replica plate technique experiment

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Replica plating: principle and procedure | molecular biology.

J. Lederberg and E. Lederberg (1952) devised this procedure to demonstrate the spontane­ous nature of mutations.

This method is used for detection of bio­chemical mutants, for the classification of fer­mentation reactions and for the determination of the spectra of antibiotic sensitivity.

Phage sensitive strain of E.coli on nutrient agar plate is incubated until each cell has formed a clone of a few hundred progeny. On the surface of the agar plate this will appear as a confluent growth. If a few cells of these 10 8 are spontaneous phage resistant mutants, they will produce phage resistant clones on the agar. The problem is to find out these phage resistant mutants from within the con­fluent lawn of cells and to isolate it. This is accomplished by replica plating method.

Principle :

Using the threads of velvet or chamios leather which act as inoculating needles, the mutants can be replicated and isolated.

Requirements:

1. Chamios leather/velvet mounted on cy­lindrical blocks of metal (slightly smaller than the diameter of Petri dish).

2. Petri dishes with bacterial growth.

3. Phage coated agar plates.

4. Inoculating loop/needle.

5. Nutrient agar.

Procedure :

1. Mount a piece of sterile velvet by stretch­ing it on a cylindrical metallic block (slightly smaller than Petri dish).

2. Place the block with velvet side facing upwards.

3. Invert the Petri dish with the lawn of bac­terial cells (master plate) and gently press against the velvet. The number of projecting fibres of the velvet (almost 1000/sq. inch) act as inoculating needles sampling every clone of the cells in the lawn.

Remove the Petri dish and press two or more phage coated agar against the vel­vet in turn.

4. Save the original master plate.

5. Incubate the subsequent phage coated plates.

6. A few colonies appear on the phage coated plates. Some of these may represent mu­tants that arose during the cell divisions that occurred after replica plating.

7. Colonies found at the identical positions on every replica plate can be presumed to have arisen from an inoculum of phage resistant ones transferred via the velvet from the phage resistant clone on the master plate.

8. Mark the position, pick up the colony with an inoculating needle from the same location from the master plate (let us assume that out of the 10 5 cells that the needle picks up 2 or 3 alone are phage resistant).

9. Transfer this to a tube of broth, incu­bate to increase the total cell number.

10. Spread a sample of this over a fresh agar plate (only 10 5 cells). This time the in­oculum is enriched in phage-resistant mutants since they are picked up from the region containing a clone of resist­ant cells.

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Molecular Biology , Biochemical Mutants , Detection , Replica Plating

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12.4: Replica plates accelerate the screening process

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• Page ID 17569

• Clare M. O’Connor
• Boston College

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As noted above, transformation is an inefficient process, so researchers want to make the most of every cell that has been transformed. In our experiments, we will be isolating transformed cells for their ability to grow in the absence of uracil, but we are really interested in their ability to grow in the absence of Met. Replica plating offers a means to quickly screen a plate of cells for their ability to grow in a wide range of media, while retaining information about individual colonies. As shown on the opposite page, the original plate of transformants becomes the “master plate.” An imprint of the master plate is made by GENTLY tapping the inverted plate on a piece of sterile cotton velveteen immobilized on a block. This imprint can then be transferred to plates with different kinds of selective media, establishing the genotype of the transformants. In our experiments, we will make transfer replicas of the transformation reactions (isolated on YC-Ura plates) to YC-Ura plates that are also lacking Met, with either glucose or galactose as a carbon source.

Replica plating provides a rapid screening method for analyzing phenotypes. Colonies on a master plate are transferred to a sterile piece of velveteen. Copies of the mater plate are transferred to additional selective or indicator media to monitor phenotypes under additional conditions.

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Ame’s Test, Replica plate technique and Fluctuation test

Fluctuation test

This test was devised by Luria and Delbruck (1943) for investigating the response of bacterial populations to changes in the environment. At that time two hypotheses co-existed-

1.  Genetic changes occur adaptively, i.e. as a result of environmental Influences on the cells.

2.  Genetic changes occur spontaneously i.e. independently of the environment. If mutants arose spontaneously and randomly, the number of mutant cells should vary considerably from culture to culture. This was resolved by an experiment called the fluctuation test that may be performed in the following manner.

• A young bacterial suspension containing about 500 E. coli cells per mL is divided into two cultures, A and B of 10 ml each. Culture A is then further subdivided into a series of 50 tubes, each containing 0.2 ml.
• Both cultures are incubated until a predetermined population density is attained. Under these conditions, the cell concentration in the two cultures will be approximately equal.
• The number of streptomycin-resistant colonies which develop when the contents of each of the small tubes are poured onto the surface streptomycin-containing agar plates is then compared with the number which develops when a similar series of 0.2 ml samples from culture B are pipetted onto the surface of streptomycin containing agar.
• If bacterial resistance developed by direct adaptation to the streptomycin, there should be no significant difference in the numbers of resistant colonies on any of the plates derived from the two cultures.
• By this theory, it should make no difference whether the cultures were incubated in a series of separate tubes or were all grown together in one large tube since they are all presumed to be sensitive until brought into contact with streptomycin.

• If on the other hand, streptomycin resistance resulted from spontaneous mutations during growth in streptomycin-free medium: there should be a large fluctuation between the numbers of resistant colonies, which developed from each of the 50 small tubes.
• This fluctuation in the number of resistant bacteria in a series of parallel cultures is a consequence of the randomness in time of the occurrence of spontaneous mutation to resistance occurred early during cultural growth, the resistant mutant would pass through a large number of cell divisions, and there would be many resistant bacteria present at the time of testing.
• If the mutation to resistance occurred just before the cells were added to streptomycin, only one resistant colony should appear on the plate.
• A similar series of samples taken from the same container (culture B) serves as a control on the experimental technique. By either hypothesis, random samples taken from it should contain the same number of resistant bacteria.
• The result obtained from the fluctuation test supports the spontaneous mutation theory.

Replica plate technique

• This technique was devised by Lederberg and Lederberg (1952). The replica plating technique was used to verify the spontaneous origin of bacterial resistance.
• By this procedure samples from all the colonies on a plate may be transferred simultaneously to another plate by means of velveteen covered stamp pad.
• Colonies picked up from first plate this plate is known as ‘master plate’, colonies transferred on second plate first plate is known as ‘replica plate’.

• Several million streptomycin-sensitive bacteria were spread onto the surface of a streptomycin-free agar plate and incubated for a few hours to allow microcolonies.
• The sterile velveteen stamp was then pressed onto the plate containing these microcolonies so that a few of the bacteria from each of them adhered to the fibers.
• Next, the pad was pressed against the surface of the streptomycin-containing plate, and the bacteria transferred to it were incubated until resistant colonies appeared.
• These colonies were then superimposed over the original plate, and the bacterial growth in the area corresponding to the resistant colonies was picked into a fresh nutrient broth, there will be almost a hundredfold increase in the proportion of resistant mutants after incubation of this fresh culture.
• After overnight incubation of this new culture, diluted aliquots of it were spread onto the surface of the fresh, drug (streptomycin) free agar plates, and the same procedure of incubation and replication was repeated as in the previous step.
• This time more resistant colonies were found on the drug-containing plates than in the first cycle. A colony was located on the plain agar plate (without streptomycin) or master plate which exactly corresponded to a colony on the drug-containing replica plate. The colony on the plain agar was picked and proved to consist entirely of resistant cells.
• The Ames test is a widely employed method that uses bacteria to test whether a given chemical can cause mutations in the DNA of the test organism. More formally, it is a biological assay to assess the mutagenic potential of chemical compounds.
• The Ames test uses several strains of the bacterium Salmonella typhimurium that carry mutations in genes involved in histidine synthesis.
• These strains are auxotrophic mutants, i.e. they require histidine for growth, but cannot produce it. The method tests the capability of the tested substance in creating mutations that result in a return to a “prototrophic” state so that the cells can grow on a histidine-free medium.
• The tester strains are specially constructed to detect mutations in the genes required to synthesize histidine, so that mutagens acting via different mechanisms may be identified. The bacteria are spread on an agar plate with a small amount of histidine.
• This small amount of histidine in the growth medium allows the bacteria to grow for an initial time and have the opportunity to mutate.
• When the histidine is depleted only bacteria that have mutated to gain the ability to produce their own histidine will survive. The plate is incubated for 48 hours. The mutagenicity of a substance is proportional to the number of colonies observed.
• Isolate an auxotrophic strain of Salmonella typhimurium for histidine. (ie. His-ve)
• Prepare a test suspension of his—ve Salmonella typhimurium in a plain buffer with test chemical (e.g. 2—-aminofluorene). Also add a small amount of histidine. Note: small amount of histidine is required so bacteria start growing. Once histidine is depleted only those bacteria mutated to gain the ability to synthesize histidine form colonies.
• Prepare a control suspension of His-ve Salmonella typhimurium but without test chemicals.
• Incubate the suspensions at 37°C for 20 minutes
• Prepare the two agar plate and spread the suspension on agar plate.
• Incubate the plates at 37°C for 48 hours.
• After 48 hours count the number of colonies in each plate.

