Overview

There are several microbiology techniques and procedures specially developed over the years to study and understand the metabolic processes, genetics, functions, and interaction of microbes with other organisms.[1]

The methodologies mostly involve techniques for culturing, identification, isolation, staining, and engineering these tiny organisms. They also have applications in other areas of biological research, including genetics, plant physiology, evolution, and molecular biology.[1]

Moreover, some of them contribute to better our health, but some cause life-threatening diseases. Others are used in food and beverage production, and to understand all this, we need to study these organisms.

Given the myriad uses of microbiology and its basic techniques, this article presents an overview of the basic microbiological laboratory techniques, how they work and are used in labs.

Categories of Basic Microbiology Techniques

The microbiology techniques are categorized based on the type of experiments. It includes:[2]

  • Culturing and Aseptic Techniques
  • Bacteria enumeration
  • Identification of Pathogens

These techniques don’t cover every microbiology technique; however, they are fundamental to all practices performed in microbiology laboratories.

1. Aseptic Techniques

Microorganisms are everywhere, and to study specific organisms, it’s essential to grow them in a strictly controlled lab environment. A complete sterile condition protects the pure microbial culture from contamination by other organisms entering through the air, water, or other unsterile sources.[2]

Some techniques used in labs to maintain a completely aseptic environment include:[2]

A. Sterilization

It’s the complete removal of all other microbial forms, including viruses, bacteria, fungus, spores, and other vegetative cells from the surface or the culture media.

Based on the purpose of the sterilization, the method is categorized into two groups:

  • Physical Methods: It involves the killing of contaminants or microbial forms using heat, sunlight, drying, filtration, or irradiation techniques (e.g., UV, infrared, gamma radiation, and X-ray).
  • Chemical Methods: It utilizes chemicals such as phenol (and any other related compounds), dyes, soaps, detergents, alcohol, gaseous compounds, and heavy metals and their compounds to destroy microorganisms.[2]

B. Disinfection

Disinfection is the process of killing microbes or inhibiting their growth from inanimate objects or surfaces by using physical or chemical agents like phenol, chlorine, alcohol, and heavy metal and their compounds.[2]

C. Sanitization

It’s the complete elimination of all pathogenic and non-pathogenic microbes from surface tops to reduce contamination. It’s also employed in daily lives to sanitize hands or in restaurants, dairies, and breweries to remove microbes and prevent infection and contamination.

It involves using chlorine-based cleaners, alcohol-based cleaners, formaldehyde, and hydrogen peroxide.[2]

2. Culturing Techniques

Before introducing the microbial strain to the culture media, the isolation and inoculation techniques are followed.[3]

A. Inoculation

It’s a basic technique used in microbiology labs to place microbial cultures onto a culture medium. It’s performed using an apparatus, called inoculation loop, made of platinum or nichrome wire with a loop at its one end. It’s mainly used in streaking and culture plate techniques.[3] The small sample picked up and transferred from the culture is known as inoculum.

B. Isolation

Isolation is a microbiological technique in which a specific microbial strain is isolated from a mixed culture of microorganisms by culturing the microbes on a selective culture media.[3] However, the procedure must be repeated several times to eliminate contamination by other microbes and achieve a pure culture of the microbial strain, which is then observed in culture plates as discrete/isolated individual colonies.

C. Culturing techniques

Microbes are grown in labs on culture media, which supply their nutritional requirements. These requirements vary for different microorganisms, thus a spectrum of culture medium recipes have been developed by scientists to obtain the desired microbial strain.[3]

  • Simple or basal media: It consists of sodium chloride, peptone, meat extracts, and water, for example, Nutrient Broth.
  • Complex media: This contains an additional special ingredient that helps to enhance a special characteristic or provide nutrients for the growth of certain microbes. It may contain extracts from plants, animals, and yeast, such as blood, yeast extracts, serum, milk, meat extracts, soybean digests, and peptone.[4]
  • Synthetic or defined media: It’s used for research purposes. They are prepared by following an exact formula and mixing distilled water with specific amounts of inorganic and organic chemicals.[4]
  • Special media: The basic medium supports the growth of a broad spectrum of microbial forms. However, a special growth condition is required for the culture and isolation of only a certain type or selected strain of bacteria. These formulated media to grow a microorganism chosen are known as special media. It’s further categorized into different groups:[5]
    • Selective media: It inhibits the growth of selected microorganisms while allowing the other to flourish. Examples include desoxycholate citrate medium for dysentery bacilli or mannitol salt agar containing 7.5% NaCl for Staphylococcus.
    • Enriched media: It contains complex organic substances like hemoglobin, serum, blood, or growth factors to support the growth of certain microbes. Examples are blood agar (widely used to grow certain streptococci and other pathogens) and chocolate agar.
    • Indicator media: It contains an indicator that changes color when a certain bacterium grows on the medium. For example, the addition of sulfite in the Wilson and Blair medium changes color to black when Salmonella typhi colonies grow on the medium.
    • Differential media: This media allows the growth of different bacterial species and distinguishes them based on their size, shape, color, or formation of gas bubbles or precipitates in the medium. Examples are MacConkey medium and blood agar.
    • Transport media: It’s a buffer solution containing peptone, carbohydrates, and other nutrients (except growth factors) to maintain the viability of the bacteria during transport without allowing their multiplication. An example is the Stuart medium for gonococci.
    • Anaerobic media: It contains ingredients that support the growth of anaerobic bacteria. An example is Robertson’s cooked meat media.

