Microbial Culture Methods

Enrichment and Isolation

Understanding the nutritional requirements of bacteria can aid their enrichment and isolation.

Learning Objectives

List the growth phases of microrganisms and the different types of growth media available to culture them

Key Takeaways

Key Points

  • The most common growth media for microorganisms are nutrient broths and agar plates.
  • Open cultures allow for a replenishment of nutrients and a reduction of waste buildup in the media.
  • Selective media are used for the growth of only selected microorganisms.
  • Differential media or indicator media distinguish one microorganism type from another growing on the same media.

Key Terms

  • closed culture: A closed culture has no additional nutrients added to the system, and waste products are not removed. Cultures in a closed system will follow a predicted growth curve.
  • Enriched media: Contains nutrients required to support the growth of a wide variety of organisms.
  • open culture: A continuous culture where periodically some of the bacterial culture is removed and added to fresh sterile medium.

Enrichment and Isolation

The most common growth media for microorganisms are nutrient broths and agar plates; specialized media are required for some microorganisms. Some, termed fastidious organisms, require specialized environments due to complex nutritional requirements. Viruses, for example, are obligate intracellular parasites and require a growth medium containing living cells.

Growth media: defined vs. undefined

An important distinction between growth media types is that of defined versus undefined media.

A defined medium will have known quantities of all ingredients. For microorganisms, this consists of providing trace elements and vitamins required by the microbe, and especially, a defined source of both carbon and nitrogen. Glucose or glycerol is 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 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.

Types of media

Enriched media contain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones. They are commonly used to harvest as many different types of microbes as are present in the specimen. Blood agar is an enriched medium in which nutritionally-rich whole blood supplements the basic nutrients. Chocolate agar is enriched with heat-treated blood (40-45°C), which turns brown and gives the medium the color for which it is named.

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.

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TSI Agar slant tubes: The agar triple-sugar iron is one of the culture media used for the differentiation of most enterobacteria.

Differential/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. The agar triple-sugar iron (TSI) is one of the culture media used for the differentiation of most enterobacteria.

Growth in closed culture systems, such as a batch culture in LB broth, where no additional nutrients are added and waste products are not removed, the bacterial growth will follow a predicted growth curve and can be modeled.

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Bacterial growth curve: Bacterial growth in batch culture can be modeled with four different phases: lag phase (A), exponential or log phase (B), stationary phase (C), and death phase (D).

Growth phases

During lag phase, bacteria adapt themselves to growth conditions. It is the period where the individual bacteria are maturing and not yet able to divide. During the lag phase of the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs.

Exponential phase (sometimes called the log or logarithmic phase) is a period characterized by cell doubling. The number of new bacteria appearing per unit time is proportional to the present population.Under controlled conditions, cyanobacteria can double their population four times a day. Exponential growth cannot continue indefinitely, however, because the medium is soon depleted of nutrients and enriched with wastes.

The stationary phase is due to a growth-limiting factor; this is mostly depletion of a nutrient, and/or the formation of inhibitory products such as organic acids.

At death phase, bacteria run out of nutrients and die.

Culture

Batch culture is the most common laboratory-growth method in which bacterial growth is studied, but it is only one of many. The bacterial culture is incubated in a closed vessel with a single batch of medium.

In some experimental regimes, some of the bacterial culture is periodically removed and added to fresh sterile medium. In the extreme case, this leads to the continual renewal of the nutrients. This is a chemostat, also known as an open or continuous culture: a steady state defined by the rates of nutrient supply and bacterial growth. In comparison to batch culture, bacteria are maintained in exponential growth phase, and the growth rate of the bacteria is known. Related devices include turbidostats and auxostats. Bacterial growth can be suppressed with bacteriostats, without necessarily killing the bacteria.

In a synecological culture, a true-to-nature situation in which more than one bacterial species is present, the growth of microbes is more dynamic and continual.

Pure Culture

A pure culture is a population of cells or multicellular organisms growing in the absence of other species or types.

Learning Objectives

Describe how pure microbial cultures can be grown in agar-based growth medium

Key Takeaways

Key Points

  • A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another.
  • Microbial cultures are foundational and basic diagnostic methods used extensively as a research tool in molecular biology.
  • The most common form of microbial cultures are liquid or solid ( agar ).

Key Terms

  • agar: A gelatinous material obtained from the marine algae, used as a bacterial culture medium, in electrophoresis and as a food additive.
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Selective Media: Geomyces destructans in culture from bat tissues. (A) Original culture tubes of Sabouraud agar supplemented with nine antibiotics and incubated at 4°C for six- or eight-weeks; notice the profuse growth of G. destructans strains. (B) Some fungal contamination on individual isolates was visible as depicted in the close-up of a culture tube. (C) Enrichment and recovery of pure fungal colonies by treating a culture contaminated with bacteria with hydrochloric acid.

