Isolation of Pure Cultures

In natural habitats microorganisms usually grow in complex, mixed populations with many

species. This presents 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.

German bacteriologist Robert Koch developed of pure culture techniques and 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 (L shaped)

rod/hockey stick 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 

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. The number of colonies developed should

be equal to the number of viable organisms in the sample, so, spread plates can be used to

count the microbial population.

 

The Pour Plate

The pour plate technique 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. Por plates can thus be used to enumerate/count the microbial population.

Colonies growing on the surface also can be used to inoculate fresh medium and prepare pure

cultures.

 

The streak plate

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 microbiology, streaking is one of the most fundamental techniques used to isolate pure colonies of microorganisms from a mixed sample. By spreading microbial cells across the agar surface in a defined pattern, the density of organisms is reduced, ultimately allowing pure, isolated individual colonies to be distinguished and studied.

Pure cultures are essential in accurate identification and can be used further for antibiotic testing & biochemical studies, Streaking is thus an essential skill for every microbiologist.

Major types of streaking techniques:

1. Quadrant Streaking The most common method. The plate is divided into four

quadrants and streaked progressively. Useful for isolating bacteria from mixed

cultures.

2. T-Streaking Plate is divided into three zones (T-shaped pattern). Provides better

separation than a simple streak and is widely used in clinical labs.

3. Continuous (Simple) Streaking Involves spreading the inoculum in one continuous

motion without turning the plate. Suitable when the culture is not highly dense.

4. Radiant (Star) Streaking Streaks radiate outward from a central point. Often used

for quick isolation and for demonstration purposes.

Streaking is an essential technique as it

 Helps in isolating pure colonies.

 Essential for identifying microorganisms.

 Forms the basis for antibiotic sensitivity testing and biochemical studies.

In both spread-plate and streak-plate techniques, successful isolation depends on spatial separation of single cells.

Spread/pour/streak 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.

Dilution plating and enrichment technique

A major practical problem is the preparation of pure cultures when microorganisms are present in large or very low numbers in a sample. Serial dilution can be used to dilute the sample if microorganisms are present in large numbers. Plating methods can be combined with the use of selective or differential media to enrich and isolate rare microorganisms.

Dilution Plating (Serial Dilution Method)

Dilution plating is a technique used to reduce the concentration of microorganisms in a sample by serial dilution so that individual colonies can be isolated and counted.

Principle

When a sample containing many microorganisms is serially diluted, the number of cells decreases progressively. After plating, each viable cell forms a separate colony (CFU –Colony Forming Unit).

Procedure

1. Prepare serial dilutions (e.g., 10⁻¹, 10⁻², 10⁻³, etc.).

2. Transfer a measured volume onto sterile agar plates.

3. Spread evenly (spread plate method) or mix with molten agar (pour plate method).

4. Incubate at suitable temperature.

5. Count colonies on plates with 30–300 colonies.

Calculation of Bacterial Count, CFU/ml= Number of colonies/ Dilution factor × Volume plated

Applications

 Estimation of bacterial load in water, milk, soil, etc.

 Isolation of pure colonies.

 Used in food microbiology and clinical microbiology.

Serial dilution, is a series of sequential dilutions performed to convert a dense solution into a more usable concentration. Serial dilution is thus, the process of stepwise dilution of a solution. In Microbiology, serial dilution reduces the concentration of cells in a culture to simplify the operation.

Objectives

 In serial dilution, the density of cells is reduced in each step so that it is easier to calculate the concentration of the cells in the original solution by calculating the total dilution over the entire series.

 The objective of the serial dilution method is to estimate the concentration (number of organisms, bacteria, viruses, or colonies) of an unknown sample by the enumeration of the number of colonies cultured from serial dilutions of the sample.

 By serial dilution, it is possible to obtain incubated culture plates with an easily countable number of colonies (around 30–100) and calculate the number of microbes present in the sample.

The serial dilution is performed as below:

Six test tubes, each with 9 ml of sterile diluents, which can either be distilled water or 0.9% saline, are taken.

1. A sterile pipette is taken.

2. 1 ml of properly mixed sample/culture is drawn into the pipette.

3. The sample is then added to the first tube to make the total volume of 10 ml. This

provides an initial dilution of 10 -1 .

