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Microbiological Culture

What are Sterile Techniques?

Microbiological culture serves as a fundamental research tool in molecular biology to study microbial organisms. The method allows the microbes to multiply on a culture media under controlled laboratory conditions. Microbiological cultures are employed in a variety of applications, such as cloning, diagnostics, and environmental monitoring. This is thanks to their versatility. The ability to grow, manipulate, and study microorganisms in culture has revolutionised scientific research and medical advancements, providing critical insights into microbial physiology, pathogenesis, and antimicrobial resistance. Because of their versatility and easy use, microbiological cultures are important in the laboratories.

Microbiological Culture


Microbiological culture involves the growth of microbial organisms. The culture serves various purposes, such as species identification via colony morphology and detecting microbial presence. In nature, microbial populations exist as mixed communities. However, in a laboratory settings, microbes can be separated into pure cultures using various techniques. Pure cultures, also known as axenic cultures, are critical for accurate microbial identification and isolation, antibiotic susceptibility testing and environmental science. A pure culture includes only one species, ensuring that observed characteristics are not influenced by other organisms. On the other hand, mixed cultures enable the study of multiple microbial species and are often used in industrial microbiological studies.

The term "culture" refers to the growth process as well as the microorganisms themselves. While culturing, it is important for the scientists to maintain an optimal physical environment, including the correct pH, temperature and pressure. On top of that, microorganisms require nutrients such as carbohydrates (for energy), proteins (for enzymatic and structural function), salts (for osmotic balance and metabolic reactions), vitamins and growth factors. Some organisms require specific nutrition and the media can be specialised if necessary.

Microbiological culture was first introduced by Louis Pasteur in 1860. His liquid media was used in Robert Koch's laboratory, which led him to develop solid media in 1881. In 1887, Julius Richard Petri refined Koch's method and introduced the Petri dish, which remains a laboratory staple until today and is widely used for fungal cultivation, antibiotic resistance, and microbiological research.

Types of Microbiological Cultures


Microbiological cultures use different techniques, including liquid broth or solid nutrient agar media. To prevent contamination, aseptic techniques, such as the use of laminar flow hood, are essential to prevent contamination.

Appearance of a prokaryote, a virus and a eukaryote.
Appearance of a prokaryote, a virus and a eukaryote.

Prokaryotic Culture

Prokaryotic cultures mainly focus on bacteria, as growing archaea is often difficult in a laboratory setting. Pure cultures originate from a single cell or colony, ensuring all cells are genetically identical. These are usually obtained from streak plate technique or serial dilution, which help isolate individual colonies. Pure cultures are necessary for the study of colony morphology without the influence of other species. Different bacteria have different growth requirements. Therefore, culture media can be enriched with specific nutrients or modified to include selective agents that inhibit the growth of unwanted species. Once a pure culture is obtained, the bacterial species can be stored for long-term study. 

Viral Culture

Viruses cannot grow independently and require host cells for replication. Viruses must infect host cells to propagate. The type of host cell depends on whether the virus infect bacterial, plant or animal cells in nature.

Viruses that infect bacterial cells are called bacteriophages. These are cultured by being introduced to a bacterial lawn on an agar plates. As the viruses infect and lyse bacterial cells, they create clear zones known as plaques, which indicate viral presence and activity. On the other hand, viruses that infect eukaryotic cells cultures are grown using suitable host cell lines. The streak plate method is often used to isolate viral strains, after which viruses are preserved in stock cultures for further research. 

Eukaryotic Cell Culture

Eukaryotic microorganisms include yeast, algae, and protozoans. Single-celled eukaryotes can be cultured using similar methods as prokaryotic cultures, such as liquid or solid media. This is also true for multicellular microscopic eukaryotes. However, macroscopic eukaryote organisms are too large to be cultured as whole organisms. Regardless, their cells can be grown for in vitro studies.

Microbiological Culture Methods


Microbiological culture advancements continue to improve microbial research. With modern developments, such as automated culture systems, microfluidics chips and synthetic media, previously uncultivated microorganisms can be studied. Established as well as new methods enhance the ability to explore microbial diversity, discover new antibiotics, and develop novel biotechnological applications.

