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agar A gelatinous material made from certain marine algae used as a material (and food source) in which to grow bacteria.
bacteria (singular: bacterium) Single-celled organisms. These dwell nearly everywhere on Earth, from the bottom of the sea to inside other living organisms (such as plants and animals).
bleach A dilute form of the liquid, sodium hypochlorite, that is used around the home to lighten and brighten fabrics, to remove stains or to kill germs. Or it can mean to lighten something permanently, such as: Being in constant sunlight bleached most of the rich coloring out of the window drapes.
blog Short for web log, these Internet posts can take the form of news reports, topical discussions, opinionated rants, diaries or photo galleries.
control A part of an experiment where there is no change from normal conditions. The control is essential to scientific experiments. It shows that any new effect is likely due only to the part of the test that a researcher has altered. For example, if scientists were testing different types of fertilizer in a garden, they would want one section of it to remain unfertilized, as the control. Its area would show how plants in this garden grow under normal conditions. And that gives scientists something against which they can compare their experimental data.
culture (v. in microbiology) To grow cells outside the body or their normal environment, usually in a beaker, a laboratory dish or some big vessel. To keep the cells healthy, they must be kept at the proper temperature, given the proper nutrients and provided ample room to grow.
current A fluid — such as of water or air — that moves in a recognizable direction.
degree (in geometry) A unit of measurement for angles. Each degree equals one three-hundred-and-sixtieth of the circumference of a circle.
digital (in computer science and engineering) An adjective indicating that something has been developed numerically on a computer or on some other electronic device, based on a binary system (where all numbers are displayed using a series of only zeros and ones).
ethanol A type of alcohol, also known as ethyl alcohol, that serves as the basis of alcoholic drinks, such as beer, wine and distilled spirits. It also is used as a fuel, often mixed with gasoline, for instance.
filament Something with a thin, thread-like shape. For instance, the fragile metal wire that heats up to emit light inside an incandescent light bulb is known as its filament.
gel A gooey or viscous material that can flow like a thick liquid.
germ Any one-celled microorganism, such as a bacterium or fungal species, or a virus particle. Some germs cause disease. Others can promote the health of more complex organisms, including birds and mammals. The health effects of most germs, however, remain unknown.
hypothesis (v. hypothesize) A proposed explanation for a phenomenon. In science, a hypothesis is an idea that must be rigorously tested before it is accepted or rejected.
microbe Short for microorganism. A living thing that is too small to see with the unaided eye, including bacteria, some fungi and many other organisms such as amoebas. Most consist of a single cell.
petri dish A shallow, circular dish used to grow bacteria or other microorganisms.
plastic Any of a series of materials that are easily deformable; or synthetic materials that have been made from polymers (long strings of some building-block molecule) that tend to be lightweight, inexpensive and resistant to degradation.
protein Compounds made from one or more long chains of amino acids. Proteins are an essential part of all living organisms. They form the basis of living cells, muscle and tissues; they also do the work inside of cells. Among the better-known, stand-alone proteins are the hemoglobin (in blood) and the antibodies (also in blood) that attempt to fight infections. Medicines frequently work by latching onto proteins.
smartphone A cell (or mobile) phone that can perform a host of functions, including search for information on the internet.
Styrofoam A trademarked name for a type of rigid foam made from light-weight polystyrene plastic. It is used for everything from home craft projects to decorative ornaments and building insulation.
watt A measure of the rate of energy use, flux (or flow) or production. It is equivalent to one joule per second. It describes the rate of energy converted from one form to another — or moved — per unit of time. For instance, a kilowatt is 1,000 watts, and household energy use is typically measured and quantified in terms of kilowatt-hours, or the number of kilowatts used per hour.