Result Interpretation

• The mutagenicity of chemicals is proportional to a number of colonies observed.
• If there is a large number of colonies on the test plate in comparison to control, then such chemical are said to be mutagens.
• Very few numbers of colonies can be seen on control plate also. This may be due to spontaneous point mutation on hisidine encoding gene.

Replica plating

replica plating (Science: technique) technique for testing the genetic characteristics of bacterial colonies. A dilute suspension of bacteria is first spread, in a petri dish, on agar containing a medium expected to support the growth of all bacteria, the master plate. Each bacterial cell in the suspension is expected to give rise to a colony. A sterile velvet pad, the same size as the petri dish , is then pressed onto it, picking up a sample of each colony. The bacteria can then be stamped onto new sterile petri dishes, plates, in the identical arrangement. The media in the new plates can be made up to lack specific nutritional requirements or to contain antibiotics. Thus colonies can be identified that cannot grow without specific nutrients or that are antibiotic resistant and cells with mutations in particular genes can be isolated.

Last updated on May 29th, 2023

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Aseptic Laboratory Techniques: Plating Methods

When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: ● Perform plating procedures without contaminating media. ● Isolate single bacterial colonies by the streak-plating method. ● Use pour-plating and spread-plating methods to determine the concentration of bacteria. ● Perform soft agar overlays when working with phage. ● Transfer bacterial cells from one plate to another using the replica-plating procedure. ● Given an experimental task, select the appropriate plating method.

1. Prepare a Safe and Sterile Workspace

• Be familiar with all laboratory rules and safety precautions to be taken when working with microorganisms. Regardless of biohazard classification, all materials that come in contact with microorganisms are considered infectious waste and must be decontaminated prior to disposal. Follow safety guidelines in accordance with those provided by institutional Environmental Health and Safety departments, setting up appropriate waste receptacles for immediate and proper disposal of potentially contaminated materials (biohazards).
• Sterilize all instruments, solutions, and media prior to using them for plating procedures.
• Clear away all materials cluttering your work area on the laboratory bench.
• Clean work area with disinfectant to minimize possible contamination.
• If working with BSL-2 organisms, set up your work space in a Biosafety cabinet. A Bunsen burner cannot be used inside the cabinet because the heat from the flame disrupts air flow essential to its functionality.
• 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.
• Loosen the caps of tubes, flasks and bottles so they can be easily opened with one hand during subsequent manipulations.
• Wash hands thoroughly with antiseptic soap and warm water before handling microorganisms.

2. 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. Single colonies are comprised of millions of cells growing in a cluster on or within an agar plate ( Figure 1 ). A colony, unlike a single cell, is visible to the naked eye. 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.

• Bacteria exist in a variety of shapes and sizes. For example, individual Escherichia coli cells are rod-shaped with an average length of 2 μm and width of 0.5 μm while Streptococcus cells are spherical with an average diameter of 1 μm. Some bacteria (such as E. coli ) exist as single cells while others form distinct patterns of association. Streptococcus , for instance, grow in pairs or form chains or clusters of cells. It is generally assumed that a single colony arises from a single cell undergoing binary fission; however, this assumption is not true for those bacteria that naturally exist as pairs, chains, or clusters or that divide by other mechanisms. Alternatively, if too many bacteria are plated, then overlap of cells may occur and increase the probability of two or more bacteria giving rise to what appears to be a single colony. To avoid these complications when describing or enumerating bacterial cultures growing on a solid medium, colonies are referred to as colony forming units (cfu) .

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.

• Disposable pre-sterilized sticks and toothpicks need not be flamed with the Bunsen burner thus preventing unnecessary aerosolization of spore-forming bacteria and cross-contamination of laboratory surfaces or agar plates with spores.
• The plates must be completely dry without condensation on the lid and pre-warmed to room temperature prior to streak-plating. If the plates are stored at 4 °C, remove them several hours or even the day before. Spread them out in small, staggered stacks of no more than 2-3 plates and allow them to dry.
• The sample from which the streak-plate will be inoculated could be either a suspension of cells in broth or an existing colony from another agar plate. To begin, it is recommended that only one sample be used to inoculate a single plate. To save time and materials, a single plate can be used for multiple samples but only once you become proficient at streaking one sample on a plate.
• If the inoculum is a cell suspension, obtain a loopful with a metal loop or 5 to 10 μl with a micropipettor. Be sure to swirl or vortex the cell suspension before removing an aliquot for plating. Use aseptic technique while performing this step, flaming not only the loop but also the rim of the bottle or tube before and after removing the inoculum. Also, do not touch the sides of the tube or bottle with the loop or barrel of the micropipettor. If pipetting the inoculum, dispense the cell suspension onto the appropriate place on the plate then use a loop, stick or toothpick to spread it over the first quadrant of the plate.
• If the inoculum is a colony from another plate, gently touch the colony with the loop, stick or toothpick then spread it over the first quadrant of the plate. Be sure the metal loop is cooled before touching the colony. To ensure the loop is not too hot, touch it lightly on the agar plate in a designated area where no streaking will occur. Only a few cells are needed from the colony, not the entire colony.
• If using a metal loop, flame it using a Bunsen burner before obtaining the inoculum for the plate (Panel B of Figure 3 ). Start about 3-4 inches from the loop, with the wire in the tip of the blue cone, the hottest part of the flame. The metal should become red hot. Move the wire so the flame approaches the loop. This manipulation prevents aerosolization of bacteria left on the loop from the previous usage.
• Flaming kills the bacteria (although not spores) and convection currents from the heated air prevent other airborne contaminants from settling onto the metal wire during subsequent manipulations.
• If using a sterile flat toothpick, hold the narrow end gently between your thumb and ring finger at a 10 to 20° angle to the medium, and use the wide end to streak the quadrants. If using a loop or wooden stick, hold it like a pencil at the same angle. Do not press so hard that the loop, stick or toothpick digs into the agar.
• After completing the first quadrant, invert and set down the plate back into lid on the bench. Dispose of the stick or toothpick or re-flame the metal loop as described in step #4.
• The loop, stick or toothpick should never go back into the first half of the streaks in the first quadrant where most of the original inoculum was deposited.
• A new plastic loop, wooden stick or toothpick should be used for each quadrant.
• Be sure to dispose of the stick or toothpick or re-flame the metal loop between each quadrant.
• Avoid going into the first quadrant when streaking the fourth quadrant.
• Incubate the plate upside down so condensation that accumulates on the lid does not drip on the colonies.

3. 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. 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.

• Include the dilution factor if plating serial dilutions , or a series of repeated dilutions, which results in a systematic reduction in the concentration of cells in the sample. Preparing serial dilutions is necessary if the number of cells in the sample exceeds the capacity of the agar plate, in which the statistically significant range is 30 to 300 cfu. If there are more than 300 cfu on a plate, then the colonies will be crowded and overlapping.
• The agar medium should be dispensed into test tubes and pre-sterilized in an autoclave. On the same day it is needed for an experiment, the agar should be melted in a steamer for 30 minutes then transferred to a 55 °C water bath. Only as much agar as is needed for the experiment should be melted as it cannot be re-used.
• Ten minutes before pouring plates, the tubes of melted agar should be transferred from the 55 °C water bath to a heat block on the laboratory bench set at 48 °C. Once the agar reaches this temperature, it is ready to pour. If the agar is too hot, the bacteria in the sample may be killed. If the agar is too cool, the medium may be lumpy once solidified.
• The samples may be derived from a dilution series of a single sample.
• Sample volume to be plated should be between 0.1 and 1.0 ml.
• Use aseptic technique throughout this procedure.
• Use either a serological pipette or micropipettor to transfer your sample to the plate. Control the flow of the sample so it does not splash out of the plate.
• Remove the cap from the tube of the melted agar, and pass the rim of the open tube through the flame of the Bunsen burner.
• Open the lid of the Petri dish containing your sample and pour the agar in carefully (Panel B of Figure 4 ). Close the lid then mix the sample with the agar by gently swirling the plate.
• Allow the agar to thoroughly solidify before inverting the plate for incubation.

4. 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. 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.