Now, the common culture techniques used in microbiology labs include:[5]

Common culture techniques
  • Streak plate method: In this technique, an inoculation loop is dipped in a diluted microorganism suspension and streaked on the solid surface of the culture medium. The plate which gets streaked is known as the streak plate. The method is generally used to obtain individual bacteria colonies from a concentrated suspension or to prepare a pure culture of a bacterial strain.
Streak plate technique illustration
Figure: An illustration of streak plate technique.[5]
  • Spread plate method: In this method, a very small volume of the liquid suspension of the microorganism is poured on the solidified surface of the media-containing plate. Then, an L-shaped glass rod is used to spread the liquid evenly all over the plate surface. This is performed to obtain individual colonies of the microorganisms but can also count the number of the microbial population.
Spread plate technique illustration
Figure: An illustration of the spread plate technique.[5]
  • Pour plate method: In this technique, a serially diluted microorganism suspension is pipetted in a sterile Petri dish. Then, a liquified, cooled culture media is poured into the plate. After the media is solidified, the culture plate is incubated for specific bacterial growth. It’s performed to estimate the viable bacterial count in a microbial suspension.
Pour plate technique illustration
Figure: An illustration of the pour plate method.[5]

D. Incubation

After the microorganisms are inoculated in plates, the culture plates are sealed from base to lid using adhesive tape to prevent contamination. Then, the plates are kept in the incubator for the required time and temperature for the growth of the organisms. Furthermore, keeping the plates in an inverted position prevents the formation and fall of water droplets into the culture media.[4]

If it’s required to store the microbial samples for later experiments, the following storage techniques are used:

  • Refrigeration
  • Deep freezing
  • Lyophilization
  • Freezing in liquid nitrogen

3. Bacteria Enumeration

Counting microbial colonies is an essential task in performing a range of experiments. Here are some enumerating techniques:[6]

  • Serial dilution: It’s used to lower the concentration of bacteria to a required amount for the purpose of experimentation to culturing processes.[5] It helps to count the number of microbial populations and perform experiments with the necessary number of microbial populations.
  • Plate counts: By applying the plate count method, it is possible to determine how many microbial colonies could potentially emerge under the given physical and chemical conditions, such as pH, temperature, available nutrients, and growth inhibitory compounds.[6]
  • Most probable number (MPN): In this statistical technique, a broth is inoculated in a 10-fold dilution, predicting the number of viable microorganisms per volume in a given sample.[6]
  • Using spectrophotometer: A spectrophotometer is used to estimate the growth of microorganisms in the culture with respect to time or at a certain time.

4. Identification of Pathogen

Pathogen identification is important for several applications. For example, it’s used to know which microbe is involved in contamination and food spoilage, which has useful applications in human lives, and which microorganism caused the particular disease for correct diagnosis and treatment in hospitals.

Microbes are identified by:[5]

  • Morphology: This is the first step in the identification process where the microorganisms are assessed with the naked eye based on colony texture, shape, and size.
  • Staining techniques: The identification is done by staining microbes with certain chemicals and observing them under a microscope to assess their cell properties.[5]
    • Simple staining: In this method, bacteria are stained using a single reagent and identified based on their size, shape, and arrangement of cells.
    • Differential staining: Here, at least three chemical reagents are used to stain bacteria, and identification is done based on the color the microorganism shows.
    • Gram staining: Here, certain chemical reagents are used to differentiate two principal groups of bacteria, gram-positive and gram-negative.
  • Microscopy techniques: Some microorganisms like viruses can not be identified using a simple microscope, as in the case of other microorganisms. Therefore, a high-throughput electron microscope is required to identify them.
  • Biochemical tests: Different tests, such as oxidase test, catalase test, indole test, and Dnase test are performed to differentiate microorganisms based on their enzymatic activities.[5]
  • Motility: The motility capacity of microorganisms is assessed to distinguish them from other microorganisms and identify their groups.
  • Microbial serology: It’s a test performed by using methods like Enzyme-linked immunosorbent assays (ELISA), western blotting, agglutination, and direct and indirect immunofluorescence to determine the bacterial or viral antibodies and antigens.[5]
  • Molecular analysis: A spectrum of techniques including plasmid profile analysis, random amplified polymorphism deoxyribonucleic acid (RAPD), pulsed-field gel electrophoresis (PFGE), multiplex polymerase chain reaction, and deoxyribonucleic acid (DNA) sequencing are used to identify, characterize, and classify microorganisms.[5]

Conclusion

Microbiology techniques are required to study microorganisms’ structure, function, metabolism, and genomics. They help understand how microbes work, interact with living organisms, cause diseases, and how they can be applied for human use.

The fundamental microbiology laboratory techniques include aseptic techniques, culturing techniques, enumerating bacteria, and identifying different classes of microorganisms. These techniques form the base of advanced research and experiments performed on microorganisms. The data obtained through these experiments also relate to other branches of biology, including molecular biology, soil science, agriculture, and the evolution of organisms.

The microbial study has helped scientists in many ways, from understanding how life began on earth to the use of microbes to control pollution. Moreover, the efficient use of these microbiological tools provides a deeper understanding of microbial life and the discovery of new species with new mysteries.

References

  1. Basic Techniques of Microbiology. Retrieved from https://labmonk.com/blog/basic-techniques-of-microbiology/
  2. Basic Microbiology Laboratory Techniques. Retrieved from http://www.biocourseware.com/iphone/vml/bmlt/index.html
  3. Microbiology Techniques: Applications And Uses. Retrieved from https://microbiologyclass.com/microbiology-techniques-applications-and-uses/
  4. Microbiological Laboratory Techniques. Retrieved from https://clinicalgate.com/microbiological-laboratory-techniques/#
  5. Varghese, Naveena & Joy, P.P.. (2014). Microbiology Laboratory Manual.
  6. What are the key activities in a Microbiology laboratory? Retrieved from https://lab-training.com/what-are-the-key-activities-in-a-microbiology-laboratory/