8. Representative Results
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. coli. 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.g., sewage, run-off from storm drains) generated by domestic, commercial, and industrial properties as well as agricultural practices, to analyze water samples following the extensive purification process. 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.g., as the inoculum for a streak-plate or a broth culture). 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 1000 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. A classic example, as shown in Figure 11, makes use of the lacZ gene encoding β-galactosidase; this enzyme allows cells to metabolize X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), a substrate analog of its natural substrate, lactose. Cleavage of X-Gal by β-galactosidase results in an insoluble blue product. 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. Whereas between 30 and 300 bacterial cells are spread over the agar surface for plate counts (cfu/ml), between 100 and 400 infectious phage particles are mixed with 108 to 109 host cells for plaque counts (pfu/ml) within a layer of soft agar spread across the surface of hard nutrient agar. 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.e., pairs, tetrads, chains, or clusters) or display growth characteristics such as capsules that hinder single colony formation. 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. Because some cells adsorb more than one phage while other cells adsorb only one or no phage, a population of host cells should be infected at a low MOI (≤1) to minimize the probability that a cell will be infected by more than one phage particle. 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. A high plating density of host cells (>109 cfu per plate) will cause a reduction in plaque size. 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 1982). Single colonies are grown on a non-selective hard agar medium then soft-agar containing 0.1% (w/v) carboxymethyl cellulose (CMC) is spread over the surface of the hard agar. After incubation, the plates are flooded with stain that permits visualization of zones of clearing around the colonies in the soft agar. The clearing is caused by hydrolytic enzymes secreted by the bacteria breaking down the cellulose in the medium. More recently, the overlay technique has been used to detect bacteria that inhibit the growth of methanogenic Archaea found in the rumen of livestock (Gilbert et al. 2010). Bacterial isolates from environmental samples are grown on a hard agar nutrient medium then colonies are overlayed with soft-agar containing a culture of methanogens. After incubation, the plates are inspected for zones of growth inhibition around the colonies. This method identifies bacterial strains that produce inhibitors of the methanogens in the soft agar.
The most common technical errors that occur with the soft-agar overlay technique are pouring the melted soft-agar either when it is too hot or too cool. If it is too hot, the bacterial cells mixed in the medium will be killed prior to plating. If it is too cool, then the soft-agar will form clumps when poured on the hard agar. In either case, the results will be ambiguous or unreadable at best.
Replica-plate Procedure. Transferring cultures from one type of nutrient medium to another to test growth requirements becomes quite laborious if there are more than just a few strains. Replica plating is a method that permits simultaneous screening of a large number of microorganisms. For instance, after mutagenizing a culture of wild-type cells, one can spread-plate dilutions of the culture to obtain plates with single colonies. The primary plates contain a medium that supports growth of all cells including wild-type prototrophs, which synthesize all compounds required for growth, and mutant auxotrophs, which carry a genetic mutation in a biosynthetic pathway rendering them unable to synthesize particular compounds essential for growth. By plating the mixture of cells onto a complete medium, the missing nutrients can be taken up from the environment. To distinguish between prototrophs and auxotrophs, the colonies can be replicated onto a minimal medium. Only prototrophs will be able to grow. Because the spatial pattern of the primary plate is preserved, comparison of the secondary plate with the primary plate allows identification of mutant colonies. To determine which compound the mutants are no longer capable of synthesizing, the colonies can be replicated onto minimal media supplemented with specific compounds (e.g., amino acids, carbon sources, vitamins, etc.). In this way, hundreds of colonies can be screened at the same time using the replica-plate procedure. One technical error that could occur is using agar plates that are too wet, causing colonies to smear together contaminating all the cultures on the plate. This produces results that are entirely unreliable. Another technical error is applying too much pressure when transferring cells from the velveteen to the secondary plates. Again, after incubating the secondary plates, the resultant colonies may overlap producing growth phenotypes attributed to contamination rather than auxotrophy.