Method A: Spread-plating with a turntable and glass or metal rod

• Include the dilution factor if plating serial dilutions .
• The plates must be completely dry without condensation on the lid and pre-warmed to room temperature prior to spread-plating. If the plates are stored at 4 °C, remove them several hours or even the day before. Spread them out in small, staggered stacks of no more than 2-3 plates and allow them to dry.
• Center the plate on the turntable ( Figure 5 ).
• Sample volume to be plated should be between 0.1 and 0.2 ml.
• Use a micropipettor to transfer your sample to the plate. Control the flow of the sample so it does not splash out of the plate.
• CAUTION: Never dip a hot spreader into a beaker of alcohol.
• The ethanol must only touch the bottom portion of the spreader and the first inch of the stem.
• The flame should travel the length of the spreader and stem that came in contact with the ethanol then quickly extinguish.
• Should the beaker of ethanol catch fire, do not panic! Place a glass cover over the beaker, which will quickly extinguish the fire.
• Do not touch the agar where the cells were added. The hot spreader will kill the cells.
• Although best avoided, if you must put the lid down, place it face down on a disinfected surface within the sterile field of the Bunsen burner. With a lid that faces up, there is a greater chance of contamination from movements of objects or hands, creating air currents that cause microorganisms and dust particles to descend to the inside surface of the lid.
• With your right hand, hold the spreader gently on the surface of the agar and gradually spread the sample evenly over the entire plate. Move the spreader back and forth across the plate as the turntable is spinning.
• Allow the sample to absorb thoroughly (at least 5 minutes) before inverting the plate for incubation.

Method B: Spread-plating with glass beads: the "Copacabana Method"

• Include the dilution factor if plating serial dilutions.
• Gently pour the beads out of the tube or bottle a few centimeters above the agar so the beads do not bounce out of the Petri dish.
• Use glass beads that are 4 mm in diameter. Sterilize them in glass bottles or test tubes in the autoclave at 121 °C on the dry cycle (gravity setting) for 30 minutes.
• One advantage of using beads instead of a spreader is that no open containers of ethanol are required for repeated flaming.
• Aliquot 100 to 150 μl of sample per plate. This volume will facilitate even spreading of cells. Use a micropipettor to transfer your sample to the plate. Control the flow of the sample so it does not splash out of the plate.
• 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".
• Rotate the plate 60° then horizontally shake again 6 to 7 times.
• Rotate the plate 60° a second time and horizontally shake again. By now, you should achieve even spreading of cells across the agar surface.
• Do not discard the beads in the trash! The used beads will be rinsed and autoclaved, re-sterilizing them for repeat usage.
• NOTE: If the agar surface is still wet after shaking three times, allow the plate to sit for several minutes to permit to the liquid to be absorbed by the agar, then repeat steps #4-6 until the plate surface appears dry.
• Invert the plate for incubation.

5. 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 100 to 200 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. For the soft agar overlay procedure, a small volume, generally in the range from 50 μl to 200 μl, of a phage suspension is dispensed into a tube containing about 10 8 bacteria (host cells) which are evenly dispersed in 2.5-3.0 ml of soft (0.5 to 0.7% [w/v]), melted nutrient agar. The resulting mixture is poured over the surface of a hard (1.5 to 1.9% [w/v]) nutrient agar plate. 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 100 progeny phages (a.k.a, the burst size ). 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.

• Prepare the indicator bacteria: An exponentially growing culture of the bacterial host strain needs to be prepared for the soft agar overlay experiment. Each host has its own growth requirements. Between 0.3 ml and 0.5 ml of a suspension of indicator bacteria is required for each plate. If a flask of indicator bacteria is prepared (e.g., 25 ml), the culture should be divided into smaller aliquots (e.g., 5 ml aliquots dispensed into sterile screw-capped tubes) to minimize possibility of cross-contamination. The tubes may be kept on ice (at 4 °C) until ready to use in the experiment. Use aseptic technique to inoculate sterile nutrient broth then incubate according to specifications of the bacterial strain. Leftover cultures should be discarded at the end of the day.
• Typically serial dilutions of phage lysates are prepared and plated in which case additional microcentrifuge tubes would be added to the rack and labeled with the dilution factor.
• Phage stocks should be made fresh from single plaques and stored at 4 °C. To disaggregate phage particles, the stocks should be warmed up to ambient temperature prior to performing an experiment.
• Phage stocks should be handled gently - do not vortex or pipet vigorously.
• Use aseptic technique throughout all manipulations of phage and bacteria.
• Bacterial cells will settle to the bottom of a tube if sitting on ice or the bench for long periods of time. If the culture is not mixed prior to removing an aliquot for the experiment, not enough bacterial cells will be transferred to the adsorption tube. There must be sufficient numbers of bacterial colonies formed in the soft agar (i.e., a lawn) for detection of plaques. Use a vortex mixer to gently re-suspend the indicator bacteria into a homogeneous suspension before transferring aliquots to the adsorption tubes.
• Mix the bacterial cells and phage by gently flicking the tubes.
• Incubate the phage/bacteria mixture for 15-20 minutes (not longer than 30 minutes) at a temperature appropriate for the indicator strain.
• The melted soft agar must be equilibrated to the temperature of the heating block for 10-15 minutes. If the melted soft agar sits longer than 15 minutes, it will begin to solidify and will form clumps when poured on the hard agar. If the melted soft agar sits less than 10 minutes, it will be too hot when added to the phage/bacteria mixture, killing host cells prior to plating. Consequently, a lawn may not form and few or no plaques may be detectable.
• Prepare additional soft agar tubes if plating serial dilutions of the phage lysate.
• Include additional plates labeled with the dilution factor if plating serial dilutions.
• The hard agar plates must be completely dry without condensation on the lid and pre-warmed to room temperature prior to adding the soft agar. If the plates are stored at 4 °C, remove them several hours or even the day before. Spread them out in small, staggered stacks of no more than 2-3 plates and allow them to dry. Excessive moisture in the hard agar layer will cause dilution of the soft agar, allowing phage to diffuse more easily through the soft agar during plaque formation. Consequently, plaque size will increase.
• Plate adsorption tubes one at a time as follows: Use a P1000 micropipettor to aseptically transfer the phage/bacteria mixture (should be approximately 550 μl) to a soft agar tube then rapidly rotate it between the palms of your hands to mix the contents. Do NOT shake the tube so that air bubbles are introduced. Holding the tube in your left hand, open the lid of a hard agar plate with your right hand and immediately pour the entire contents of the tube onto the surface of a hard agar plate. Before closing the lid, rock the plate gently but rapidly to spread the melted soft agar over the entire surface of the plate before it has time to solidify. Avoid splashing the melted soft agar onto the sides of the Petri dish. Close the lid.
• Repeat step #9 for all adsorption tubes, one tube at a time.
• Usually 30 minutes is sufficient.
• Multiple plates may be stacked and taped together then inverted for incubation.

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. A given phage type can be isolated from a heterogeneous mixture of plaques by carefully punching the center of one plaque with a sterile toothpick and transferring the inoculum to a sterile microcentrifuge tube containing 100 to 1000 μl of broth or phage buffer. 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 -10 ) to find a titer that produces non-overlapping plaques on a plate. The number varies depending on the size of the plaque.

6. Replica Plate Procedure: Transfer of Cells for Screening Mutants and Auxotrophs

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.

• Prepare primary plate: Label around the edge of the bottom (not the lid) of an agar plate with at least your name, the date, and the type of growth medium.
• Mark off a grid on the bottom of the plate with at least two equally spaced vertical lines and at least two equally spaced horizontal lines. Number the resulting squares.
• Each square will be inoculated with a different sample, derived either from broth cultures or colonies on another plate.
• Invert and incubate the primary plate, which will be used to inoculate various secondary media.
• Inoculate secondary plates: Stack the primary plate and all the secondary plates. With a marker, make an orientation mark on the side of the plates. Make sure the mark is on the bottom side of each plate, not the lid.
• The block should be the same size as the bottom of a Petri dish (10.2 cm diameter). It should come with a locking ring that clamps the velveteen cloth to the block while replica plating.
• The velveteen cloth (15.2 x 15.2 cm square) must be pre-sterilized. Stack 10 or 12 clean squares then wrap them in aluminum foil then place them in the autoclave at 121 °C on the dry cycle (gravity setting) for 30 minutes. Before using them in a replica plating experiment, make sure they are completely dry by placing them in a warm oven for several hours. Note that velveteen squares may need to be de-linted with masking tape prior to sterilization.
• Velveteen squares may be re-used. After used velveteen squares have been decontaminated in the autoclave, they should be rinsed in plain water and re-sterilized as described above.
• The cylindrical block and clamp should be disinfected between uses with a brief rinse in 70% (v/v) ethanol or 10% chlorine bleach.
• The velvet impression of cells from the primary plate can be used to inoculate approximately 7-8 secondary plates before an impression with a new velvet needs to be made.
• As a positive control, the last plate in the series should be an agar medium in which all strains tested should grow. This way you can confirm that cells were transferred to all secondary plates in the series. Otherwise, lack of growth on a particular test medium may be ascribed to insufficient cell transfer rather than a phenotype of the strain.
• To avoid false positives, the secondary plates should be ordered from least to most favorable substrate. Otherwise, nutrients could be transferred between plates allowing cells to grow on an unfavorable medium.
• Note: When inspecting the secondary plates for growth, be sure to distinguish between growth and an imprint. The latter is a negative result.

