What is streak plate

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. 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.

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. 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. 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. 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 inches from the loop.

Leave it long enough for the wire to become red hot. Be sure the metal loop is cooled before touching the inoculum. Figure 4. Pour-plate technique. A A small volume of sample between 0. 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.

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. 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.

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. Phage T4 produces plaques that are approximately 1 mm in diameter.

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. 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.

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 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 Example result using pour-plate technique. Then melted but cooled YTA was poured into the dish with the sample. After gently swirling to mix, the plate was set upon a flat surface and the agar was allowed to solidify completely. 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.

Example result using spread-plate technique. The "Copacabana Method" was used to plate a mixture of E. Example result of plaque assay using soft-agar overlay technique. 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.

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. Example result using replica-plate procedure. Four Pseudomonas strains P. A The primary plate is a complete medium YTA inoculated with the four strains as indicated. 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.

Both are negative results in comparison to the growth patterns exhibited by P. 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. The MSA plates were supplemented with a single carbon source as indicated.

Culturing microorganisms involves a number of plating methods, all of which require that aseptic technique be maintained throughout the manipulation of cells and media. Five different procedures were described in this protocol. Although these plating techniques are routinely used to manipulate bacteria and phage, they also can be applied to mammalian cell culture and eukaryotic microorganisms commonly used in molecular genetics such as yeast i.

There also are numerous and even more sophisticated variations of each plating method depending on the experimental goal or organism under study. Thus, it is important to not only select the most appropriate technique for a given experiment or target microorganism but also to tailor the methodology such that the experimental outcomes suitably address the research question or problem.

Some of the most current applications of the plating techniques discussed in this protocol involve technological advances that yield high-throughput results for screening and drug discovery experiments. For example, genome sequencing centers use the "Copacabana Method" for spread-plating clone libraries, which are E. Because dozens of large plates called bioassay trays are prepared at once, an automated plate shaker is used to shake the glass beads for the entire batch of trays.

Furthermore, when selecting colonies from these plates following incubation, a robotic colony picker is used to collect cells from appropriate colonies as the inoculum for LB broth in well microplates. For this high-throughput screening assay, the procedural principles of the spread-plate technique apply but technology allows various steps to be automated and scaled to permit a large number of samples to be analyzed simultaneously and within a short time frame. Biotechnology and pharmaceutical companies invest considerable resources in the development of high-throughput technology for the most basic techniques in microbiology and molecular genetics.

For instance, there are multi-channel micropipettors to perform volume transfers for up to 8 or 12 samples at once. There even are robotic workstations that maneuver a channel pipette head! These efforts involve multi-disciplinary teams of scientists, pairing biologists that possess methodological expertise with engineers and computer programmers who can develop the instrumentation needed to perform the mechanical operations associated with the experiments.

Regardless of the research application, the goal shared by companies developing these technologies is the same - to automate laboratory processes, tools, systems and instruments, making them less labor intensive and more efficient.

National Center for Biotechnology Information , U. J Vis Exp. Published online May Erin R. Sanders 1. Author information Copyright and License information Disclaimer. Correspondence to: Erin R.

Sanders at ude. This article has been cited by other articles in PMC. Abstract Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory.

Keywords: Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions.

Download video file. Protocol 1. Prepare a Safe and Sterile Workspace Be familiar with all laboratory rules and safety precautions to be taken when working with microorganisms. Clear away all materials cluttering your work area on the laboratory bench. Clean work area with disinfectant to minimize possible contamination. Place the Bunsen burner to your right on the bench.

Place agar plates or Petri dishes to your left. Arrange cell cultures, tubes, flasks and bottles in the center of the bench. Streak Plate Procedure: Isolation of Bacterial Colonies Using the Quadrant Method The streak-plate procedure is designed to isolate pure cultures of bacteria, or colonies, from mixed populations by simple mechanical separation.

A new plastic loop, wooden stick or toothpick should be used for each quadrant. Repeat step 6 twice for the third and fourth quadrants.

Avoid going into the first quadrant when streaking the fourth quadrant. Pour Plate Procedure: Enumeration of Bacterial Cells in a Mixed Sample 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.

Obtain a tube containing 18 ml of melted agar medium. The samples may be derived from a dilution series of a single sample. Sample volume to be plated should be between 0. Use aseptic technique throughout this procedure. Allow the agar to thoroughly solidify before inverting the plate for incubation.

Spread Plate Procedure: Formation of Discrete Bacterial Colonies for Plate Counts, Enrichment, Selection, or Screening 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.

Method A: Spread-plating with a turntable and glass or metal rod Label around the edge of the bottom not the lid of an agar plate with at least your name, the date, the type of growth medium, and the type of organism to be plated on the medium.

Include the dilution factor if plating serial dilutions. Center the plate on the turntable Figure 5. Drain and ignite excess ethanol by passing it through the flame of a Bunsen burner.

Method B: Spread-plating with glass beads: the "Copacabana Method" Label around the edge of the bottom not the lid of an agar plate with at least your name, the date, the type of growth medium, and the type of organism to be plated on the medium. Do not swirl the beads or else all the cells will end up at the plate edge. HINT: If done properly, the procedure sounds like "shaking maracas". Invert the plate for incubation. Soft Agar Overlay Procedure: Formation of Plaques for Isolation and Enumeration of Phage Plaque Assay This technique is commonly used to detect and quantify bacteriophage phage , or bacterial viruses that range in size from to nm.

Phage stocks should be handled gently - do not vortex or pipet vigorously. 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.

Learning Objectives Recall aseptic technique, dilution series, streaking and spreading plates. 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. Figure: Serial Dilution : Example of Serial dilution of bacteria in five steps. The diluted bacteria were then spread plated. Figure: Streak plate : Four streak plates. Allow beans to cool completely then remove to a paper towel-lined plate to dry.

Miller traces his irreverent and subversive streak to a psychedelic experience during the particularly sweltering summer of The young man had the presence of mind to tail Gaylard Williams out of the park and jot down his license plate. Well, the pudding moment arrived, and a huge slice almost obscured from sight the plate before us.

He conducts this ceremony with the greatest solemnity, occasionally pronouncing these incantatory words, "Plate or shell, sah? We sat down pell-mell, anywhere, I next to Liszt, who kept putting things on my plate.

He silenced her with a gesture, and, leaving a piece of toast half-eaten on his plate, he got up and went into his study.