Microbial cultures are foundational and basic diagnostic methods used extensively as a research tool in molecular biology. It is often essential to isolate a pure culture of microorganisms. A pure (or axenic) culture is a population of cells or multicellular organisms growing in the absence of other species or types. A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another. For the purpose of gelling the microbial culture, the medium of agarose gel (agar) is used. Agar is a gelatinous substance derived from seaweed. A cheap substitute for agar is guar gum, which can be used for the isolation and maintenance of thermophiles.

Microbiological cultures can be grown in petri dishes of differing sizes that have a thin layer of agar-based growth medium. Once the growth medium in the petri dish is inoculated with the desired bacteria, the plates are incubated at the best temperature for the growing of the selected bacteria (for example, usually at 37 degrees Celsius for cultures from humans or animals or lower for environmental cultures). Another method of bacterial culture is liquid culture, in which the desired bacteria are suspended in liquid broth, a nutrient medium. These are ideal for preparation of an antimicrobial assay. The experimenter would inoculate liquid broth with bacteria and let it grow overnight (they may use a shaker for uniform growth). Then they would take aliquots of the sample to test for the antimicrobial activity of a specific drug or protein (antimicrobial peptides). As an alternative, the microbiologist may decide to use static liquid cultures. These cultures are not shaken and they provide the microbes with an oxygen gradient.

Preserving Bacterial Cultures

Bacteria can be stored for months or years if they are stored at -80C and in a high percentage of glycerol.

Learning Objectives

Describe how bacterial cultures can be stored for a long time at -80C in glycerol

Key Takeaways

Key Points

  • Preserve your selected bacteria so you always have something to go back to if something goes wrong.
  • While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.
  • Ensure a pure culture is being preserved by picking a single colony of the bacteria off a plate for cryopreservation.

Key Terms

  • cryogenic: of, relating to, or performed at low temperatures
  • logarithmic growth phase: exponential phase (sometimes called the log phase or the logarithmic phase) is a period characterized by cell doubling.
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Bacteria in liquid media: An erlenmeyer containing a bacterial culture. Bacteria that have been preserved in glycerol stocks can be grown overnight in liquid media to promote propagation.

Three species of bacteria, Carnobacterium pleistocenium, Chryseobacterium greenlandensis, and Herminiimonas glaciei, have reportedly been revived after surviving for thousands of years frozen in ice. As a practical matter, as a researcher, you will want to preserve your selected bacteria so you can go back to it if something goes wrong.

Whenever you successfully transform a bacterial culture with a plasmid or whenever you obtain a new bacterial strain, you will want to make a long term stock of that bacteria. Bacteria can be stored for months and years if they are stored at -80C and in a high percentage of glycerol.

In order to ensure a pure culture is being preserved, pick a single colony of the bacteria off a plate, grow it overnight in the appropriate liquid media, and with shaking. Take the overnight culture and and mix an aliquot with 40% glycerol in sterile water and place in a cryogenic vial. It is important to label the vial with all the relevant information (e.g. strain, vector, date, researcher, etc.). Freeze the glycerol stock and store at -80C. At this point you should also record the strain information and record the location.

While it is possible to make a long term stock from cells in the stationary phase, ideally your culture should be in logarithmic growth phase. Certain antibiotics in the medium should be removed first as they are supposedly toxic over time, e.g. Tetracycline. To do this, spin the culture down and resuspend it in the same volume of straight LB medium.

The FISH Technique

FISH is a hybridization technology which allows the labeling of target RNAs with a fluorescent probe.

Learning Objectives

Describe how fluorescent in situ hybridization (FISH) is used in clinical and biomedical studies to detect and localize the presence or absence of specific DNA sequences and to identify pathogens

Key Takeaways

Key Points

  • FISH can be used in a clinical setting to identify pathogens or DNA / RNA targets of interest.
  • FISH is used to detect and localize the presence or absence of specific DNA or RNA sequences in tissue or cells.
  • FISH can also be used to compare the genomes of two biological species, such as in ecological studies, where a bacteria may not be culturable, it can be identified using FISH.

Key Terms

  • fluorescence: The emission of light (or other electromagnetic radiation) by a material when stimulated by the absorption of radiation or of a subatomic particle
  • hybridize: To combine complementary subunits of multiple biological macromolecules.
  • FISH: Fluorescence in situ hybridization is a cytogenetic technique used to detect and localize thespecific DNA or RNA sequences.

FISH (fluorescence in situ hybridization ) is a cytogenetic technique developed by biomedical researchers in the early 1980s. It is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes bind to those targets that show a high degree of sequence complementarity. FISH can be used to detect RNA or DNA sequences of interest. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets, including mRNAs, in cells. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.

