Can you culture viruses in nutrient broth

Bodily fluids and tissues should be placed in a sterile container. Upon receipt, the specimen is inoculated into several different types of cell culture depending on the nature of the specimen and the clinical presentation. The maintenance media should be changed after one hour or the next morning. The inoculated tubes should be incubated at o C in a rotating drum. Rotation is optimal for the isolation of respiratory viruses and result in an earlier appearance of the cytopathic effects CPE for many viruses.

If stationary tubes are used, it is critical that the culture tubes be positioned so that the cell monolayer is bathed in nutrient medium. Learning Objectives Discover the use of, and reasons for, culturing animal viruses in host cells.

In theory, all the cells in a colony are derived from a single bacterium initially deposited on the plate and thus are referred to as a clone, or cluster of genetically identical cells. With the streak-plate procedure, a mixture of cells is spread over the surface of a semi-solid, agar-based nutrient medium in a Petri dish such that fewer and fewer bacterial cells are deposited at widely separated points on the surface of the medium and, following incubation, develop into colonies.

The quadrant method for isolating single colonies from a mixture of cells will be described here. This method often is used to count the number of microorganisms in a mixed sample, which is added to a molten agar medium prior to its solidification. The process results in colonies uniformly distributed throughout the solid medium when the appropriate sample dilution is plated. This technique is used to perform viable plate counts, in which the total number of colony forming units within the agar and on surface of the agar on a single plate is enumerated.

Viable plate counts provide scientists a standardized means to generate growth curves, to calculate the concentration of cells in the tube from which the sample was plated, and to investigate the effect of various environments or growth conditions on bacterial cell survival or growth rate.

This technique typically is used to separate microorganisms contained within a small sample volume, which is spread over the surface of an agar plate, resulting in the formation of discrete colonies distributed evenly across the agar surface when the appropriate concentration of cells is plated. In addition to using this technique for viable plate counts, in which the total number of colony forming units on a single plate is enumerated and used to calculate the concentration of cells in the tube from which the sample was plated, spread-plating is routinely used in enrichment, selection, and screening experiments.

The desired result for these three experiments is usually the same as for plate counts, in which a distribution of discrete colonies forms across the surface of the agar. However, the goal is not to ensure all viable cells form colonies. Instead, only those cells within a population that have a particular genotype should grow. The spread plate procedure may be employed over the pour plate technique for an enumeration experiment if the end goal is to isolate colonies for further analysis because colonies grow accessibly on the agar surface whereas they become embedded in the agar with the pour plate procedure.

There are two strategies described here for the spread plate procedure. The first Method A involves use of a turntable and glass or metal rod shaped like a hockey stick. The second Method B , referred to as the "Copacabana Method", involves shaking pre-sterilized glass beads. Both facilitate even spreading of cells across the agar surface. This technique is commonly used to detect and quantify bacteriophage phage , or bacterial viruses that range in size from to nm.

An electron microscope is needed to see individual phage particles. However, the presence of infectious phage particles can be detected as plaques on an agar plate Figure 7A. Phages cannot replicate outside their host bacterial cells, so propagation and detection requires mixing phages and host cells together prior to plating. The resulting mixture is poured over the surface of a hard 1. The plate is rocked sufficiently to ensure that the soft agar covers the entire surface of the hard agar. Then the plate is placed on a level surface until the top agar layer has had time to solidify and subsequently can be placed in the incubator.

Over time, a cloudy suspension of bacterial cells, referred to as a lawn , becomes visible throughout the soft agar medium Figure 7B. Plaques form if a phage infects one of the bacterial cells, replicates within the cell, then lyses the cell releasing as many as progeny phages a. The new phage particles diffuse into the soft agar, infecting bacteria in the area surrounding the lysed bacterial cell. After multiple cycles of infection and lysis, the cloudy bacterial cell suspension in the soft agar disappears, leaving a zone of clearing called a plaque.

Each plaque contains more than 10 9 phage particles, all genetically identical to the original infectious phage particle. Because a plaque arises from a single phage particle, the resulting number of plaque forming units pfu may be counted and the original concentration, or titer , of the phage suspension may be calculated.

This type of experiment, called a plaque assay, also provides scientists a standardized means to generate one-step growth curves, to investigate host range specificity, and to transduce bacterial cells for genetic experiments. Following incubation, the plates may be inspected for plaques. The negative control should have only a lawn of bacteria no holes indicative of plaques. Plaques vary in terms of size, shape and overall appearance. This lysate can be plated using the same procedure described above.

At least 3 to 6 successive single-plaque isolations are necessary to ensure that a pure phage has been obtained. Often the lysate must be diluted over a large range 10 -1 to 10 to find a titer that produces non-overlapping plaques on a plate.