Method Description Uses and Advantages
Liquid/Broth Cultures Organisms are inoculated into a flask of liquid media Large-scale culture, antimicrobial assays, bacterial differentiation
Agar Plates Organisms are transferred into Petri dishes containing solid (agar) media Provides a solid growth surface, compact and stackable
Agar-Based Dipsticks Miniature agar plates in dipstick form Diagnostic applications, portable, cost-effective
Selective and Differential Media Media that select for or differentiate between microorganisms Identifies unknown organisms, purifies cultures
Stab Cultures Organisms are inoculated into solid agar in a test tube Short-term storage, bacterial differentiation

Liquid Cultures

Liquid cultures include a nutrient-rich medium, resulting in rapid and large-scale microbial growth. These cultures are useful when large number of cells is required for experiments. Liquid cultures are also important for antimicrobial resistance assay, where bacterial growth in the presence of antibiotics is monitored to assess susceptibility and resistance pattern.

The methods used in liquid cultures can be refined based on the experiment's requirements. Shaken or aerated cultures provide oxygenation, promoting aerobic growth, while static cultures create oxygen gradients, allowing researchers to study facultative anaerobes and microaerophiles. More advanced culturing techniques include bioreactors, chemostats, and batch or continuous culture systems. These enable precise control over parameters such as nutrient supply, pH, temperature, dissolved oxygen, and waste removal. These systems are essential in industrial microbiology, where large-scale microbial cultivation is required.

Agar Plates

Agar plates provide a solid growth surface for microbiological cultures. Once inoculated, the plates are incubated under optimal conditions required by the specific microorganism. 

Selective and Differential Media

Selective and differential media are specialised types of culture media. They are used to study microbial characteristics based on growth patterns and biochemical traits. These media are crucial in clinical diagnostics, food safety testing, and environmental microbiology, as they help identify pathogens and beneficial microorganisms efficiently.

Selective Media

Selective media encourage the growth of specific microorganisms while inhibiting others using antimicrobial agents, dyes, salts, or specific nutrients. These media are essential for isolating target organisms from mixed microbial populations.

  • Eosin Methylene Blue (EMB) Agar selectively allow Gram-negative bacteria to grow while suppressing Gram-positive bacteria.
  • Mannitol Salt Agar (MSA) supports the growth of Staphylococcus species due to its high salt concentration, inhibiting most other bacteria.
  • Sabouraud Dextrose Agar (SDA) is designed for fungal isolation, as its low pH discourages bacterial growth. 

Differential Media

Differential media distinguish microorganisms based on biochemical properties. These include enzyme activity, carbohydrate fermentation, or haemolysis.

  • MacConkey Agar (MAC) differentiates lactose-fermenting bacteria from non-fermenters.
  • Blood Agar distinguishes bacterial species based on their haemolytic activity.

Multitarget Panels

Multitarget panels automate microbial identification and antibiotic susceptibility testing. Bacteria are distributed into wells containing biochemical test ingredients. Automated analysis compares results with a database to identify bacterial species.

Stab Cultures

Stab cultures involve inoculating bacteria into a solid agar in a test tube. These cultures are primarily used for short-term storage and transport. They also help identify microbial motility and oxygen requirements. Additionally, stab cultures provide valuable insights into microbial motility and oxygen requirements:

  • Motile bacteria spread away from the stab line, producing a diffused growth pattern.
  • Non-motile bacteria remain confined to the stab line.
  • Oxygen-sensitive organisms grow at varying depths depending on their aerobic or anaerobic preferences.

Solid Plate Culture for Thermophilic Microorganisms

Thermophilic microorganisms, thriving at temperatures between 50-70°C, are cultured on solid media using low acyl clarified gellant gum. This provides a more heat-stable and chemically defined medium, as traditional agar-based media can degrade at high temperatures, reducing their efficacy.

These media are essential in industrial microbiology, where thermophiles are used for:

  • Biodegradation of waste at high temperatures
  • Enzyme production
  • Biofuel generation from thermophilic microbial fermentation

Learn More


Colony Morphology Colony Morphology

Colony morphology describes the characteristics of cultures. It refers to characteristics visible to the naked eye of both bacterial and fungal colonies growing on an agar plate. It helps researchers to choose the appropriate confirmatory tests. While the methods might look simple, careful observation and examination of colony characteristics are essential in microbiology.

Read more...
Laminar Flow Hood Uses in Microbiology Laminar Flow Hood Uses in Microbiology

Laminar flow hoods are especially useful when you need to maintain a clean and germ-free environment for your experiments. These systems create a controlled environment by filtering the air to remove any airborne contaminants. Laminar flow hoods are especially useful in experiments that involve safe and non-harmful substances, requiring aseptic environment.

Read more...

References


  1. A.C. Ogodo, D.I. Agwaranze, M. Daji, R.E. Aso Microbial techniques and methods: basic techniques and microscopy Analytical Techniques in Biosciences, Pages 201-220, 2022

Contributors


Written by

Linda Vidova

Scientific Writer

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