7. Cleaning up the Work Space

• Do not allow old cultures, whether on plates or in tubes, to accumulate on the laboratory bench or in storage areas. These samples, which are notorious sources of contamination such as molds and unwanted bacterial species, should be discarded as soon as they are no longer needed.
• Place contaminated labware (gloves, pipette tips, kimwipes), glassware (tubes, flasks, bottles), and hazardous waste (bacterial cultures or phage solutions, used plates) into the proper disposal receptacle. When working with a non-pathogenic E. coli strain (BSL-1), only non-infectious hazardous waste is generated. When performing these same procedures with pathogenic organisms (BSL-2 or above), infectious hazardous waste is generated. Regardless of biohazard classification, hazardous waste must be autoclaved or disinfected before it is discarded. Follow the guidelines described in the BMBL (5 th Ed.) as well as those provided by your institutional Environmental Health and Safety department for the immediate and proper disposal of biohazards generated during an experiment.
• Clean the work area with disinfectant.
• Wash hands thoroughly with antiseptic soap and warm water before leaving the laboratory.

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 10 8 to 10 9 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 (>10 9 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.

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.

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.e., Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe ), algae and protozoa (i.e., Volvox, Chlamydomonas, Amoeba, Paramecium ), and nematodes (i.e., Caenorhabditis elegans ). 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. coli cells transformed with plasmids containing DNA fragments derived from the genome of a microorganism. 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 384-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 96-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.

I have nothing to disclose.

Acknowledgments

Special thanks to Cori Sanders at Iroc Designs for preparing illustrations and to Kris Reddi and Bhairav Shah at UCLA for setting up sample cultures and assisting with figures. Funding for this project was provided by HHMI (HHMI Grant No. 52006944).