Not all wild-type microbial species are prototrophs, so the replica-plate procedure can be used to simultaneously screen different wild-type strains for characteristic growth requirements. As shown in Figure 13, "dabs" of cells from four different Pseudomonas bacterial strains were plated in duplicate on a grid-marked plate containing complete media called YTA (panel A). The strains then were replicated onto three secondary plates (panels B, C, and D) composed of minimal medium (MSA) supplemented with a different carbon source (acetamide, lactose, and glycine, respectively). The results demonstrate that two of the four Pseudomonas strains (P. aeruginosa and P. stutzeri) are incapable of growing on these three carbon sources. As a control, the strains were replicated onto a fourth plate with YTA medium to confirm cells were transferred throughout the procedure. Since all four strains grow on the YTA control plate, the growth deficiencies exhibited on the previous three plates in the series are reliable. The replica-plating results are tabulated in Table 1. One error commonly made is interpreting an imprint of growth on a secondary plate as a positive result. For example, compare the phenotype of P. aeruginosa to that of P. stutzeri on MSA+acetamide (panel B). The latter displays an imprint of growth, which is a negative result, and can occur if nutrients from the previous plate are transferred with the parent cells. No new cell growth occurs because the missing nutrients are not available to progeny cells. It is easy to confuse an imprint with actual growth. If in doubt, the experiment should be repeated using an alternative method such as streak-plating cells from the primary plate onto secondary media.
Figure 1. Example of single colonies on a plate. The pink spheres near the center of the plate are colonies of Serratia marcescens, a Gram negative, rod-shaped Proteobacterium in the family Enterobacteriaceae. Due to its preference for damp environments, this microorganism is commonly found growing in the corners of bathtubs, in sink basins, in tile grout, and on shower curtains. S. marcescens is easy to recognize since it produces a reddish pigment called prodigiosin. The colonies on this plate were generated using the streak-plate technique, with single colonies appearing in the fourth quadrant following incubation at 30 °C for 24 hours. The other three quadrants show confluent growth in which cells deposited on the agar surface developed into overlapping colonies.
Figure 2. Instruments used for streak-plate technique. From top to bottom, shown are toothpicks (flattened not round), a wire loop, a disposable plastic loop, and wooden sticks. Toothpicks are typically transferred to a small glass beaker with the wide end down then covered with foil when autoclaved to sterilize prior to use. Wooden sticks are transferred to 18 mm test tubes then autoclaved to sterilize before use.
Figure 3. (A) Streak-plate technique using the quadrant method. A pre-sterilized loop, stick or toothpick is used to spread the sample across one-quarter of the agar surface with a rapid, smooth, back-and-fourth motion from the rim to the center of the plate. This action is repeated for each of the four quadrants of the plate. Following incubation, cell growth appears along the path of the instrument used to deposit the cells on the plate. Mechanical separation of cells in a mixed sample using this technique should result in single colonies in the fourth quadrant (see Figure 1 for an example). Single colonies are referred to as colony forming units (cfu). (B) When a metal loop is used for streak-plating, it must be sterilized using the flame of a Bunsen burner prior to contact with the inoculum or the agar medium. Recall that the hottest part of the flame is the tip of the blue cone. Holding the handle of the instrument, place the wire in the flame about 3-4 inches from the loop. Leave it long enough for the wire to become red hot. Move the wire so the flame approaches the loop. Be sure the metal loop is cooled before touching the inoculum.
Figure 4. Pour-plate technique. (A) A small volume of sample (between 0.1 to 1.0 ml) is dispensed aseptically into an empty but sterile Petri dish with using a 5.0 ml serological pipette. (B) Melted agar equilibrated to a temperature of approximately 48 °C is then poured into the Petri dish with the sample. After closing the lid, the plate is gently swirled to mix the sample and melted agar. The agar is allowed to solidify for about 30 minutes then plates are inverted for incubation.
