obtained by transferring cells to a medium in which carbon source, glucose has been exhausted to fresh glucose salt medium. This lag is regularly observed before the first division. The transition from resting phase to exponential growth under certain conditions can be made to above with a fair degree of division synchrony. A combination of chilling and glucose starvation was used to obtain two rather than synchronized division cycles. Pretreatment may cause fractionation of cells to keep division at a certain time.
II) Selection of cells
1) Filtration (Hemstelfer Cumming’s)
This method involves the continuous collection of young cells coming from the lower surface of a filter onto which a parent population is adsorbed. In this Millipore cellulose acetate filter of the same pore size is used. The pore size is so adjusted that it permits selectively passage of the smallest cells of the same size. The synchronous culture is observed on the filter paper pad, then the filter is removed and placed in such a way that bacteria facing to lower side, fresh medium is supplied, the loosely attached cell will be washed out and attached cells remain and divide. Thus filtrate collects young cells of the same size and age. But filtrate should be collected within 1 minute otherwise cell division will start and a large age difference may occur.
2) Centrifugation (sucrose gradient)
When concentrated cell suspension of E.coli is layered on top of sucrose solution of specific gravity, 1.245 column then centrifuge, the smallest cell tends to accumulate at the interface, immediately after centrifugation, large cell fraction is nonviable due to osmotic pressure but incubation in small without glucose for 20-minute result in partial recovery subsequent addition of glucose produces a fairly synchronous growth after a lag of 20 minutes. Because of the osmotic injury suffered during centrifugation, however, this proved
Significance of synchronous culture
- Measurement made on randomly dividing population. Do not permit any conclusion about the growth behavior of individual cells, because the distribution of individual cells size and age is completely random but synchrony maintains the entire population uniformly with respect to the growth phase. Thus measurements made on such culture are similar to the measurement made on individual cells.
- Synchrony facilitates to analyze growth behavior i.e. differentiation, organization, macromolecule synthesis of uniform culture which also implies the same at individual cells level thus time course pattern of various macromolecules (DNA/protein synthesis) synthesis can be studied by removing portions of synchronously dividing culture and analyzing the cells for the component or enzyme activity under investigation.
- Usually, synchrony is short-lived but can be maintained indefinitely for a longer time.
Applications of synchronous culture
1) Studies on kinetics of biosynthesis of macromolecules – Such induction to obtain synchronous growth is used in the study of the kinetics of biosynthesis of macromolecules.
2) Studies on the effect of disinfectant on macromolecules – Such culture can also be used in the study of certain disinfectant effects on the protein. DNA, RNA, and other macromolecules.
3) Metabolism and research studies – Synchronous culture is useful for metabolism and research studies.