Chapter 3 Pure Culture and Growth
1. Pure culture
A culture containing only one strain of microorganism, which is usually derived from a single colony.
1.1 Methods of isolating pure cultures
1.1.1 The streak-plate technique
By meansof a transfer loop, a loop of the mixed culture is placed on the surface of an agar medium and streaked across the surface. This manipulation “ thins out” the bacteria on the agar surface so that some individual bacteria are separated from each other.
1.2 The pure-plate and spread plate Techniques
Notes: The pure-plate technique has certain disadvantages:
1) Some of the microorganisms are trapped beneath the surface of the medium when it gels.
2) The microorganism being isolated must be able to withstand temporary exposure to the 45℃ temperature of the liquid agar medium, For instance, the pure-plate method would be unsuitable for isolating psychrophilic microorganisms.
In the spread-plate technique, I ml of the diluted sample is placed onto the surface of an agar plate, and spread evenly over the surface by means of a steril, bent glass rod. In contrast to the pure-plate method, only surface colonies develop; moreover, the microorganismbeing isolated is not required to withstand the 45℃ temperature of liquid agar.
1.1.3 Micromanipulator technique
A device called the micromanipulator can be used in conjunction with a microscope to pick up a single cell ( or spore) from a mixed culture. This technique permits the operator to control the movements of a micropipette or a microprobe (a fine needle) so that a single cell (or sproe) can be isolated. This technique requires a skilled operator.
1.2 Miaintenance and preservation of pure culture
Most microbiology laboratories maintain a large collection of strains, frequently referred as a stock-culture collection. These sterains are needed for research work and for practical usage. The two international famous culture collections are ATCC and USDA.
1.2.1 The objective of maintenanceand preservation of pure culture
● Preservation at 4℃
The strainsare stored in nomal refrigerator. Periodic transfer to fresh media is needed.
Advantage: simple, convenient to use.
Disadvantage: It can not prevent changes in their characteristics due to genetic mutations.
● Freeze-Drying method
Most microorganisms die if cultures become dry, although spore-and cyst-formers can remain viable for many years. However, freeze-drying can satisfactorily preserve many kinds of microorganisms that would be killed by ordinary drying. In this process a dense cell suspension is placed in small vials and frozen at –60~78℃. The vials are then connected to a high-vacuum line for dehydration. The vials are sealed off under a vacuum and stored in a refrigerator. The freeze-drying cultures can be revived by opening the vials, adding liquid medium and transferring to a suitable growth medium.
Disadvantage: The method is complicate. There may be small damage to cell structure.
Advantage: The storage time can be very long. Many species preserved by this method have remained viable and unchanged in their characteristics for more than 30 years.
It is convenient for exchange between different laboratories and organizations.
● storage at -70℃~80℃
20% glycerol is added to the liquid medium to prevent cell damage due to ice crystal formation
● storage using liquid nitrogen (-150℃)
● storage using sand
● storage using oil
2. Quantitative measurement of microbial growth
2.1 Enumeration of cell numbers
2.1.1 Total count
● Direct Microscopic Count
2.1.2 Viable count
● The plate-count method
● Most probable number, MPN
2.2 Determination of cell biomass and cell activity
● Determination of the dry weight of cells
●Determination of nitrogen content, DNA, ATP
● Determination of metabolic activity.
A. The lag phase
The addition of inoculum to a new medium is not followed immediately by a doubling of the population. The population remains temporarily unchanged. There is a lag in cell division. But this does not mean that the cells are dormant. On the contrary, during this stage the individual cells are very active, and synthesizing enzymes and molecules required for cell division. Time for adjustment in the new environment is needed. At the end of the lag phase, cells begin to divide. However, since not all cells complete the lag phase simultaneously, there is gradual increase in the population until the end of this period.
B. The logarithmic or exponential phase
During this period the cells divide steadily at a constant rate, and the log of the number of cells plotted against time results in a straight line. The increase in population is by geometric progression.
Moreover, the population is most nearly uniform in terms of chemical composition of cells, metabolic activity, and their physiological characteristics, so log-phase cultures are commonly used for studies of microbial metabolism.
Generation time (G): the time required for the population to double. The generation time can be calculated by the following formula:
N: the number of generations;
N the viable number of bacterial cells at time o;
Nt: the viable number of bacterial cells at log phase and at time t.
Growth rate ( R ): the number of generations per hour.
C. The stationary phase
The viable number of bacterial cells remains constant for a time. The reproduction rate is balanced by an equivalent death rate. This can be attributed to a variety of circumstances, particularly the exhaustion of some nutrients, and the production of toxic products during growth.
D. The decline or death phase
Following the stationary phase the bacteria die faster than new cells are produced, the number of viable cells decreases exponentially, essentially the inverse of growth during the log phase. A variety of conditions contribute to bacterial death, but the most important are the depletion of essential nutrients and the accumulation of inhibitory products, such as acids.
Batch culture: A closed-system microbial culture of fixed volume.
4 Synchronous Growth
◆ Growth in a cell population in which all cells divide at the same time.
◆ The significance
It is not feasible to analyze a single bacterium because of its small size. However, if all the cells in a culture were to be in the same stage of the division cycle, the result from analysis of the cell crop could be interpreted as that for a single cell.
◆ Methods to obtain synchronous culture
① The most common method of synchronization takes advantage of the fact that the smallest cells in a log-phase culture are those which have just divided. When those cells are separated out by filtration or by differential centrifugation, they are reasonably well synchronized with each other.
② A cell population can be synchronized by manipulating the physical environment or the chemical composition of the medium. For example, the cells may be inoculated into a medium at a suboptimal temperature. If they are kept in this condition for some time, they will metabolize slowly but will not divide. When the temperature is subsequently raised, the cells will undergo a synchronized division.
5 Continuous culture
◆ In both experimental research and in industrial processes, it is often desirable to maintain a bacterial population growing at a particular rate in the exponential or log phase. This condition is known as steady-state growth. The culture volume and the cell concentration are both kept constant by allowing fresh sterile medium to enter the culture vessel at the same rate that “ spent ” medium, containing cells, is removed from the growing culture. Under these conditions, the rate at which new cells are produced in the culture vessel is exactly balanced by the rate at which cells are being lost through the overflow from the culture vessel.
◆ One type of system that is widely emploged for continous cultivation is the chemostat. Another type is the turbidostat.
◆ The batch culture and continous culture.