Culturing Bacteria

Culture Media

Culture media is the food used to grow and control microbes.

Learning Objectives

Classify culture media

Key Takeaways

Key Points

  • Culture media contains the nutrients needed to sustain a microbe.
  • Culture media can vary in many ingredients allowing the media to select for or against microbes.
  • Glucose or glycerol are often used as carbon sources, and ammonium salts or nitrates as inorganic nitrogen sources in culture media.

Key Terms

  • culture: The process of growing a bacterial or other biological entity in an artificial medium.
  • lysogeny broth: Lysogeny broth (LB) is a nutritionally-rich medium; primarily used for the growth of bacteria.

Microbiological Cultures

Culture medium or growth medium is a liquid or gel designed to support the growth of microorganisms. There are different types of media suitable for growing different types of cells. Here, we will discuss microbiological cultures used for growing microbes, such as bacteria or yeast.

NUTRIENT BROTHS AND AGAR PLATES

These are the most common growth media, although specialized media are sometimes required for microorganism and cell culture growth. Some organisms, termed fastidious organisms, need specialized environments due to complex nutritional requirements. Viruses, for example, are obligate intracellular parasites and require a growth medium containing living cells. Many human microbial pathogens also require the use of human cells or cell lysates to grow on a media.

The most common growth media nutrient broths (liquid nutrient medium) or LB medium (Lysogeny Broth) are liquid. These are often mixed with agar and poured into Petri dishes to solidify. These agar plates provide a solid medium on which microbes may be cultured. They remain solid, as very few bacteria are able to decompose agar. Many microbes can also be grown in liquid cultures comprised of liquid nutrient media without agar.

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Microbial pathogen growing on blood-agar plate: Red blood cells are used to make an agar plate. Different pathogens that can use red blood cells to grow are shown on these plates. On the left is staphylococcus and the right streptococcus.

DEFINED VS UNDEFINED MEDIA

This is an important distinction between growth media types. A defined medium will have known quantities of all ingredients. For microorganisms, it provides trace elements and vitamins required by the microbe and especially a defined carbon and nitrogen source. Glucose or glycerol are often used as carbon sources, and ammonium salts or nitrates as inorganic nitrogen sources. An undefined medium has some complex ingredients, such as yeast extract, which consists of a mixture of many, many chemical species in unknown proportions. Undefined media are sometimes chosen based on price and sometimes by necessity – some microorganisms have never been cultured on defined media.

There are many different types of media that can be used to grow specific microbes, and even promote certain cellular processes; such as wort, the medium which is the growth media for the yeast that makes beer. Without wort in certain conditions, fermentation cannot occur and the beer will not contain alcohol or be carbonated (bubbly).

COMMON BROADLY-DEFINED CULTURE MEDIA

Nutrient media – A source of amino acids and nitrogen (e.g., beef, yeast extract). This is an undefined medium because the amino acid source contains a variety of compounds with the exact composition being unknown. These media contain all the elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory-culture collections.

Minimal media – Media that contains the minimum nutrients possible for colony growth, generally without the presence of amino acids, and are often used by microbiologists and geneticists to grow “wild type” microorganisms. These media can also be used to select for or against the growth of specific microbes. Usually a fair amount of information must be known about the microbe to determine its minimal media requirements.

Selective media – Used for the growth of only selected microorganisms. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing.

Differential media – Also known as indicator media, are used to distinguish one microorganism type from another growing on the same media. This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism. This type of media is used for the detection and identification of microorganisms.

These few examples of general media types provide some indication only; there are a myriad of different types of media that can be used to grow and control microbes.

Complex and Synthetic Media

In defined media all the chemical compounds are known, while undefined media has partially unknown chemical constituents.

Learning Objectives

Differentiate complex and synthetic medias

Key Takeaways

Key Points

  • Defined media is made from constituents that are completely understood.
  • Undefined media has some part of which is not entirely defined.
  • The presence of extracts from animals or other microbes makes a media undefined as the entire chemical composition of extracts are not completely known.

Key Terms

  • recombinant: This term refers to something formed by combining existing elements in a new combination. Thus, the phrase recombinant DNA refers to an organism created in the lab by adding DNA from another species.
  • serum: The clear yellowish fluid obtained upon separating whole blood into its solid and liquid components after it has been allowed to clot. Also called blood serum.

There are many types of culture media, which is food that microbes can live on. Two major sub types of media are complex and synthetic medias, known as undefined and defined media.

