What is Microbial Isolation in Microbiology?

Isolation of the microorganisms in microbiology is the process of developing a pure culture by separating one species of microbe from a mixture of many other species. A pure culture is a culture that contains only one species of microbe.

Sources of Microorganisms

Natural sources, such as water and soil, and bodily fluids, such as blood, bile secretions, urine, and stomach fluid, can be used to isolate microorganisms of interest. Archaea, bacteria, fungi, and protozoa are all examples of soil microorganisms. In the air, viruses, bacteria, fungus, yeasts, and protozoans can be discovered.

The bacterium E.coli forms an important part of the gut microbiota, and some of it is excreted along with the undigested food in the feces. Microbial isolation can be utilized to isolate E.coli bacterium from the feces. Similarly, other microbial organisms can be isolated from bodily fluids, such as blood. By extracting blood with a sterilized hypodermic needle and treating the blood with anticoagulants such as heparin and potassium oxalate, a pure culture of the bacterium can be obtained.

Importance of isolation and identification of microorganisms

There are around 10,000 microbe species that have been identified. For every known species, there are thought to be 10,000 to 100,000 additional unidentified species. The capacity to detect microorganisms is vital in many different aspects of microbiology. For example, in food microbiology, the ability to reliably identify food spoilage microorganisms is critical. The identification of microorganisms aids in the characterization of biodiversity in microbial ecology. The major goal of medical microbiology, a branch of microbiology that studies pathogenic microorganisms, is to isolate, detect, and study disease-causing microorganisms.

Identification of microorganisms aids in determining the organism's name to the genus or species level, which can assist in determining whether the organism is beneficial or harmful and whether it causes disease. The initial step in identifying the microbe, for example, bacteria, that may be responsible for a disease is the isolation of a single bacterial species. The identification is needed so that an appropriate antibiotic can be administered to treat the disease or infection caused by this bacterium.

Culture media in microbial isolation

Several microbiological experiments require culture media in order to gain pure cultures, develop and quantify microbial cells, and nurture and identify microorganisms, among other things. A microbiological growth medium is a chemical that helps microorganisms grow, survive, and thrive. Nutrients, growth regulators, energy sources, buffer salts, minerals, metals, and suspending agents are all contained in the growth medium.

Nutrient broths and agar plates are the most frequent microbe growth media. Selective medium is the medium that allows only some bacteria to grow. Differential media, also known as indicator media, are used to differentiate one microorganism strain from the other that grows on the same medium.

The methods of microbial isolation

Pure culture

The ability to culture a microbe in the laboratory is the initial step for isolation. Microbial colonies do not isolate themselves by species in nature, but rather coexist alongside a variety of other cell types. These populations can be sorted into pure cultures in the laboratory. Pure cultures contain only one type of microorganism and are ideal for studying their biochemical, morphological, and cultural characteristics.

Spread plate culture technique

The spread plate culture is one of the most commonly used methods for isolating microorganisms. It involves plating a sample mixture containing microorganisms so that they may be counted and isolated easily. An effective spread plate will contain a quantifiable number of isolated microbial or bacterial colonies dispersed equally around the plate.

In this process, a serially diluted sample (mixed culture) containing two or more bacteria or microbes is spread as a thin layer over solidified agar plates using a sterilized L-shaped glass rod or spreader while the agar plate is rotating on a platter. The theory behind such a procedure is that single cells are spread onto the surface of the agar media by using a bent glass rod while the media plate rotates. Some of the cells in the diluted specimen will be sufficiently separated from one another for self-contained colonies to form.

Pour-plate method

The pour-plate method is the most familiar method for calculating the total viable count. The advantage of this method is that it does not require pre-prepared plates, and it is frequently used to test for bacterial contamination in foods.

