Pure culture techniques-Streak, Spread, pour plate methods

Isolation of Pure Cultures

In natural habitats microorganisms usually grow in complex, mixed populations with many species. This is a problem for microbiologists because a single type of microorganism cannot be studied adequately in a mixed culture. A pure culture which is a population of cells arising from a single cell is needed to characterize an individual species. 

Pure cultures are so important that the development of pure culture techniques by the German bacteriologist Robert Koch transformed microbiology. Within about 20 years after the development of pure culture techniques most pathogens responsible for the major human bacterial diseases had been isolated.

There are several ways to prepare pure cultures. Pure cultures usually are obtained by isolating individual cells with any of three plating techniques: the spread-plate, streak-plate and pour-plate methods.

The spread-plate and pour-plate methods usually involve diluting a culture or sample and then plating the dilutions. In the spread-plate technique, a specially shaped rod is used to spread the cells on the agar surface; in the pour-plate technique, the cells are first mixed with cooled agar-containing media before being poured into a petri dish.

The streak-plate technique uses an inoculating loop to spread cells across an agar surface.

 

The Spread Plate and Streak Plate

    If a mixture of cells is spread out on an agar surface at a relatively low density, every cell grows into a completely separate colony, a macroscopically visible growth or cluster of microorganisms on a solid medium. Because each colony arises from a single cell, each colony represents a pure culture.

 The spread plate is an easy, direct way of achieving this result. A small volume of dilute microbial mixture containing around 30 to 300 cells is transferred to the center of an agar plate and spread evenly over the surface with a sterile bent-glass rod. The dispersed cells develop into isolated colonies. Because the number of colonies should equal the number of viable organisms in the sample, spread plates can be used to count the microbial population.

 

    Pure colonies also can be obtained from streak plates. The microbial mixture is transferred to the edge of an agar plate with an inoculating loop or swab and then streaked out over the surface in one of several patterns. After the first sector is streaked, the inoculating loop is sterilized and an inoculum for the second sector is obtained from the first sector. A similar process is followed for streaking the third sector, except that the inoculum is from the second sector. Thus this is essentially a dilution process. Eventually, very few cells will be on the loop, and single cells will drop from it as it is rubbed along the agar surface. These develop into separate colonies. In both spread-plate and streak-plate techniques, successful isolation depends on spatial separation of single cells.

 

 

The Pour Plate

Extensively used with procaryotes and fungi, a pour plate also can yield isolated colonies. The original sample is diluted several times to reduce the microbial population sufficiently to obtain separate colonies when plating. Then small volumes of several diluted samples are mixed with liquid agar that has been cooled to about 45°C, and the mixtures are poured immediately into sterile culture dishes. Most bacteria and fungi are not killed by a brief exposure to the warm agar. After the agar has hardened, each cell is fixed in place and forms an individual colony. 

Like the spread plate, the pour plate can be used to determine the number of cells in a population. Plates containing between 30 and 300 colonies are counted. The total number of colonies equals the number of viable microorganisms in the sample that are capable of growing in the medium used. Colonies growing on the surface also can be used to inoculate fresh medium and prepare pure cultures.

These techniques require the use of special culture dishes named petri dishes or plates after their inventor Julius Richard Petri, a member of Robert Koch’s laboratory; Petri developed these dishes around 1887. They consist of two round halves, the top half overlapping the bottom. Petri dishes are very easy to use, may be stacked on each other to save space, and are one of the most common items in microbiology laboratories.