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Undefined Media: Luria Broth as shown here is made with yeast extract, as yeast extract is not completely chemically defined Luria Broth is therefore an undefined media. By Lilly_M [GFDL (http://www.gnu.org/copyleft/fdl.html), CC-BY-SA, via Wikimedia Commons

An undefined medium has some complex ingredients, such as yeast extract or casein hydrolysate, which consist of a mixture of many, many chemical species in unknown proportions. Undefined media are sometimes chosen based on price and sometimes by necessity – some microorganisms have never been cultured on defined media.A defined medium (also known as chemically defined medium or synthetic medium) is a medium in which all the chemicals used are known, no yeast, animal, or plant tissue is present. A chemically defined medium is a growth medium suitable for the culture of microbes or animal cells (including human) of which all of the chemical components are known. The term chemically defined medium was defined by Jayme and Smith as a ‘Basal formulation which may also be protein-free and is comprised solely of biochemically-defined low molecular weight constituents.

A chemically defined medium is entirely free of animal-derived components (including microbial derived components such as yeast extract) and represents the purest and most consistent cell culture environment. By definition chemically defined media cannot contain either fetal bovine serum, bovine serum albumin, or human serum albumin as these products are derived from bovine or human sources and contain complex mixes of albumins and lipids. The term ‘chemically defined media’ is often misused in the literature to refer to serum albumin-containing media. Animal serum or albumin is routinely added to culture media as a source of nutrients and other ill-defined factors, despite technical disadvantages to its inclusion and its high cost. Technical disadvantages to using serum include the undefined nature of serum, batch-to-batch variability in composition, and the risk of contamination. There are increasing concerns about animal suffering inflicted during serum collection that add an ethical imperative to move away from the use of serum wherever possible. Chemically defined media differ from serum-free media in that bovine serum albumin or human serum albumin with either a chemically defined recombinant version (which lacks the albumin associated lipids) or synthetic chemical such as the polymer polyvinyl alcohol which can reproduce some of the functions of serums.

Selective and Differential Media

Selective media allows for the growth of specific organisms, while differential media is used to distinguish one organism from another.

Learning Objectives

Compare selective and differential media

Key Takeaways

Key Points

  • Selective media generally selects for the growth of a desired organism, stopping the growth of or altogether killing non-desired organisms.
  • Differential media takes advantage of biochemical properties of target organisms, often leading to a visible change when growth of target organisms are present.
  • Differential media, unlike selective media, does not kill organisms. It indicates if a target organism is present.

Key Terms

  • recombinant: This term refers to something formed by combining existing elements in a new combination. Thus, the phrase recombinant DNA refers to an organism created in the lab by adding DNA from another species.
  • gene: A unit of heredity; a segment of DNA or RNA that is transmitted from one generation to the next. It carries genetic information such as the sequence of amino acids for a protein.
  • allele: One of a number of alternative forms of the same gene occupying a given position on a chromosome.

There are many types of media used in the studies of microbes. Two types of media with similar implying names but very different functions, referred to as selective and differential media, are defined as follows.

Selective media are used for the growth of only selected microorganisms. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing. Media lacking an amino acid such as proline in conjunction with E. coli unable to synthesize it were commonly used by geneticists before the emergence of genomics to map bacterial chromosomes. Selective growth media are also used in cell culture to ensure the survival or proliferation of cells with certain properties, such as antibiotic resistance or the ability to synthesize a certain metabolite. Normally, the presence of a specific gene or an allele of a gene confers upon the cell the ability to grow in the selective medium. In such cases, the gene is termed a marker. Selective growth media for eukaryotic cells commonly contain neomycin to select cells that have been successfully transfected with a plasmid carrying the neomycin resistance gene as a marker. Gancyclovir is an exception to the rule as it is used to specifically kill cells that carry its respective marker, the Herpes simplex virus thymidine kinase (HSV TK). Some examples of selective media include:

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Non-selective versus selective media.: The non-selective media on the right allows of the growth of several different bactarial species and is overgrown with bacteria (whitish lines). While the plate on the right selectively only allows the bacteria Neisseria gonorrhoeae, to grow (white dots).

  • Eosin methylene blue (EMB) that contains methylene blue – toxic to Gram-positive bacteria, allowing only the growth of Gram negative bacteria.
  • YM (yeast and mold) which has a low pH, deterring bacterial growth.
  • MacConkey agar for Gram-negative bacteria.
  • Hektoen enteric agar (HE) which is selective for Gram-negative bacteria.
  • Mannitol salt agar (MSA) which is selective for Gram-positive bacteria and differential for mannitol.
  • Terrific Broth (TB) is used with glycerol in cultivating recombinant strains of Escherichia coli.
  • Xylose lysine desoxyscholate (XLD), which is selective for Gram-negative bacteria buffered charcoal yeast extract agar, which is selective for certain gram-negative bacteria, especially Legionella pneumophila.

