Pure culture techniques- streak, spread and pour plate methods-Dr C R Meera

 

In natural habitats microorganisms usually grow in complex, mixed populations containing several species. This presents a problem for the microbiologist because a single type of microorganism cannot be studied adequately in a mixed culture. One needs a “pure culture”- that's a population of cells arising from a single cell to characterize an individual species. 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 major human bacterial diseases had been isolated.

 A single bacterial cell on suitable culture media will give rise to a number of genetically identical daughter cells by binary fission. These are called “clones” of cells forming into a colony on culture media. A pure culture is usually made up of a succession of cultures and is often derived from a single colony. All strains of bacteria isolated and identified till now are maintained in American Type Culture Collection (ATCC) and Microbial Type Culture Collection (MTCC).  Each strain is designated by an identifying number and its history is recorded (the source from which the isolation was made, name of the person who made the isolation, date of isolation and the culture collection in which the strain is maintained and from which it can be obtained for study).

A variety of techniques have been developed whereby isolation into pure culture can be accomplished. Each technique has certain advantages and limitations, and there is no one method that can be used for all bacteria. Most commonly used pure culture techniques include streak plate method, spread plate method and pour plate method. Streak plate is considered only as a qualitative method. Unlike the streak plate technique, the pour plate and spread plate techniques may be performed in a quantitative manner to determine the number of bacteria present in a specimen.

 Methods of isolating pure cultures

 A)  The streak plate technique

The streak culture or surface plating method is routinely employed for the isolation of bacteria in pure culture from clinical specimens. A platinum loop is charged with the specimen to be cultured.  Owing to the high cost of platinum, loops for routine work are made of nichrome resistance wires. One loopful of specimen is transferred onto the surface of a well dried agar plate on which it is spread over a small area at the periphery. It is called the primary inoculum. The primary inoculum is then distributed over the plate by streaking it with the loop in a series of parallel lines in different segments of the plate. The loop should be flamed and cooled between the different sets of streaks. At some point in the process, single cells dropped from the loop as it is rubbed along the agar surface would develop into separate colonies on incubation. Growth may be confluent at the site of original inoculation but become progressively thin and well separated colonies are obtained over the final series of streaks. Common streaking methods include T- streaking, Zig-zag streaking,  Continuous streaking and Quadrant streaking (Figure 1).

 

Quadrant Streaking

Zig-zag Streaking

T-Streaking

Radiant streaking

Continuous streaking

Fig 1. Different streaking patterns

A)  The pour plate technique

 

A pour plate can yield isolated colonies and extensively used with the bacteria and fungi. The original sample is diluted several times to reduce the microbial population sufficiently to obtain separate colonies when plating (Figure 2). Serial dilution is the method commonly used to dilute the original sample. For serial dilution, a series of tubes containing a definite volume of sterile liquid, usually water or physiological saline is prepared. Suppose we prepare a series of tubes containing 9 ml of sterile distilled water. For carrying out serial dilution of the original sample we can add 1 ml of the original sample into the first tube which contains 9 ml of sterilized distilled water. Now the final volume of the first tube becomes 10. So the dilution of the first tube is 1/10 which can be also written as 10^-1.  To continue the serial dilution,  from the first tube we can add 1 ml to the second tube. This is continued in the following tubes also till we reach the final tube. From the final tube, remove 1ml of the media, so that the volume will be 9 ml in all the tubes. From these diluted tubes small volumes of several diluted samples are mixed with liquid Agar that has been cooled to about 45 degrees celsius. The mixtures are then 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  individual colony. Plates containing between 30 to 300 colonies are counted. The total number of colonies equals the number of viable microorganisms in the diluted sample. Colonies growing on the surface can be used to inoculate fresh medium and to prepare pure cultures. This is considered as a quantitative as well as qualitative method. This is a preferred quantitative method for urine cultures. Surface colonies formed by pour culture technique are circular and subsurface colonies could be lenticular or lens shaped.

Disadvantages

1.    Some of the organisms are trapped beneath the surface of the medium during pour plate technique and therefore both surface and the subsurface colonies develop.  The subsurface colonies can be transferred to fresh media only by first digging them out of the agar with a sterile instrument. So, there is more chance for contamination.

2.    The organisms being isolated must be able to withstand temporary exposure to 45 degree celsius, temperature of the liquid agar medium. So, this method would be unsuitable for isolating psychrophilic bacteria. (Psychrophiles are the bacteria that grow well at 20⁰ C degree and have an optimum growth temperature of 15⁰ C or lower and maximum around 20⁰ C)

Figure- 2. The pour plate technique- The original sample is diluted several times to thin out the population sufficiently. The most diluted samples are then mixed with warm agar and poured into petri dishes. Isolated cells grow into colonies and can be used to establish pure cultures.

CFU/ml (Colony Forming Unit/ml)= (No: of colonies X Dilution factor)  / Volume of sample plated

Dilution factor = Reciprocal of dilution or Final volume/ sample volume

(If 1 ml sample is added to 9 ml, its dilution = 1/10= 10 -1; Dilution factor is 10)

C) Spread plate technique

The spread plate is an easy, direct way of achieving pure cultures. This method is also considered as a quantitative as well as qualitative method.  In this method also, serial dilution has to be conducted initially. The original culture is diluted in a series of tubes containing sterile liquid, usually water or physiological saline. After that, a small volume of dilute microbial mixture containing around 30- 300 cells is transferred to the center of a dry Agar plate. Then it is spread evenly over the surface of agar media with a sterile  glass rod or the L-rode. The dispersed cells develop into isolated colonies. The number of colonies developed on the plates would be equal to the number of viable organisms, as each viable cell develops into a colony on incubation. In contrast to the pour plate technique, only surface colonies develop in the pour plate technique. Moreover the organisms are not required to withstand the temperature of liquid agar as in the case of pour plate technique.

 Preparation of spread plate

●      Pipette a small volume (0.01ml) of sample on to the center of an agar medium plate

●      Dip  the glass spreader into a beaker of ethanol

●      Briefly flame the ethanol soaked spreader and allow it to cool

●      Spread the sample evenly over the agar surface with the sterilized spreader and incubate (Fig 3)

 

Figure 3. Spread Plate Technique