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6.3E: Special Culture Techniques

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    9166
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    LEARNING OBJECTIVES

    • Evaluate special culture techniques

    Microbiologists would prefer to use well-defined media to grow a microbe, making the microbe easier to control. However, microbes are incredibly varied in what they use as a food source, the environments they live in, and the danger levels they may have for humans and other organisms they may compete with. Therefore they need special nutrient and growth environments. To grow these difficult microbes, microbiologists often turn to undefined media which is chosen based on price and more-so in this case by necessity as some microorganisms have never been cultured on defined media. Some special culture conditions are relatively simple as demonstrated by microaerophile.

    A microaerophile is a microorganism that requires oxygen to survive, but requires environments containing lower levels of oxygen than are present in the atmosphere (~20% concentration). Many microphiles are also capnophiles, as they require an elevated concentration of carbon dioxide. In the laboratory they can be easily cultivated in a candle jar. A candle jar is a container into which a lit candle is introduced before sealing the container’s airtight lid. The candle’s flame burns until extinguished by oxygen deprivation, which creates a carbon dioxide-rich, oxygen-poor atmosphere in the jar. Many labs also have access directly to carbon dioxide and can add the desired carbon dioxide levels directly to incubators where they want to grow microaerophiles.

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    Candle jar: A candle is lit in a jar with a culture plate. The lid is put on, as the burns it increases the carbon dioxide levels in the jar.

    Animals can often be used to culture microbes. For example, armadillos are often used in the study of leprosy. They are particularly susceptible due to their unusually low body temperature, which is hospitable to the leprosy bacterium, Mycobacterium leprae. The leprosy bacterium is difficult to culture and armadillos have a body temperature of 34°C, similar to human skin. Likewise, humans can acquire a leprosy infection from armadillos by handling them or consuming armadillo meat. Additionally, Syphillis which is caused by the bacteria Treponema pallidum is difficult to grow with defined media, so rabbits are used to culture Treponema pallidumTreponema pallidum belongs to the Spirochaetesphylum of bacteria.

    To date Spirochaetes are very difficult if not impossible to rear in a controlled laboratory environment. This also includes other human pathogens like the bacterium that causes Lyme disease. Using animals to culture human-pathogens has problems. First, the use of animals is always difficult for technical and ethical reasons. Also, a microbe growing on animal other than a human may behave very differently from how that same microbe will behave on a human. Some human pathogens are grown directly on cells cultured from humans. Exemplified by the bacteria Chlamydia trachomatis, the bacteria responsible for the sexually transmitted infection (STI) in humans known as Chlamydia. As Chlamydia trachomatis only grows in humans. The human cell culture known as McCoy cell culture is used to culture this bacteria.

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    Chlamydias bacteria group: Light microscope view of cells infected with chlamydiae as shown by the brown inclusion bodies.

    A large concern of microbiology is trying to find ways in which humans can avoid or get rid of microbrial infections. As typified by some of the above examples, some microbes have to be grown in the lab, and some of them can infect humans. To deal with this, microbiologists use a classification of biosafety levels. A biosafety level is the level of the biocontainment precautions required to isolate dangerous biological agents in an enclosed facility. The levels of containment range from the lowest biosafety level 1 (BSL-1) to the highest at level 4 (BSL-4). In the United States, the Centers for Disease Control and Prevention (CDC) have specified these levels.

    • Biosafety Level 1: This level is suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, with minimal potential hazard to laboratory personnel and the environment.
    • Biosafety Level 2: This level is similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It includes various bacteria and viruses that cause only mild disease to humans or are difficult to contract via aerosol in a lab setting such as chlamydia.
    • Biosafety Level 3: This level is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents that may cause serious or potentially lethal disease after inhalation. It includes various bacteria, parasites, and viruses that can cause severe to fatal disease in humans, but for which treatments exist (eg. yellow fever).
    • Biosafety Level 4: This level is reserved for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections, agents that cause severe to fatal disease in humans for which vaccines or other treatments are not available, such as Bolivian and Argentine hemorrhagic fevers, Marburg virus, and the Ebola virus. Very few laboratories are biosafety level 4.

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    Positive pressure suit: A scientist puts on a positive pressure suit, something needed to work with the most dangerous human pathogens in a biosafety level 4 laboratory.

    Key Points

    • Microbes, often those that we know little about, have to be cultured with undefined media or growth conditions.
    • The use of animals to culture animals is sometimes necessary as no simple media can be used, this presents technical and ethical issues.
    • As human pathogens are often studied by microbiologists, special safety conditions know as biosafety levels are used to keep researches free of infection from the pathogens they study.

    Key Terms

    • yellow fever: An acute febrile illness of tropical regions, caused by a flavivirus and spread by mosquitoes, characterized by jaundice, black vomit, and the absence of urination.
    • Lyme disease: Infection by a bacterium of the genus Borrelia which is transmitted by ticks. Symptoms include a rash followed by fever, joint pain, and headaches.

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