The number varies depending on the size of the plaque. This technique permits comparison of cell growth on a primary plate to secondary plates, generating a means to screen cells for a selectable phenotype. First a primary, or master, plate is inoculated with cells either by spread-plating a dilution that produces single colonies or by transferring them to a plate in a spatial pattern specified by grid markings.

Secondary plates containing media with growth inhibitors or media that lacks a particular nutrient are inoculated with cells from colonies on the primary plate. The spatial pattern of colonies is reproduced first by pressing a piece of velvet to the primary plate. Bacterial cells adhere to the velvet because they have greater affinity for the velvet than for the agar. The imprint of cells on the velvet then is transferred to multiple secondary plates with cell growth reflecting the same colony pattern as that of the primary plate.

In other words, it is like having a rubber stamp, replicating the growth pattern from one plate to another. This technique is advantageous because it allows a relatively large number of colonies to be screened simultaneously for many phenotypes in a single experiment. Streak-plate Technique. A sample application for streak plating is shown in Figure 1. This procedure is used for isolating bacterial colonies from mixed cell cultures and is by far one of the most important techniques to master in microbiology and molecular genetics.

Each colony represents a population of cells that are genetically identical. For many downstream applications it is imperative to start with either a single colony or a pure bacterial culture generated by inoculating media with cells from a single colony. For instance, the morphology of individual cells within a colony can be inspected using a light microscope. Genetic identity can be assigned by sequencing the small subunit ribosomal RNA gene from genomic DNA isolated from a cell culture started with a single colony.

And metabolic characteristics can be described by subjecting cells to various biochemical and physiological assays. Only by performing such experiments with pure cultures can one be certain of the properties ascribed to a particular microorganism.

The results are not obscured by the possibility that the culture is contaminated. Technical errors may occur if the sterility of the instrument used to streak the cells across the plate is not maintained throughout the procedure. Forgetting to flame a loop or retrieve a fresh toothpick between quadrants make it difficult to obtain single colonies. Some bacterial species cannot be isolated in pure culture as they are dependent on a cooperative association with another bacterial species for certain growth requirements.

Referred to as syntrophs, these organisms may only be grown under co-culture conditions, so colonies if formed always will be comprised of two or more species. Another challenge encountered in the laboratory when performing the streak-plate procedure with bacteria derived from environmental samples is that cells exhibit growth characteristics that deviate from traditional laboratory strains such as E. Such bacterial strains may produce colonies that are filamentous as opposed to tight clusters of cells with branches that spread over a large section of an agar plate, calcified and thus refractory to penetration by a streak-plate instrument, or surrounded by a sticky capsule so that individual colonies cannot be discerned.

These characteristics make it difficult to purify single colonies by the streak-plate technique. Pour-plate Technique. With the pour-plate technique, the colonies form within the agar as well as on the surface of the agar medium thus providing a convenient means to count the number of viable cells in a sample.

This procedure is used in a variety of industrial applications. For instance, it is critical for a wastewater treatment plant, which is responsible for cleaning up liquid waste e. Treated wastewater non-potable water is reused in a variety of ways - for irrigation of non-food crops in agriculture, for sanitary flushing in residences, and in industrial cooling towers - so it must be free of chemical and microbial contamination. Drinking water potable water must be purified according to EPA standards and is tested using microbiological plating methods that allow enumeration of specific human pathogens.

Shown in Figure 10 are bacterial colonies resulting from bacteria cells present in a water sample collected from a public drinking fountain. It is unlikely bacterial pathogens produced these colonies given the purification measures for potable water; however, microbes are everywhere and contamination by even non-pathogenic strains can be only minimized, not eliminated entirely. As another example, a pharmaceutical company needs to assess the degree of microbial contamination, or bioburden, of a new drug during production, storage and transport.

By sampling the drug during various phases of the process and plating samples using the pour-plate procedure, the microbial load, or number of contaminating bacteria, can be readily determined.

Precautionary measures then can be devised to minimize or eliminate microbial contamination. One of the most common technical errors that occurs when performing the pour-plate technique is insufficient mixing of the sample with the melted agar causing colonies to clump together thereby making plate counts inaccurate.

Another frequent error is pouring the melted agar when it is too hot, killing many of the bacterial cells in the sample. This mistake also will affect accuracy of plate counts giving numbers that under-represent the total number of colony forming units in the sample.

Spread-plate Technique. The spread-plate technique is analogous to the pour-plate procedure in its utility as a means to perform viable plate counts. However, because the colonies that form using the spread-plate technique are evenly distributed across the surface of the agar medium, cells from individual colonies can be isolated and used in subsequent experimental manipulations e.

Three common applications in which the spread-plate technique is an important component are enrichment, selection and screening experiments. In all three applications, the desired cell type can be separated from the mixture and later subjected to any number of biochemical, physiological, or genetic tests. An enrichment experiment involves plating a mixed culture on a medium or incubating plates in environmental conditions that favor growth of those microorganisms within the sample that demonstrate the desired metabolic properties, growth characteristics, or behaviors.