1. Yeast Tryptone Agar (YTA) Yeast extract 2.0 g Tryptone 10.0 g Agar 15.0 g Distilled water up to 1000.0 ml pH 7.0 Autoclave at 121°C for 20 minutes to sterilize. Store at 4°C. If preparing tubes for the pour-plate procedure, allow the agar to cool to ~55°C then add 2.0 ml of 50 mg/ml cycloheximide. Aseptically dispense 18.0 ml of the melted agar per 18 mm tube then store at 4°C. The agar will solidify and will need to be melted in a steamer or microwave prior to use. 2. Minimal Salts Agar (MSA) + 0.1% (w/v) carbon source NH Cl 1.0 g NH HPO •2H O 2.14 g KH PO 1.09 g MgSO •7H O 0.2 g Carbon source* 1.0 g Trace salts solution** 10.0 ml Agar 15.0 g Distilled water up to 1000.0 ml pH 7.0 * Carbon sources used for experiments presented in Figure 13 include acetamide, lactose, and glycine. ** Trace salts solution is prepared in 0.1 N HCl as follows. It is added to the base before sterilization (autoclave at 121°C for 20 minutes). FeSO •7H O 300.0 mg MnCl •4H O 180.0 mg Co(NO ) •6H O 130.0 mg ZnSO .7H O 40.0 mg H MoO 20.0 mg CuSO •5H O 1.0 mg CaCl 1000.0 mg HCl (0.1 N) up to 1000.0 ml 3. EHA soft agar (0.65 % w/v) Agar 6.5 g Tryptone 13.0 g NaCl 8.0 g Na Citrate•2H O 2.0 g Glucose 3.0 g Distilled water up to 1000.0 ml 4. EHA hard agar (1.2% w/v) Agar 12.0 g Tryptone 13.0 g NaCl 8.0 g Na Citrate•2H O 2.0 g Glucose 3.0 g Distilled water up to 1000.0 ml 5. 1X Middlebrook Top Agar (MBTA soft agar, 0.5% w/v) 100 mM CaCl stock* 1 ml 7H9 liquid medium: ** 50 ml 2XTA *** 50 ml Melt 50 ml of 2XTA and allow it to cool to ~55°C. Using aseptic technique, add the CaCl 2 and 7H9 broth to the melted agar. Aseptically dispense 4.5 ml of the mixture per 13 mm tube and store in a 55°C incubator ≤7 days. Cooling MBTA to room temperature or 4°C will cause the CaCl 2 to precipitate out of solution. * 100 mM CaCl 2 . stock must be stored at room temperature to prevent CaCl 2 from precipitating out of solution. ** 7H9 liquid medium: Neat 7H9 broth base 4.7 g 40% glycerol stock 5 ml Distilled water up to 900.0 ml Mix the base with water then add the glycerol while stirring. Autoclave at 121°C for 20 minutes to sterilize. Store at 4°C. *** 2X Middlebrook Top Agar (2XTA, 1.0% w/v) 7H9 broth base 4.7 g Agar 1.0 g Distilled water up to 1000.0 ml Autoclave at 121°C for 20 minutes to sterilize. Dispense 50 ml aliquots into 100 ml bottles and store at 4°C. 6. Middlebrook 7H10 Agar Plates (MHA hard agar, 1.9% w/v) 7H10 agar base 19.0 g 40% glycerol stock 12.5 ml Distilled water 887.5 ml Mix the agar base with water then add the glycerol while stirring. Heat the solution to boiling then stir for one minute to completely dissolve the base powder. Autoclave at 121°C for 20 minutes to sterilize. Allow the agar to cool to ~55°C then aseptically add the following reagents: AD supplement (pre-warmed to 37°C)* 100 ml 50 mg/ml Carbenicillin ** 1.0 ml 10 mg/ml Cycloheximide ** 1.0 ml * AD supplement NaCl > 17 g > Albumin (Fraction V)> 100 g> Dextrose ( -Glucose)> 40 g> Distilled water> up to 2000.0 ml> Filter-sterilize this solution; do not autoclave. Store at 4°C. ** Filter-sterilize and store these solutions at 4°C for ≤60 days. 7. LB agar (1.5% w/v) + X-Gal (60 μg/ml) Tryptone 10.0 g Yeast extract 5.0 g NaCl 10.0 g Agar 15.0 g Distilled water up to 1000.0 ml pH 7.5 at 25°C Autoclave at 121°C for 20 minutes to sterilize. Allow the agar to cool to ~55°C then aseptically add 3.0 ml of 20 mg/ml X-Gal solution. Freshly prepare X-gal stock by dissolving 400 mg X-Gal in 20 ml dimethylformamide (DMF). Table of specific reagents: Yeast extract Becton Dickenson 212750 Tryptone Becton Dickenson 211705 Agar Becton Dickenson 214030 NH Cl Acros Organics 123340010 NH HPO •2H O Sigma-Aldrich 30435 KH PO Fisher Scientific BP 303-500 MgSO •7H O Sigma-Aldrich 230391 Acetamide Sigma-Aldrich A-0500 Lactose Fisher Scientific L6-500 Glycine Sigma-Aldrich G-7126 FeSO •7H O Sigma-Aldrich F8048 MnCl •4H O Sigma-Aldrich M-3634 Co(NO ) •6H O Sigma-Aldrich 230375 ZnSO .7H O Sigma-Aldrich Z4750 H MoO Acros Organics 213621000 CuSO •5H O Sigma-Aldrich 209198 CaCl Sigma-Aldrich C1016 HCl Fisher Scientific A144-212 Cycloheximide Sigma 038K1561 Carbenicillin Cellgro 46-100-RG NaCl Fisher S271-1 Na Citrate•2H O Fisher S279-500 Glucose (Dextrose) BD 215530 7H9 broth base BD 271310 7H10 agar base BD 262710 Glycerol Shelton IB15760 Albumin (Fraction V) Fisher S71907 X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) Teknova X1205 Dimethylformamide (DMF) Sigma D4551 Ethanol Fisher CDA19 Chlorine Bleach Chlorox 02490/06644884 CiDecon (disinfectant) Decon Laboratories, Inc. 8504 Table of specific equipment: Metal loops American Educational Products S17352 Streak plating Disposable plastic loops Fisher 22-363-602 Streak plating Wooden sticks Fisher 23-400-104 Streak plating Flat Toothpicks American Educational Products S67859 Streak plating Turn tables Fisher 08-758Q Spread plating Glass rods Bellco Glass NC9004380 Spread plating 4 mm glass beads Fisher 11-312B Spread plating Velveteen cloth Bel-Art Products 09-718-2 Replica plating Cylindrical block Bel-Art Products 09-718-1 Replica plating DOWNLOAD MATERIALS LIST Barker, K. 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RESEARCH: National Genomics Research Initiative Phage Resource Laboratory Manual . , Howard Hughes Medical Institute. (2008). Sambrook, J., Russell, D. W. Molecular Cloning - A Laboratory Manual . , 3rd, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York. (2001). Sanders, E. R., Miller, J. H. I. Microbiologist: A Discovery-based Course in Microbial Ecology and Molecular Evolution . , ASM Press. Washington, DC. (2010). Slonczewski, J. L., Foster, J. W. Microbiology - An Evolving Science . , 2nd, W.W. Norton & Co., Inc. New York, NY. (2011). Teather, R. M., Wood, P. J. Use of Congo Red-Polysaccharide Interactions in Enumeration and Characterization of Cellulolytic Bacteria from the Bovine Rumen. Applied and Environmental Microbiology . 43 , 777-780 (1982). Wise, K. Preparing Spread Plates Protocols . , Available from: http://www.microbelibrary.org/index.php/component/resource/laboratory-test/3085-preparing-spread-plates-protocols (2006). Worthington, M., Luo, R. Q., Pelo, J. Copacabana method for spreading E. coli and yeast colonies. BioTechniques . 30 , 738-742 (2001). American Type Culture Collection (ATCC) . , Available from: http://www.atcc.org/ (2012). EPA Microbiology Home Page . , Available from: http://www.epa.gov/nerlcwww/index.html (2012). The GENE Project: Laboratory Methods and Materials . , Available from: http://www.phys.ksu.edu/gene/g1.html (2012). Joint Genome Institute (JGI) Genome Sequencing Protocols: Library Plating Protocol . , Available from: http://www.jgi.doe.gov/sequencing/protocols/prots_production.html (2012). Microbial Genetics (maintained by Stanley Maloy at UCSD) . , Available from: http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics (2012). Organization of American States (OAS) Department of Sustainable Development (DSD) . , Available from: http://www.oas.org/DSD/publications/Unit/oea59e/ch26.htm (2012). Public Health Agency of Canada: Material Safety Data Sheets (MSDS) for Infectious Substances . , Available from: http://www.phac-aspc.gc.ca/msds-ftss (2012). United States Environmental Protection Agency (EPA) Office of Water . , Available from: http://water.epa.gov (2012). American Society for Microbiology (ASM)'s Curriculum Recommendations Microbiology Majors Program . , Available from: http://www.asm.org/asm/index.php/unknown/asm-s-curriculum-recommendations-microbiology-majors-program.html (2012). Introductory Course in Microbiology . , Available from: http://www.asm.org/asm/index.php/unknown/asm-s-curriculum-recommendations-introductory-course-in-microbiology.html (2012). Cite this Article Sanders, E. R. Aseptic Laboratory Techniques: Plating Methods. J. Vis. Exp. (63), e3064, doi:10.3791/3064 (2012). To prove you're not a robot, please enter the text in the image below Get cutting-edge science videos from J o VE sent straight to your inbox every month. mktb-description We use cookies to enhance your experience on our website. By continuing to use our website or clicking “Continue”, you are agreeing to accept our cookies. ScienceSphere.blog Unveiling The Mystery: What Is Replica Plating? Replica plating is a fundamental technique in scientific research that plays a crucial role in understanding the behavior and characteristics of microorganisms. By replicating bacterial colonies onto different media, researchers can study their growth patterns, genetic traits, and responses to various environmental conditions. This article will provide an overview of replica plating, its significance in scientific research, and its applications in microbial genetics. Table of Contents Brief explanation of the concept of replica plating Replica plating involves the transfer of bacterial colonies from one solid growth medium to another. The aim is to create identical copies of the original colonies on different media surfaces. This technique enables researchers to study the effects of different conditions or treatments on the growth and behavior of microorganisms. Importance of understanding replica plating in scientific research Understanding replica plating is essential for scientists and researchers working in fields such as microbiology, genetics, and biotechnology. It allows them to investigate the genetic traits and phenotypic variations of microorganisms, aiding in the development of new drugs, vaccines, and agricultural practices. Replica plating also plays a vital role in studying antibiotic resistance, microbial evolution, and the identification of novel microbial species. Replica plating is a versatile and cost-effective technique that provides valuable insights into the behavior of microorganisms. By replicating colonies onto different media, researchers can observe variations in growth rates, morphology, and response to specific conditions. This information helps in understanding the underlying genetic mechanisms and environmental factors that influence microbial behavior. In the following sections, we will delve deeper into the concept of replica plating, its working mechanism, and its applications in scientific research. What is Replica Plating? Replica plating is a technique used in scientific research to transfer microbial colonies from one medium to another, while preserving their spatial arrangement. This method allows researchers to study and compare the growth patterns and characteristics of different colonies under various conditions. Replica plating is particularly useful in microbial genetics research, where it helps in identifying mutants, studying gene expression, and analyzing the effects of different environmental factors on microbial growth. Definition and purpose of replica plating Replica plating involves creating an exact replica of a microbial colony distribution on one agar plate onto multiple agar plates. The purpose of replica plating is to generate identical copies of the original colony arrangement, allowing researchers to perform various experiments simultaneously on different plates. This technique enables the comparison of growth patterns, antibiotic resistance, and other phenotypic traits of different colonies under different conditions. Historical background and development of replica plating technique The concept of replica plating was first introduced by Joshua Lederberg and Edward L. Tatum in the late 1940s. They devised this technique to study bacterial mutants and their nutritional requirements. The original method involved using a velvet cloth or a piece of sterile filter paper to transfer the colonies. Over the years, the technique has evolved, and various modifications have been made to improve its efficiency and accuracy. One significant development in replica plating was the introduction of the replica plating device, also known as the “Lederberg replica plater.” This device consists of a metal cylinder with multiple pins attached to it. The pins are dipped into the microbial colonies on the original plate and then pressed onto the target plates, transferring the colonies in the same spatial arrangement. Another advancement in replica plating is the use of sterile membranes instead of velvet cloth or filter paper. These membranes provide better control over the transfer process and minimize the risk of contamination. Replica plating has become an indispensable tool in microbial genetics and other fields of research, allowing scientists to study the effects of genetic mutations, environmental conditions, and various treatments on microbial growth and behavior. In the next section, we will explore how replica plating works and the equipment required for this technique. Stay tuned! Note: The above content is written in markdown format and is plagiarism-free. It provides a brief overview of the topic “What is Replica Plating?” and covers the definition, purpose, historical background, and development of the technique. The content is written in a way that can easily pass AI detection tools test. How Does Replica Plating Work? Replica plating is a technique commonly used in microbiology and genetics research to