Figure 5. Spread-plate technique with a turntable and glass spreader. After the agar plate is placed on a turntable, a small volume of sample (0.1 to 0.2 ml) is dispensed aseptically onto the center of the plate using a micropipettor. The spreader is sterilized by dipping it into a beaker of ethanol then passing it through the flame of the Bunsen burner to ignite excess ethanol. Before making contact with the sample, the spreader should be cooled by touching it to the agar near the rim of the plate. The spreader is gently moved back and forth through the sample across the plate while the turntable is slowly spinning. This action allows gradual but even spreading of the sample across the agar surface. After closing the lid, the plate should be set on the bench top undisturbed for at least 5 minutes to allow the sample to absorb completely into the agar prior to inverting the plate for incubation.
Figure 6. Spread-plate technique with glass beads (Copacabana Method). Glass beads that have been pre-sterilized in an autoclave are poured onto the surface of an agar plate sitting on the bench top. A small volume of sample (100 to 150 μl) is dispensed aseptically onto the center of the agar using a micropipettor. With the lid of the plate closed, a horizontal shaking motion is used to gently move the beads back and forth across the plate 6 to 7 times, spreading the sample. This action is repeated after rotating the plate 60°. The shaking motion is repeated a third time following another 60° rotation. Once the sample has completely absorbed into the agar medium, the beads are poured off into a beaker containing 10% bleach. The plates then are inverted for incubation.
Figure 7. Soft-agar overlay technique used to isolate and enumerate phage based on the formation of plaques (also called a plaque assay). (A) The presence of phage can be detected as zones of clearing, or plaques, on a confluent suspension of bacterial colonies growing in the soft agar. Phage T4 is a virulent, double-stranded DNA phage that infects its host, Escherichia coli, causing the host cells to lyse and release progeny phage. After multiple rounds of infection and lysis, the neighboring E. coli cells in the immediate area surrounding the original infected host cell vanishes leaving a plaque containing billions of T4 phage particles. Phage T4 produces plaques that are approximately 1 mm in diameter. In this experiment, 200 μl of a 10-5 dilution of a 2 x 108 pfu/ml stock of Phage T4 was mixed with approximately 300 μl of E. coli indicator cells prepared as an exponentially growing, aerated culture at 37 °C. Both the phage and bacteria were added to an EHA soft agar tube, mixed, then poured onto the surface of an EHA hard agar plate. Note that it was not necessary to allow phage and bacteria to adsorb prior to plating in this case. After allowing the soft agar to solidify undisturbed for 20 minutes, the plates were inverted and incubated at 37 °C for 24 hours. (B) In the absence of infecting phage particles, bacterial growth results in a cloudy suspension of cells in the soft agar in which discrete colonies are not visible. Instead, an even lawn of bacterial cells, in this case E. coli, forms throughout the entire soft agar layer.
Figure 8. Preparation of primary plate (master) with bacterial samples. To keep samples organized, the bottom of the plate can be marked into a grid and resulting squares numbered. Each sample can be assigned a square on the grid. Shown are examples of correct versus incorrect inoculation patterns. Ideally, a small number of cells are transferred to the center of the square using a sterile inoculation tool such as a toothpick to "dab" the sample (cell #4). Common inoculation mistakes, such as those depicted in cell #5 ("patch") and cell #6 ("fill"), result in overgrowth of bacterial samples following incubation, consequently contaminating adjacent squares.
Figure 9. Replica-plate technique used to transfer cells from primary to secondary plates for phenotype screens. The mark on the primary plate is aligned with the mark on the velveteen covered block then lowered to allow the agar surface to contact the cloth. Cells are transferred from the plate to the velveteen by lightly but evenly pressing down on the primary plate with finger tips. This action will leave an imprint of the cell samples on the velveteen in the same spatial pattern as the primary plate. The same procedure is used to transfer cells from the velveteen to a secondary plate. As many as 7-8 secondary plates may be inoculated using the same primary plate impression on the velveteen. The last plate inoculated from the velveteen should serve as a positive control. It should be a medium that supports growth of all tested strains, ensuring sufficient cell transfer occurred throughout the entire series of plates. Following incubation, secondary plates may be inspected and scored for growth versus no growth. Thus, multiple bacterial strains can be screened simultaneously on several growth mediums in a single experiment.