This strategy does not inhibit the growth of other organisms but results in an increase in the number of desired microorganisms relative to others in the culture.

Thus, the colonies that form on an enrichment plate likely exhibit phenotypic properties that reflect the desired genotype. For instance, if your goal is to cultivate nitrogen-fixing bacteria from an environmental sample containing a mixture of more than different bacterial species, then plating the sample on a nitrogen-deficient medium will enrich for those bacteria that can produce this compound from the atmosphere using metabolic capabilities provided by a suite of genes required for fixing nitrogen.

A selection experiment involves plating a mixed culture on a medium that allows only those cells that contain a particular gene or set of genes to grow. This type of experiment is common in molecular biology laboratories when transforming bacterial strains with plasmids containing antibiotic-resistance genes.

If your goal is to cultivate only recombinant cells, or those that successfully took up the plasmid, then plating the sample on a medium that has been supplemented with an appropriate concentration of the antibiotic will select for those cells that exhibit resistance to this particular drug. A screening experiment involves plating a mixed culture on a medium that allows all viable cells to grow; however, the cells with the desired genotype can be distinguished from other cells based on their phenotype.

Again, this type of experiment is common in molecular biology laboratories when performing mutagenesis assays or cloning genes into plasmids. Thus, if a medium contains X-gal, and a sample containing cells with either a wild-type functional or mutant non-functional lacZ gene are plated on this medium, then following incubation wild-type cells harboring a functional lacZ gene will appear as blue-pigmented colonies while mutant cells with a non-functional lacZ gene will appear as unpigmented "white" colonies.

A technical problem encountered most frequently when first learning how to perform the spread-plate technique is uneven spreading of cells across the agar surface.

When using a turntable and glass rod, the sample may be absorbed too quickly such that the colonies form only near the center of the plate. When doing the "Copacabana Method", the glass beads are swirled rather than shaken across the agar surface. Consequently, many colonies grow along the outer rim of the plate. In either case the resulting distribution of colonies does not take advantage of the complete surface area available so cells may clump together and grow into overlapping colonies making plate counts inaccurate or distinction of cell types unfeasible.

Soft Agar Overlay Technique. A procedure akin to the spread-plate technique used to count bacterial colonies can be used to tally the number of phage.

Unless demonstrated otherwise, it is generally assumed that a single bacterial cell divides and accumulates large numbers of genetically identical cells in a single cluster called a colony. As discussed previously, this assumption is not valid when cells grow in bunches i. A similar assumption is made for plaque formation, in that each plaque represents activity of a single phage. This statement is true only if one phage infects one bacterium.

What happens if multiple phage particles infect a single bacterium? This problem relates to an important statistical parameter that must be considered when performing experiments with phage - multiplicity of infection MOI - describing the ratio of infectious phage particles to the number of host cells in a sample.

Employing the plaque forming unit pfu as a functional definition avoids these complications when performing plaque counts to calculate the titer of a phage stock.

As shown in Figure 12 , plaque morphology varies for different phage. Some phage generate small plaques panel A while others give rise to large plaques panel B. A number of variables affect plaque size. There are technical reasons that contribute to this variability. For instance, complete media and thick hard agar support development of larger plaques because host cells can sustain phage growth for a longer period of time.

Using lower concentrations of soft agar will increase the rate of phage particle diffusion in the soft agar and thereby increase the size of plaques. Recall that this increased diffusion rate can occur unintentionally if the hard agar plates are not completely dry such that condensation or excess moisture in the dish dilutes the soft agar in the overlay. This technical oversight will produce inconsistent results with respect to plaque size for a particular phage.

Plaque size also is related to a number of host cell events including the efficiency of adsorption, the duration of the latent period the time span from phage adsorption to lysis of the host cell , and the burst size the number of progeny released by a single infection. A heterogeneous mixture of plaque sizes may be observed if phage particles infect host cells at different phases of bacterial growth.

For instance, those that adsorb during early exponential phase make larger plaques with more progeny phage than those that adsorb in late exponential phase. As a general rule, lytic phage produce clear plaques while lysogenic phage form turbid plaques. However, some lytic phage produce interesting patterns such as the "bull's eye" plaque shown in Figure 12B.

These clear plaques are surrounded by a turbid halo because those cells at the edge of the plaque are not fully lysed or may be resistant to phage infection. A "bull's eye" pattern observed with temperate phage is a plaque with a turbid center surrounded by a clear ring. This morphology reflects the MOI and the physiology of the host cell with respect to the lysis-lysogeny decision. When cells are first infected with phage, the MOI is low and cells grow rapidly because nutrients are abundant; together this facilitates lytic growth.