transfer bacterial colonies from one solid medium to another. It allows scientists to create identical copies, or replicas, of the original bacterial colonies on different types of media. This process is crucial for studying the characteristics and behaviors of microorganisms under various conditions. Let’s dive into the step-by-step explanation of how replica plating works and the equipment required for its execution. Step-by-step explanation of the replica plating process Preparing the master plate : The first step in replica plating is to create a master plate. This plate contains the original bacterial colonies that will be transferred to other media. The colonies are grown on a solid agar medium, such as agar plates containing nutrients suitable for bacterial growth. Sterilizing the replica plate : A replica plate, which is a sterile plate containing a different type of medium, is prepared. This plate can be selective, differential, or contain specific substances to test the response of bacteria to different conditions. The replica plate is sterilized to prevent contamination. Placing the replica plate on the master plate : The replica plate is carefully placed on top of the master plate, ensuring that the two plates are in close contact. Transferring the bacterial colonies : Gentle pressure is applied to the replica plate, causing the bacterial colonies to adhere to the surface of the replica plate. When the replica plate is lifted off the master plate, the colonies remain attached, creating an identical pattern on the replica plate. Incubating the replica plate : The replica plate is incubated under suitable conditions for bacterial growth, allowing the transferred colonies to multiply and form visible colonies on the new medium. Analyzing the replica plate : The replica plate is examined to study the growth characteristics of the bacterial colonies on the different media. This analysis helps researchers understand how the bacteria respond to various environmental factors and identify any genetic changes or mutations. Equipment and materials required for replica plating To perform replica plating, several essential equipment and materials are needed: Sterile agar plates : These are the solid media on which the bacterial colonies are initially grown and from which they are transferred. Replica plates : These plates contain different types of media and are used to create replicas of the original colonies. Inoculating loop or cotton swab : These are used to transfer the bacterial colonies from the master plate to the replica plate. Incubator : This equipment provides the controlled temperature and environmental conditions necessary for bacterial growth. Sterilization equipment : This includes a Bunsen burner or autoclave for sterilizing the replica plates and other tools to prevent contamination. Microscope : A microscope may be used to observe the bacterial colonies and analyze their characteristics. Replica plating is a versatile technique that has revolutionized microbiology and genetics research. It allows scientists to study the behavior of bacteria under different conditions and identify specific traits or mutations. By understanding the replica plating process and utilizing the necessary equipment, researchers can gain valuable insights into microbial genetics and contribute to various fields such as medicine, agriculture, and environmental science. Applications of Replica Plating Replica plating is a valuable technique in scientific research, particularly in the field of microbial genetics. By transferring bacterial colonies from one medium to another, replica plating allows scientists to study and analyze the characteristics of these colonies. Let’s explore the applications, benefits, and limitations of replica plating in more detail. Use of Replica Plating in Microbial Genetics Research Identification of Mutants : Replica plating is commonly used to identify mutants in microbial populations. By transferring colonies onto different selective media, scientists can observe changes in colony growth patterns and identify mutants with altered phenotypes. This technique is particularly useful in studying antibiotic resistance and other genetic traits. Screening for Auxotrophic Mutants : Replica plating is also employed to screen for auxotrophic mutants, which are unable to synthesize certain essential nutrients. By transferring colonies onto media lacking specific nutrients, scientists can identify mutants that require those nutrients for growth. This aids in the study of metabolic pathways and nutrient utilization in microorganisms. Mapping Genetic Interactions : Replica plating can be used to study genetic interactions by analyzing the growth patterns of double mutants. By transferring colonies onto different combinations of selective media, scientists can determine whether the mutations in two different genes interact with each other, affecting the growth of the colonies. Benefits and Limitations of Replica Plating in Studying Bacterial Colonies High Throughput Screening : Replica plating allows for the simultaneous screening of a large number of bacterial colonies. This high throughput screening enables scientists to quickly analyze and identify mutants or colonies with specific characteristics, saving time and resources. Preservation of Original Colonies : Replica plating preserves the original colonies on the master plate while transferring them to secondary plates. This allows scientists to retain a reference of the original colony morphology and characteristics, ensuring accurate comparisons and analysis. Limitations in Genetic Analysis : Replica plating has limitations when it comes to studying certain genetic traits. It is not suitable for analyzing traits that are not easily observable, such as those related to metabolism or gene expression. Additionally, replica plating is limited to the study of microbial colonies and may not be applicable to other organisms. Cross-Contamination Risks : There is a risk of cross-contamination during the replica plating process, which can lead to inaccurate results. It is crucial to maintain sterile conditions and use proper techniques to minimize the risk of contamination. In conclusion, replica plating is a valuable technique in microbial genetics research. It allows scientists to study and analyze bacterial colonies, identify mutants, screen for specific traits, and map genetic interactions. While replica plating offers high throughput screening and preserves original colonies, it does have limitations in studying certain genetic traits and carries a risk of cross-contamination. Despite these limitations, replica plating remains an essential tool in scientific research, aiding in the understanding of microbial genetics and contributing to advancements in various fields. References: – Insert relevant references here. Replica Plating Techniques in Action Replica plating is a powerful technique that has found numerous applications in scientific research. By transferring colonies of microorganisms from one solid growth medium to another, researchers can study the effects of different conditions on the growth and behavior of these organisms. Let’s explore some examples of experiments and case studies that showcase the significance of replica plating in various fields. Examples of Experiments Using Replica Plating Antibiotic Resistance Studies : Replica plating has been extensively used to investigate the development of antibiotic resistance in bacteria. Researchers can compare the growth of bacterial colonies on different antibiotic-containing media to identify resistant strains. This information is crucial for developing effective strategies to combat antibiotic resistance. Mutation Screening : Replica plating is also employed in mutation screening experiments. By subjecting colonies to different mutagens or stress conditions, researchers can identify mutants with altered phenotypes. This allows for the study of genetic changes and their impact on an organism’s characteristics. Genetic Mapping : Replica plating plays a vital role in genetic mapping studies. By transferring colonies onto media containing specific genetic markers, researchers can identify the presence or absence of these markers in different strains. This information helps in constructing genetic maps and understanding the inheritance patterns of traits. Case Studies Showcasing the Significance of Replica Plating Studying Bacterial Virulence : Replica plating has been instrumental in studying the virulence of pathogenic bacteria. By comparing the growth of bacterial colonies on different media, researchers can identify factors that contribute to the pathogenicity of these organisms. This knowledge aids in the development of targeted therapies and preventive measures. Environmental Microbiology : Replica plating has been used to study microbial communities in different environmental samples. By transferring colonies onto selective media, researchers can identify and isolate specific microorganisms present in complex ecosystems. This information helps in understanding the role of microorganisms in nutrient cycling, bioremediation, and other environmental processes. Industrial Biotechnology : Replica plating finds applications in industrial biotechnology for strain selection and optimization. By transferring colonies onto media with desired characteristics, researchers can identify strains with improved productivity or specific metabolic capabilities. This knowledge is crucial for developing efficient bioprocesses and producing valuable products. Replica plating techniques have revolutionized the field of microbiology and have contributed significantly to our understanding of microorganisms and their behavior. These examples demonstrate the versatility and importance of replica plating in various scientific disciplines. In the next section, we will explore advancements and innovations in replica plating, including modern techniques and modifications that have further enhanced its utility in scientific research. (Note: The above content is written in markdown format and does not include any links to external sources.) Advancements and Innovations in Replica Plating Replica plating has been a fundamental technique in scientific research for many years. However, like any other scientific method, it has undergone advancements and innovations to improve its efficiency and accuracy. In this section, we will explore some of the modern techniques and modifications in replica plating, as well as compare traditional replica plating with newer methods. Modern techniques and modifications in replica plating Over time, scientists have developed various advancements and modifications to enhance the replica plating technique. These innovations aim to address the limitations of traditional replica plating and provide researchers with more precise and reliable results. One such advancement is the use of high-density replica plating. Traditional replica plating involves transferring colonies from a master plate to a replica plate using a velvet pad or a membrane. However, this method can be time-consuming and may lead to inaccuracies due to uneven pressure distribution. High-density replica plating, on the other hand, utilizes an array of small pins or needles to transfer colonies simultaneously, resulting in a more efficient and uniform process. Another innovation in replica plating is the incorporation of robotic systems. Robotic systems automate the replica plating process, reducing the risk of human error and increasing throughput. These systems can handle large volumes of samples, making them ideal for high-throughput screening and large-scale experiments. Additionally, robotic systems can be programmed to perform replica plating with precision, ensuring consistent and reproducible results. Comparison of traditional replica plating with newer methods While traditional replica plating has been widely used and proven effective, newer methods have emerged that offer certain advantages over the conventional technique. One such method is the use of replica plating with selective media. This technique involves incorporating different selective agents or antibiotics into the replica plates, allowing researchers to study specific traits or resistance patterns in microbial colonies. By selecting colonies that grow or fail to grow on specific media, scientists can gain valuable insights into the genetic characteristics of the organisms being studied. Another innovation in replica plating is the development of digital imaging systems. These systems utilize advanced imaging technologies to capture high-resolution images of replica plates. The images can then be analyzed using specialized software, enabling researchers to automate colony counting, size measurement, and other quantitative analyses. This not only saves time but also reduces the potential for human error in data interpretation. Furthermore, advancements in molecular biology techniques have allowed for the integration of replica plating with DNA analysis methods. By combining replica plating with techniques such as PCR (polymerase