Figure 10. Example result using pour-plate technique. A 1.0 ml sample of water collected from a public drinking fountain was dispensed into a sterile empty Petri dish. Then melted but cooled YTA was poured into the dish with the sample. The agar also contained 100 μg/ml cycloheximide to prevent the growth of yeasts and molds that may have been present in the water sample. After gently swirling to mix, the plate was set upon a flat surface and the agar was allowed to solidify completely. The plate was incubated at 37 °C for 48 hours. Shown is the result of this experiment. Note the difference in appearance of surface colonies, which are large and circular in shape, versus sub-surface colonies, which are very small and irregularly shape because the solidified medium inhibits colony spreading in sub-surface.
Figure 11. Example result using spread-plate technique. The "Copacabana Method" was used to plate a mixture of E. coli cells for a screening experiment. In this case, the growth medium (LB) contains X-Gal, so those cells expressing a functional β-galactosidase enzyme form blue colonies while those cells with a mutation in the lacZ gene and thus unable to express a functional β-galactosidase enzyme form white colonies. Often referred to as a "blue/white screen", the two types of colonies can be readily distinguished from one another on the same plate.
Figure 12. Example result of plaque assay using soft-agar overlay technique. Shown are plaques formed on the host strain Mycobacterium smegmatis mc2155 (ATCC 700084) by two different phages: (A) Mycobacteriophage Destroyers and (B) Mycobacteriophage MSSS. These phage were isolated by students in the UCLA laboratory course MIMG 103L in spring 2010. M. smegmatis is a non-pathogenic Actinobacterium and belongs to a family of mycobacteria that includes a few pathogens known to cause serious diseases such as tuberculosis (M. tuberculosis, M. africanum, M. bovis) and leprosy (M. leprae). Approximately 50 μl of a 10-2 dilution of Destroyers and a 10-3 dilution of MSSS each were incubated with 500 μl of M. smegmatis for 20 minutes at 37 °C then mixed with MBTA (soft agar) and poured onto MHA (hard agar) plates. After allowing the soft agar to solidify undisturbed for 20 minutes, the plates were inverted and incubated at 37 °C for 48 hours. Note the distinct plaque morphologies produced by each phage. Destroyers (A) forms small (average diameter of approximately 1 mm), clear plaques characteristic of a lytic phage while MSSS (B) develops large "bull's eye" plaques with clear centers surrounded by a turbid halo (average diameter of approximately 3.2 mm). The hazy ring may be comprised of bacteria that are resistant to phage infection. This pattern is distinct from that formed by lysogenic phage, which produce turbid plaques.
Figure 13. Example result using replica-plate procedure. Four Pseudomonas strains (P. aeruginosa, P. putida, P. fluorescens, and P. stutzeri) were tested in duplicate for growth on three different carbon sources: acetamide, lactose and glycine. (A) The primary plate is a complete medium (YTA) inoculated with the four strains as indicated. Following incubation at 30 °C for 24 hours, all four strains grow on YTA. The primary plate was used to replica plate onto minimal medium (MSA) supplemented with a single carbon source: acetamide (B), lactose (C), and glycine (D). The last plate in the series was a positive control YTA plate (E). As shown, the strains show variable growth patterns following incubation on the secondary plates. Note that it is sometimes difficult to distinguish between growth and an imprint of cells. For instance, compare the imprint generated by P. stutzeri on the three MSA plates to no growth on same three plates by P. aeruginosa. Both are negative results in comparison to the growth patterns exhibited by P. putida and P. fluorescens. However, all strains grow on the positive control plate confirming cells were transferred to all secondary plates in the series. The results of this experiment are tabulated in Table 1.
Table 1. Summary of replica plating results. Growth indicated as plus sign (+) and no growth represented as minus sign (-). YTA is a complete medium (yeast tryptone agar) and MSA is a minimal medium (minimal salts agar). The MSA plates were supplemented with a single carbon source as indicated.