As more and more cells are lysed, the MOI increases and a clear plaque forms. Lysogens in the center of the plaque, however, continue to grow because they are immune to lysis giving rise to a clear plaque with a turbid center. The overlay technique can be modified for plaque assays with eukaryotic viruses. In the same way bacteriophage form plaques on a lawn of bacterial cells in soft agar, eukaryotic viruses form plaques on a monolayer of cells covered by a gel.

A monolayer is a confluent sheet of cells growing side by side on the surface of a culture dish, touching each other but not growing on top of one another.

To carry out this type of plaque assay, aliquots of virus are added to susceptible monolayers of eukaryotic cells. Then the monolayer is covered with an agarose-based nutrient medium - this gel restricts the spread of progeny viruses released from infected cells to adjacent cells in the monolayer. Accordingly, a spherical area, or plaque, is produced that contains cells damaged by release of virions. To aid visualization of the plaques, dyes that stain living cells can be applied to the cell culture providing contrast between infected and uninfected cells.

The soft-agar overlay technique is used for experiments other than plaque assays. First, it is significant to remember that the hard nutrient agar is a support matrix that permits growth of bacteria. Second, the soft-agar used for the overlay can have a different nutrient composition than the hard agar. In this way, the soft-agar can serve as a means to assay bacterial strains for various growth characteristics or metabolic properties.

For instance, the overlay technique is used to screen bacteria for the ability to degrade cellulose Teather and Wood Single colonies are grown on a non-selective hard agar medium then soft-agar containing 0. Micro-organisms affect every aspect of life on Earth. Some microbes cause disease but the majority are completely harmless. More on About Microbiology. The dirtiest spots in the kitchen are dishcloths, cutting boards, sponges, and sink handles.

Surprisingly, the floor is often cleaner than the sink! Micro-organisms can be used to demonstrate principles of biology and to model industrial processes, as well as offering opportunities for teaching across the curriculum. More on Teachers. Some dentists recommend that a toothbrush should be kept at least 2 metres away from a toilet to avoid air-borne particles resulting from the flush — what a large bathroom!

More on Students. Keeping up with the latest news and research about microbes is easy with Microbiology Online — your one-stop shop for microbial science education. More on What's new. If you pick up a handful of garden soil you will be holding hundreds if not thousands of different kinds of microbes. The Microbiology Society is a professional body for scientists who work in all areas of microbiology.

It has over 4, members worldwide who are based in universities, industry, hospitals and research institutes. More on About Us. The method for the preparation of basic microbiology media is given below. In situations where preparation is uneconomic in time, prepared, sterilized media liquid and solid are available from the major school science equipment suppliers. Swabs should be put in a vial containing virus transport medium. Bodily fluids and tissues should be placed in a sterile container.

Upon receipt, the specimen is inoculated into several different types of cell culture depending on the nature of the specimen and the clinical presentation. The maintenance media should be changed after one hour or the next morning.

The inoculated tubes should be incubated at o C in a rotating drum. Rotation is optimal for the isolation of respiratory viruses and result in an earlier appearance of the cytopathic effects CPE for many viruses.

If stationary tubes are used, it is critical that the culture tubes be positioned so that the cell monolayer is bathed in nutrient medium. Yellow fever virus : A micrograph of the yellow fever virus. Viruses are obligate intracellular parasites and cannot grow on inanimate media.

They need living cells for replication, which can be provided by inoculation in live animals among other methods used to culture viruses cell culture or inoculation of embryonated eggs. Inoculation of human volunteers was the only known method of cultivation of viruses and understanding viral disease. In , Reed and his colleagues used human volunteers for their work on yellow fever. Due to serious risk involved, human volunteers are recruited only when no other method is available and the virus is relatively harmless.

Smallpox was likely the first disease people tried to prevent by purposely inoculating themselves with other infections and was the first disease for which a vaccine was produced. Today, studying viruses via the inoculation of humans would require a stringent study of ethical practices by an institutional review board.

In the past few decades, animal inoculation has been employed for virus isolation. The laboratory animals used include monkeys, rabbits, guinea pigs, rats, hamsters, and mice. The choice of animals and route of inoculation intracerebral, intraperitoneal, subcutaneous, intradermal, or intraocular depends largely on the type of virus to be isolated. Handling of animals and inoculation into various routes requires special experience and training.

In addition to virus isolation, animal inoculation can also be used to observe pathogenesis, immune response, epidemiology, and oncogenesis. Growth of the virus in inoculated animals may be indicated by visible lesions, disease, or death. Sometimes, serial passage into animals may be required to obtain visible evidence of viral growth.

Animal inoculation has several disadvantages as immunity may interfere with viral growth, and the animal may harbor latent viruses. The genetic material within virus particles varies considerably between different types of viruses.