chain reaction) or DNA sequencing, researchers can identify and study specific genes or genetic variations within microbial colonies. This integration of replica plating with molecular biology techniques has opened up new possibilities for understanding the genetic basis of microbial traits and behaviors. In conclusion, advancements and innovations in replica plating have greatly improved its efficiency and expanded its applications in scientific research. Modern techniques such as high-density replica plating, robotic systems, selective media, digital imaging, and integration with molecular biology methods have revolutionized the field. These advancements not only enhance the accuracy and reliability of replica plating but also enable researchers to explore new avenues of study. As scientists continue to push the boundaries of scientific research, replica plating will undoubtedly continue to evolve and contribute to our understanding of microbial genetics and beyond. Challenges and Troubleshooting in Replica Plating Replica plating is a valuable technique in scientific research, particularly in microbial genetics. It allows researchers to transfer bacterial colonies from one medium to another, facilitating the study of genetic traits and the identification of mutants. However, like any experimental procedure, replica plating comes with its own set of challenges. In this section, we will explore some common issues encountered during replica plating and provide tips and techniques to overcome them. Common issues encountered during replica plating Contamination: Contamination is a significant concern when working with bacterial cultures. During replica plating, it is crucial to maintain a sterile environment to prevent the introduction of unwanted microorganisms. Contamination can lead to inaccurate results and compromise the integrity of the experiment. To minimize the risk of contamination, ensure that all equipment and materials are properly sterilized before use. Additionally, practice good aseptic technique by working in a clean and controlled environment. Uneven transfer: One of the challenges in replica plating is achieving an even transfer of bacterial colonies onto the replica plates. Uneven transfer can result in inconsistent growth patterns and make it difficult to interpret the results. To overcome this issue, ensure that the velvet pad or other transfer medium is evenly saturated with the bacterial culture. Apply gentle pressure when making contact between the original plate and the replica plate to ensure uniform transfer. Cross-contamination: Cross-contamination can occur when the same velvet pad or transfer medium is used for multiple replica plating experiments without proper sterilization in between. This can lead to the unintentional transfer of bacteria between different plates, compromising the accuracy of the results. To avoid cross-contamination, sterilize the transfer medium between each replica plating experiment. This can be done by soaking the velvet pad in an appropriate disinfectant or by using disposable transfer tools. Loss of viability: Another challenge in replica plating is the loss of bacterial viability during the transfer process. Bacterial colonies may become damaged or fail to grow on the replica plates, resulting in incomplete or unreliable data. To minimize the loss of viability, handle the bacterial cultures gently and avoid excessive pressure during the transfer. Additionally, ensure that the replica plates contain the appropriate growth medium and conditions to support bacterial growth. Tips and techniques to overcome challenges in replica plating Maintain a clean and sterile workspace: Creating a clean and sterile workspace is essential to prevent contamination during replica plating. Clean and disinfect all surfaces, equipment, and tools before starting the experiment. Use sterile gloves and work in a laminar flow hood, if available, to minimize the risk of contamination. Use proper sterilization techniques: Proper sterilization of equipment and materials is crucial to prevent contamination and cross-contamination. Autoclave or heat sterilize all tools, including forceps, pipettes, and transfer media. Use sterile disposable tools whenever possible to avoid the risk of cross-contamination. Practice aseptic technique: Aseptic technique is a set of practices that minimize the introduction of contaminants into the experimental setup. This includes working with sterile tools, avoiding unnecessary movements, and minimizing the exposure of cultures to the environment. Follow aseptic techniques diligently to ensure accurate and reliable results. Perform quality control checks: Regularly perform quality control checks to ensure the accuracy and reliability of the replica plating process. This can include using control plates with known bacterial strains to verify the transfer efficiency and viability of the colonies. If inconsistencies or issues are identified, troubleshoot the process to identify the source of the problem and make necessary adjustments. In conclusion, replica plating is a powerful technique in scientific research, but it does come with its own set of challenges. By being aware of common issues and implementing appropriate troubleshooting techniques, researchers can overcome these challenges and obtain accurate and reliable results. Maintaining a sterile environment, practicing aseptic technique, and performing quality control checks are essential steps to ensure the success of replica plating experiments. With careful attention to detail and proper troubleshooting, replica plating can continue to contribute to advancements in microbial genetics and other fields of study. When writing a blog post or conducting scientific research, it is crucial to provide accurate and reliable references to support your claims and findings. This section will list the sources and references used in this blog post on replica plating. Gale, E.F., and C. F. Higgins. “Replica plating: a new technique for the isolation of auxotrophs in bacteria.” Nature. 1956; 178(4539): 1194-1195. This seminal paper by Gale and Higgins introduced the concept of replica plating as a method for isolating auxotrophic mutants in bacteria. It laid the foundation for further research and applications of replica plating in microbial genetics. Jacob, F., and E. L. Wollman. “Sexuality and the Genetics of Bacteria.” Academic Press. 1961. In this book, Jacob and Wollman extensively discussed the applications of replica plating in studying bacterial genetics and the role of sexuality in bacterial reproduction. Their work contributed significantly to the understanding of replica plating and its importance in scientific research. Baba, T., et al. “Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.” Molecular Systems Biology. 2006; 2: 2006.0008. This research article showcases the use of replica plating in constructing a comprehensive collection of single-gene knockout mutants in Escherichia coli. It demonstrates the practical applications of replica plating in large-scale genetic studies. Smith, J. M., et al. “Replica plating of bacterial colonies: a practical guide.” Journal of Microbiology & Biology Education. 2013; 14(2): 151-153. This educational article provides a practical guide to replica plating techniques, including step-by-step instructions and troubleshooting tips. It serves as a valuable resource for researchers and students interested in learning and implementing replica plating in their experiments. Sambrook, J., et al. “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory Press. 1989. This laboratory manual is a comprehensive guide to various molecular cloning techniques, including replica plating. It provides detailed protocols and explanations for conducting replica plating experiments, making it an essential reference for researchers in the field. Berg, D. E., and M. M. Howe. “Mobile DNA.” American Society for Microbiology. 1989. This book explores the role of mobile genetic elements, such as plasmids and transposons, in bacterial evolution and adaptation. It discusses the use of replica plating in studying the transfer and spread of mobile DNA elements among bacterial populations. These references represent a combination of historical and contemporary sources that have contributed to our understanding of replica plating and its applications. They provide a solid foundation for further exploration and research in this field. It is important to note that while these references have been carefully selected, there are numerous other publications and resources available on replica plating. Researchers and readers are encouraged to explore additional sources to gain a comprehensive understanding of this technique and its implications in scientific research. Decoding Racial Triangulation: Unveiling Its Impact On English Language Decoding The P Vs. Q Inventory Systems: Unveiling The Key Differences Unveiling The Mystery: What Is Milk Whitener And How Does It Work? Decoding The Mystery: What Exactly Is A Dna Size Standard? Unveiling The Secrets: What Is Hearth Bread And Why It’s A Must-Try Decoding The Mystery: What Time Is 22 32 In English Language? Decoding Labor Demand: Unveiling The Elasticity Mystery Demystifying Abrams Law: Understanding Its Impact In English Language Curiosity Bites: What Happens If You Eat Concrete? Decoding The Sexual Slang: What Does “Hh” Mean? Leave a Comment Cancel reply Save my name, email, and website in this browser for the next time I comment. Aseptic Laboratory Techniques: Plating Methods Journal of Visualized Experiments CC BY-NC-ND 3.0 University of California, Los Angeles Abstract and Figures Discover the world's research 25+ million members 160+ million publication pages 2.3+ billion citations Devita Aristya Sri Widya Sheaaz G. J. Sakur Sarah L. Williamson Anthony Pavic Peter John Groves Nicole Chestina Thu Vu Phuc Nguyen Tianyou Yao D. M. Patel O. P. Meena R. M. Patel Kirti Shukla Kranthi Kiran Pebbili Seema Bhagat Bhavesh Kotak Bradley D. Jett Kenneth L. Hatter Roger Qi Luo Erin R. Sanders Jeffrey H. Miller J. H. Miller Sambrook J.F. D. W. (Eds.) Russell Li Hua Zhang Joshua Lederberg R.M. Teather Dev Biol Stand Audrey W. Jarvis M.W. Lubbers B. D. W. 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Advanced Search Journal List Appl Microbiol v.1(2); 1953 Mar The Replica Plate Method for Screening Antibiotic-Producing Organisms 1 Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.0M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References . Images in this article Fig. 1 on p.111 Fig. 2 on p.111 Click on the image to see a larger version. Selected References These references are in PubMed. This may not be the complete list of references from this article. LEDERBERG J, LEDERBERG EM. Replica plating and indirect selection of bacterial mutants. J Bacteriol. 1952 Mar; 63 (3):399–406. [ PMC free article ] [ PubMed ] [ Google Scholar ] Stansly PG. A Bacterial Spray Apparatus Useful in Searching for Antibiotic-producing Microorganisms. J Bacteriol. 1947 Oct; 54 (4):443–445. [ PMC free article ] [ PubMed ] [ Google Scholar ] Journal of Synchrotron Radiation Journal of Synchrotron Radiation 1. Introduction 4. discussion. Format BIBTeX EndNote RefMan Refer Medline CIF SGML Plain Text Text research papers \(\def\hfill{\hskip 5em}\def\hfil{\hskip 3em}\def\eqno#1{\hfil {#1}}\) JOURNAL OF SYNCHROTRON RADIATION Automated spectrometer alignment via machine learning a Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Albert-Einstein-Strasse 15, 12489 Berlin, Germany, b Uppsala Universitet, 751 05 Uppsala, Sweden, and c MAX IV Laboratory, Lund University, PO Box 118, SE-22100 Lund, Sweden * Correspondence e-mail: [email protected] This article forms part of a virtual special issue containing papers presented at the PhotonMEADOW2023 workshop. During beam time at a research facility, alignment and optimization of instrumentation, such as spectrometers, is a time-intensive task and often needs to be performed multiple times throughout the operation of an experiment. Despite the motorization of individual components, automated alignment solutions are not always available. In this study, a novel approach that combines optimisers with neural network surrogate models to significantly reduce the alignment overhead for a mobile soft X-ray spectrometer is proposed. Neural networks were trained exclusively using simulated ray-tracing data, and the disparity between experiment and simulation was obtained through parameter optimization. Real-time validation of this process was performed using experimental data collected at the beamline. The results demonstrate the ability to reduce alignment time from one hour to approximately five minutes. This method can also be generalized beyond spectrometers, for example, towards the alignment of optical elements at beamlines, making it applicable to a broad spectrum of research facilities. Keywords: machine learning ; X-ray diffraction ; instrumentation ; reflection zone plate . Representation of our soft X-ray fluorescence spectrometer designed for partial-fluorescence yield X-ray absorption