Central to FISH are the use of probes. The probe must be large enough to hybridize specifically with its target but not so large as to impede the hybridization process. They are anti-sense to the target mRNA or DNA of interest, thus they hybridize to targets. The probe can be tagged directly with fluorophores, or with targets for flourescently labelled antibodies or other substrates. Different types of tags can be used, therefore different targets can be detected in the same sample simultaneously (multi-colour FISH). Tagging can be done in various ways, such as nick translation, or PCR using tagged nucleotides. Probes can vary in length from 20 to 30 nucleotides to much longer sequences.

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Dual label FISH image: Here is an example of FISH being used to differentiate Bifidobacteria (red) and other bacteria (green)

FISH is often used in clinical studies. If a patient is infected with a suspected pathogen, bacteria from the patient’s tissues or fluids, are typically grown on agar to determine the identity of the pathogen. Many bacteria, however, even well-known species, do not grow well under laboratory conditions. FISH can be used to directly detect the presence of the suspect on small samples of the patient’s tissue. FISH can also be used to compare the genomes of two biological species, to deduce evolutionary relationships. A similar hybridization technique is called a zoo blot. Bacterial FISH probes are often primers for the 16s rRNA region. FISH is widely used in the field of microbial ecology, to identify microorganisms. Biofilms, for example, are composed of complex (often) multi-species bacterial organizations. Preparing DNA probes for one species and performing FISH with this probe allows one to visualize the distribution of this specific species within the biofilm. Preparing probes (in two different colors) for two species allows to visualize/study co-localization of these two species in the biofilm, and can be useful in determining the fine architecture of the biofilm.

Coupling Specific Genes to Specific Organisms Using PCR

PCR allows for the amplification and mutation of DNA and allowing researchers to study very small samples.

Learning Objectives

Describe how polymerase chain reaction (PCR) allows for the amplification and mutation of DNA and enables researchers to study very small samples

Key Takeaways

Key Points

  • PCR allows for identification of an infectious agent without the need for culturing.
  • Researchers can use PCR as a method of searching for specific genes and/or mutations.
  • PCR, coupled with other biochemical techniques, allows us to analyze the very core of organisms and the processes by which they function.

Key Terms

  • polymerase chain reaction: A technique in molecular biology for creating multiple copies of DNA from a sample; used in genetic fingerprinting etc.

Polymerase chain reaction (PCR) is a useful technique for scientists, because it allows for the amplification and mutation of DNA. Through PCR, small quantities of DNA can be replicated by orders of magnitude, not only essentially preserving the sample if successful, but allowing for study on a much larger scale.. Without PCR, the studies we perform would be limited by the amount of DNA we were able to isolate from samples. Through PCR, the original DNA is essentially limitless, allowing scientists to induce various mutations in different genes for further study.

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Polymerase chain reaction: a schematic of the polymerase chain reaction

Through site-directed mutagenesis or customized primers, individual mutations in DNA can be made. By changing the amino acids transcribed from DNA through individual mutations, the importance of those amino acids with respect to gene function can be analyzed. However, this process can be difficult, particularly when genes act in concert (with varying expression with respect to gene activity). The length of time it takes to run a successful PCR and perform other techniques before additional studies can be done (protein expression, isolation, and purification, for example), makes biochemical research time-consuming and difficult. However, PCR, coupled with other biochemical techniques, allows us to analyze the very core of organisms and the processes by which they function. Common PCR protocols in labs today include knockout genotyping, fluorescence genotyping and mutant genotyping. Researchers can use PCR as a method of searching for genes by using primers that flank the target sequence of the gene along with all other necessary components for PCR. If the gene is present, the primers will bind and amplify the DNA, giving a band of amplified DNA on the agarose gel that will be run. If the gene is not present, the primers will not anneal and no amplification will occur.

The ability to identify specific genes to specific organisms has increased the use of PCR and has allowed it to be more specific and eliminate the possibility of cross contaminants. The identification of specific genes to specific organisms has important medical diagnostic value.

PCR is a reliable method to detect the presence of unwanted genetic materials, such as infections and bacteria in the clinical setting. It can even allow identification of an infectious agent without culturing. For example, in diagnosis of diseases like AIDS, PCR can be used to detect the small percentage of cells that are infected with HIV by utilizing primers that are specific for genes specialized to the HIV virus. PCR can reveal the presence of HIV in people who have not mounted an immune response to this pathogen, which may otherwise be missed with an antibody assay). Additionally, PCR is used for identifying bacterial species, such as Mycobacterium tuberculosis in tissue specimens. With the use of PCR, as few as 10 bacilli per million human cells can be readily detected. The bacilli are identified by using Mycobacterium tuberculosis specific genes.