Differential media or indicator media distinguish one microorganism type from another growing on the same media. This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism. This type of media is used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria. Examples of differential media include:

  • Blood agar (used in strep tests), which contains bovine heart blood that becomes transparent in the presence of hemolytic.
  • Streptococcuseosin methylene blue (EMB), which is differential for lactose and sucrose fermentation.
  • MacConkey (MCK), which is differential for lactose fermentationmannitol salt agar (MSA), which is differential for mannitol fermentation.
  • X-gal plates, which are differential for lac operon mutants.

Aseptic Technique, Dilution, Streaking, and Spread Plates

Microbiologists rely on aseptic technique, dilution, colony streaking and spread plates for day-to-day experiments.

Learning Objectives

Recall aseptic technique, dilution series, streaking and spreading plates

Key Takeaways

Key Points

  • Aseptic technique is basically the mindset of keeping things free of contamination, as the world we live in has so many microbes that can interfere with experiments.
  • Colony streaking leads to to the isolation of individual colonies, which are a group of microbes that came from one single progenitor mircrobe.
  • Spread plates allow for the even spreading of bacteria onto a petri dish; allowing for the isolation of individual colonies, for counting or further experiments.

Key Terms

  • colony: A bacterial colony is defined as a visible cluster of bacteria growing on the surface of or within a solid medium, presumably cultured from a single cell.
  • bunsen burner: A small laboratory gas burner whose air supply may be controlled with an adjustable hole.

Microbiologists have many tools, but four relatively simple techniques are used by microbiologists daily, these are outlined here.

Aseptic technique or sterile technique is used to avoid contamination of sterile media and equipment during cell culture. Sterile technique should always be employed when working with live cell cultures and reagents/media that will be used for such cultures. This technique involves using flame to kill contaminating organisms, and a general mode of operation that minimizes exposure of sterile media and equipment to contaminants.

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Serial Dilution: Example of Serial dilution of bacteria in five steps. The diluted bacteria were then spread plated. By Leberechtc (Own work) [GFDL (http://www.gnu.org/copyleft/fdl.html) and CC-BY-3.0 (http://creativecommons.org/licenses/by/3.0)], via Wikimedia Commons

When working with cultures of living organisms, it is extremely important to maintain the environments in which cells are cultured and manipulated as free of other organisms as possible. This requires that exposure of containers of sterilized culture media to outside air should be minimized, and that flame is used to “re-sterilize” container lids and rims. This means passing rims and lids through the flame produced by a Bunsen burner in order to kill microorganisms coming in contact with those surfaces.

Sterile technique, in general, is a learned state-of-being, or mantra, where every utilization of any sterile material comes with the caveat of taking every precaution to ensure it remains as free of contaminants as possible for as long as possible. Heat is an excellent means of killing microorganisms, and the Bunsen burner is the sterile technician’s best friend.

A serial dilution is the step-wise dilution of a substance in solution. Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion. A ten-fold serial dilution could be 1 M, 0.1 M, 0.01 M, 0.001 M… Serial dilutions are used to accurately create highly-diluted solutions as well. A culture of microbes can be diluted in the same fashion. For a ten-fold dilution on a 1 mL scale, vials are filled with 900 microliters of water or media, and 100 microliters of the stock microbial solution are serially transferred, with thorough mixing after every dilution step. The dilution of microbes is very important to get to microbes diluted enough to count on a spread plate (described later).

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Streak plate: Four streak plates. Successful streaks lead to individual colonies of microbes.

In microbiology, streaking is a technique used to isolate a pure strain from a single species of microorganism, often bacteria. Samples can then be taken from the resulting colonies and a microbiological culture can be grown on a new plate so that the organism can be identified, studied, or tested.The streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop. This is dipped in an inoculum such as a broth or patient specimen containing many species of bacteria.The sample is spread across one quadrant of a petri dish containing a growth medium, usually an agar plate which has been sterilized in an autoclave. Choice of which growth medium is used depends on which microorganism is being cultured, or selected for. Growth media are usually forms of agar, a gelatinous substance derived from seaweed.

Spread plates are simply microbes spread on a media plate. Microbes are in a solution, and can be diluted. They are then transferred to a petri dish with media specific for the growth of the microbe of interest. The solution is then spread uniformly through a number of possible means, the most popular is the use of sterile glass beads that are shook on top of the media, spreading the microbe-containing liquid evenly on the plate. Also common is the use of a bent-glass rod, often referred to as a hockey stick, due to its similar shape. The glass rod is sterilized and used to spread the microbe-containing liquid uniformly on the plate.

Special Culture Techniques

Many microbes have special growth conditions or require precautions to grow in a laboratory setting, leading to special culture techniques.

Learning Objectives

Evaluate special culture techniques

Key Takeaways

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.

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 pallidum. Treponema 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.