4. The dilution is thoroughly mixed by emptying and filling the pipette several times.

5. The pipette tip is discarded, and a new pipette tip is attached to the pipette.

6. Now, 1 ml of mixture is taken from the 10 -1  dilution and is emptied into the second

tube. The second tube now has a total dilution factor of 10 -2 .

7. The same process is then repeated for the remaining tube, taking 1 ml from the

previous tube and adding it to the next 9 ml diluents.

8. As six tubes are used, the final dilution for the bacteria/cells will be 10 -6  (1 in

1,000,000).

Applications8

1. Serial dilution is used in microbiology to estimate the concentration or number of cells/organisms in a sample to obtain an incubated plate with an easily countable number of colonies.

2. In biochemistry, serial dilution is used to obtain the desired concentration of reagents and chemicals from a higher concentration.

Limitations

Even though serial dilution is a useful technique in laboratories, it faces some challenges.

Some of which are:

1. Errors and transfer inaccuracies lead to less accurate and less precise transfer. This

results in inaccuracies.

2. Serial dilution only allows the reduction of bacteria/cells but not the separation of

bacteria/cells like in other techniques like flow cytometry.

3. This technique also requires highly trained microbiologists and aseptic procedures.

Enrichment Technique

Enrichment technique is used to increase the number of a desired microorganism in a mixed culture by providing favourable growth conditions for it while suppressing others.

Specific nutrients, incubation conditions, or selective agents are used to promote growth of target organism and/or to inhibit unwanted organisms

Types

1. Enrichment Culture – Uses special media to enhance desired organism growth.

2. Selective Enrichment – Contains inhibitory substances to suppress competing flora.

Examples

 Selenite F broth for isolating Salmonella.

 Alkaline peptone water for Vibrio cholerae.

Applications

 Isolation of pathogens from stool samples.

 Environmental microbiology studies.

 Detection of low-number pathogens.

A good example is the isolation of bacteria that degrade the herbicide 2,4- dichloro phenoxy acetic acid (2,4-D). Bacteria able to metabolize 2,4-D can be obtained with a liquid medium containing 2,4-D as its sole carbon source and the required nitrogen, phosphorus, sulfur, and mineral components. When this medium is inoculated with soil, only bacteria able to use 2,4-D will grow. After incubation, a sample of the original culture is transferred to a fresh flask of selective medium for further enrichment of 2,4-D metabolizing bacteria. A mixed population of 2,4-D degrading bacteria will arise after several such transfers. Pure cultures can be obtained by plating this mixture on agar containing 2,4-D as the sole carbon source. Only bacteria able to grow on 2,4-D form visible colonies and can be sub-cultured. This same general approach is used to isolate and purify a variety of bacteria by selecting for specific physiological characteristics.

Culture Techniques

Stab Culture

A stab culture is prepared by inserting (stabbing) an inoculating needle containing microorganisms deep into a solid medium, usually in a test tube with agar.

Purpose:

 To study oxygen requirements of bacteria (aerobic vs anaerobic growth).

 To observe motility of bacteria.

 For maintenance and storage of bacterial strains.

Uses:

 Detecting motility in organisms like Escherichia coli.

 Growing anaerobic bacteria such as Clostridium tetani.

Growth Pattern:

 Growth only at top → Aerobic organism

 Growth throughout → Facultative anaerobe

 Growth at bottom → Obligate anaerobe

 

        

  

        Stab Culture                                                                      Stroke Culture

Stroke Culture (Slant Culture)

A stroke culture is made by streaking microorganisms over the surface of a solid agar slant in a test tube using an inoculating loop.

Purpose:

 To maintain bacterial cultures.

 To observe colony characteristics.

 For short-term preservation and transport.

Common Organisms Grown:

 Staphylococcus aureus

 Salmonella typhi

Growth Pattern:

 Growth appears along the slanted surface in a zigzag or continuous streak.

 Lawn Culture

A lawn culture is prepared by spreading bacteria evenly across the entire surface of an agar plate to produce a uniform, confluent growth. Used in the Kirby–Bauer antibiotic susceptibility test. Thick, uniform bacterial growth covering the whole plate surface.

Purpose:

 Antibiotic sensitivity testing.

 Bacteriophage typing.

 Testing disinfectants.

Growth Pattern:

Growth apperas as uniform/lawn like confluent growth on the surface of media.