A loop or pipette is used to dilute the mixed culture in the pour-plate procedure serially. A Petri dish containing a predetermined amount of the diluted sample is filled with molten agar that has been cooled to 45 °C. The cover is replaced when the molten-then-cooled agar is added, then the plates are gently rotated in a circular motion to achieve uniform microbe distribution. For all dilutions to be plated, repeat this procedure.

It is easy to execute. It will recognize lower levels than the surface spread technique due to the large sample volume. The pour plate approach takes longer to prepare than the streak plate and spread plate method. Heat-sensitive microorganisms that come into touch with heated agar lose their viability.

Streak plate method

The streak plate method is the most common approach to obtain isolated colonies from microbial culture. The method works by diluting the number of microorganisms and lowering their concentration. Microbiologists can use it to identify and isolate specific bacterial colonies. A colony is an observable bacterial aggregation. Inside a single colony, all the bacteria come from the same bacterial cell. As a result, single colonies are known as "pure" colonies. The separated pure culture of one species of bacterium is moved to another plate for better isolation.

Two types of Streak plate method

The T-streak approach: The T-streak approach has three portions: an upper half and two lower sections of equal size. The first inoculum is put in the plate's top half. Bacteria are moved from the top to one of the lower portions, and from that bottom part to the next.

The quadrant approach: involves streaking four equal-sized sections. The continuous streaking approach involves inoculating the top half of the plate, turning it 180 degrees, then inoculating the second half of the plate without sterilizing the loop or dragging microorganisms from the preceding area.

Bacterial isolation

Bacterial isolation is the way to separate one strain of bacterium from a mixed culture of bacteria using various plating techniques such as spreading, pouring, streaking, and serial dilution. Bacterial growth can be observed on both the solid nutrient medium and the liquid broth medium. Bacterial isolation is a useful technique for studying and classifying bacteria based on microscopic characteristics such as color, size, and shape.

Method of seriel dilution

This approach is well-known for bacterial isolation and culture. Take the bacterial suspension and dilute it sequentially in subsequent test tubes using the serial dilution procedure. Add 1 ml of the sample to the adjacent test tube in a series of 10 times, 100 or 1000 times diluted, and so on, following the serial dilution. We can inoculate the bacterial culture using one of three methods: pouring, spreading, or streaking, following the serial dilution of the bacterial suspension. Isolating bacterium from a smaller bacterial population is simple using serial dilution.

Context and Applications

This topic is significant in the exams of school, graduate, and post-graduate levels especially for Bachelors in Zoology/botany/microbiology/biotechnology and Masters in Zoology/botany/microbiology/biotechnology.

Practice Problems

Question 1: Which is the source of E.coli for E.coli isolation?

1. Blood
2. Soil
3. Mammalian faeces
4. Air

Answer: Option 3 is correct.

Explanation: Bacterium such as E. coli is primarily found in mammals' gastrointestinal systems and is shed into the environment via feces.

Question 2: What is pure culture?

1. Cultures of mixed population
2. Cultures of one type of species
3. Cultures of bacteria
4. None of the above

Answer: Option 2 is correct.

Explanation: A pure culture is a culture that contains only one species of microbe.

Question 3: Purpose of isolation of microbes includes…..?

1. Detection of pathogens
2. Identification of  microbes
3. Characterization of biodiversity
4. All of the above

Answer: Option 4 is correct.

Explanation: The identification of microorganisms aids in the characterization of biodiversity in microbial ecology. The major goal of medical microbiology, is the isolation, detection, and research of infectious disease-causing microbes.

Question 4: The …………… method is the isolation method that not requiring pre-prepared plates.

1. Streak plate method
2. Pour plate method
3. Spread plate method
4. None of the above

Answer: Option 2 is correct.

Explanation: The advantage of the pour plate method is, that not requiring pre-prepared plates.

Question 5: Quadrant approach is a type of --------------.

1. Pour plate method
2. Spread plate method
3. Serial dilution
4. Streak plate method

Answer: Option 4 is correct.

Explanation: The quadrant approach involves streaking four areas of equal size.