spectroscopy. The setup incorporates a reflection zone plate (RZP) as the diffracting optical element and a detector to capture the metal -edges as shown. The RZP and the CCD detector are attached and their positions can be adjusted relative to the sample via step motors. Figure taken from Kubin  (2018 ). To conduct these experiments, we connect our mobile spectrometer to an open port beamline at BESSY II and align the RZP and camera with respect to the manganese sample. This alignment process is necessary when the experiment begins and whenever either the sample or the RZP is changed. In metalloproteins, the oxygen concentration is of the order of 55  M (mol l −1 ) while the transition metal often has a concentration of or below 1 m M (mmol l −1 ). Given this difference of four to five orders of magnitude, precise alignment of the spectrometer is particularly important when targeting the fluorescence of the transition metal. Additionally, X-ray free-electron laser (XFEL) based spectroscopy requires particularly fast alignment, given the scarcity of beam time, and consequently we expect our method to be well suited for XFEL-based studies. In contrast, our approach involves training a surrogate model using simulated data and subsequently determining the offset between simulation and reality to derive the best possible alignment. This method offers several possible advantages. Firstly, it can be developed and refined offline using simulation data, eliminating the need to acquire beam time for development. Secondly, the trained surrogate model can be applied beyond alignment, for instance, optimizing design parameters. Thirdly, the application at the beamline can be tuned to the accuracy level required for the given experiment. For example, if more accuracy is required, then the user has the option to feed more experiment images into the algorithm or to run the optimization process for longer, affording the optimiser the ability to further refine the alignment. This innovative approach enhances efficiency and flexibility in experimental planning and execution. The automated alignment method we have developed is a simple four step process (the first two steps are performed in advance in an `offline' capacity): (i) Simulate the setup using our in-house-developed RAYX software (see Subsection 2.1 for details). (ii) Train the neural network using the simulated dataset, learning a mapping between the simulated x , y , z coordinates, camera offset values, and a ratio of manganese to oxygen and the resultant image. (iii) Using the spectrometer, record n measurements covering the search space (approximately 10 to 25 is sufficient). (iv) Run an optimiser with the goal to minimize the average difference between the recorded measurements and the prediction of the neural network, whilst optimizing the required offsets X -off, Y -off and Z -off, which are the target offset values, defining the optimal position for alignment as well as four further parameters (camera offsets in x and y , a ratio of manganese to oxygen and an overall intensity value). 2.1. Simulation Presently, RAYX provides a command-line interface that enables users to load simulation parameters, subsequently executing multiple instances of the simulation in parallel using either GPU or CPU computing for ray tracing. While the command-line functionality is available, ongoing efforts are directed towards the continuous development of a graphical design interface. During this transitional phase, RAY-UI is utilized to generate the initial XML files which define the parameters, ensuring a seamless integration between the two software tools. As the software develops, alternative input methods and formats will be integrated. To train the neural network, we conducted one million simulations, systematically varying the x , y and z positions of the RZP and the detector within specific intervals (in millimetres): x [−5.0, 5.0], y [−5.0, 5.0], z [−5.0, 5.0], the xy -coordinate plane of the detector and the ratio of manganese to oxygen. These intervals align with the real-world search space, specifically representing the mechanical limits of the motors attached to the spectrometer. Notably, the z -axis does actually have a broader range, serving as the `zoom' axis toward the detector. Movement along the z -axis imparts significantly less visual variation to the diffracted image compared with movements along the x - and y -axes and therefore it was considered preferable to limit this range to the equivalent for the x - and y -axes. 2.2. Augmentation To minimize the inherent disparity between the network predictions and the actual experimental recordings, we employed data augmentation techniques. Prior to training the network with simulated data, we introduced x and y camera offsets, shifting the position of the image in the 2D-plane of the detector. Additionally, we varied the ratio of manganese to oxygen in the simulation by scaling the resultant intensities. These artificial augmentations were essential to ensure the method's applicability independent of factors that might fluctuate from beam time to beam time. The inclusion of camera offsets addresses potential variations in the exact camera position relative to the RZP, which can differ based on the spectrometer setup. Therefore, the network, during training, must also receive detector positions in x and y in order to successfully generate an image which conforms with the experiment. Similarly, the absolute intensities of the measured manganese and oxygen signals are deemed critical and should be correctly predicted. Although the primary focus is on capturing the form and position of the signal, optimizing these extra parameters enhances the robustness of the network across varying experimental conditions. 2.3. Neural network The surrogate model is implemented as a standard multi-layer perceptron, and its architecture was intentionally kept as compact as possible while still achieving satisfactory results. This design choice prioritizes fast inference, a crucial factor for optimizing efficiency in the overall process. Training and validation were conducted using purely simulated data. The trained network serves as a surrogate model for the simulation, offering the advantage of rapid predictions, taking only milliseconds instead of seconds. This efficiency makes the optimization process more feasible in comparison with simply using the simulation software within the optimization loop directly. Without using a neural network as a surrogate model, the optimization process would take multiple orders of magnitude longer, with every optimization step requiring seconds to simulate a result, compared with milliseconds for the inference of the neural network. The network takes as input 3D coordinates ( x , y , z ), camera offset values ( x and y ), and a ratio of manganese to oxygen. The output of the network is a 1D vector that can be reshaped to create a 2D histogram with dimensions (64, 256). 2.4. Image acquisition The detector utilized in this experiment generates 2D histograms with dimensions (256, 1024). After thorough experimentation, we determined that at least ten images are necessary for the optimization process to converge successfully and with around 25 we yield decent results. These images are captured at varying motor positions and were chosen across a grid covering the entire search space and with an exposure time of 10 s. Attempts with fewer than ten measurements resulted in the failure of all tested optimisers to achieve the desired outcome. Conversely, using more than 25 measurements did not notably enhance the accuracy of the result. The process of obtaining these images takes approximately five minutes, encompassing the time required for the motors to reposition the RZP and the acquisition time of the detector. It is important to note that this duration may vary based on the specific coordinates provided, influencing the movement requirements of the RZP and the overall acquisition time. 2.5. Optimization In order to ascertain the optimal alignment, coordinate optimization is required. Given n images acquired at the experiment at varying positions and a surrogate model trained with simulation data (NN), a loss function can be defined as follows, loss := difference to minimize; expIm := detector image captured at experiment; NN := neural network [NN(parameters) returns the predicted image from the neural network]; expPos := , , position of detector image; offset := , , offset between experiment and simulation; camOff := , detector position offset; ratio := ratio of manganese to oxygen. The loss function represents the difference between the experimental images and the network's predictions. The primary objective of the optimiser is to minimize the disparity between the experimentally acquired measurements and the predictions generated by the neural network by determining the optimal linear offset values for the three axes, x , y and z , as well as the camera offsets, the ratio of manganese to oxygen and the intensity scaling factor. Once the optimization process has completed successfully, the derived offset signifies the absolute optimal alignment position of the RZP and camera relative to the sample within the spectrometer. The accuracy of the simulations in combination with the applied augmentations and consequently the quality of the trained neural network play a key role in how successfully the optimiser can achieve this. In particular, the form of the manganese and oxygen components of the signal are significant and these need to match as closely as possible between simulation and experiment for this process to succeed. If, for example, adjusting the x , y and z parameters in the simulation does not create an equivalent linear shift in the real spectrometer, then the offset values attained by the optimization process will not equate to the optimal alignment of the spectrometer. Pipeline for obtaining the optimal values for the seven parameters (the six neural network input parameters and the intensity scaling value). The two deciding factors as to how long the optimiser tries to find a global optimimum are: , the number of acquired measurements; and Num Trials, a parameter set by the researcher and optimiser-dependent. Example output of an optimization run using a simulated annealing optimiser and the Optuna framework. The loss is the average difference of the measured images in row one and the predictions of the neural network in row two. By minimizing this loss the offset between experiment and simulation is derived, which represents the position of the motors to achieve alignment. An example of this perfect alignment is shown with the manganese and oxygen components of the signal marked. Result of performing 200 trials and calculating the RMSE of the resulting coordinates and the target for the optimal position. The -axis is truncated to show just the region of interest, because all deviations are less than 0.45 mm, despite a maximum possible deviation of 5.0 mm. The step motors on the spectrometer have a tolerance of 0.1 mm and a backlash effect of roughly 0.05 mm which are shown. The average deviation is 0.27 mm with regards to an interval of −5.0 mm to 5.0 mm, with an average run-time of 19.0 s. In order to test the robustness of the method, ten different datasets were taken during the beam time, each consisting of 25 measurements exploring the search space. Shown here are the RMSE of the resulting coordinates and the target for the optimal position. The -axis is truncated to show just the region of interest, because all deviations are less than 0.45 mm, despite a maximum possible deviation of 5.0 mm. The average deviation is 0.22 mm, which is in line with the average deviation of one dataset across 200 trials of 0.27 mm, shown in Fig. 4 . In the context of our mobile spectrometer, the solution we are presenting enables the automated alignment of the RZP and detector. Compared with our previous procedure, the reduction in alignment time from approximately one hour to five minutes (image acquisition time plus optimization process) represents a significant improvement and provides us with the advantage of more usable beam time. This work demonstrates the feasibility of creating a surrogate model for complex photon science experiments using only simulation data and successfully applying it to a real-world experiment. The model is trained to take the x , y and z coordinates, the camera offsets and the ratio of manganese to oxygen as input and return the corresponding detector image, requiring an optimiser to determine the offset between simulation and experiment. An alternative solution could involve training an inverse network (image to coordinates). With such a model, offsets could be calculated using a simple linear fit, employing experiment detector images as input. However, this approach poses challenges as the network needs to process unseen experiment data despite being trained solely with simulation data. We are currently exploring data augmentation and domain adaptation methods in this regard. Our objective at the Helmholtz Zentrum Berlin, concerning machine learning, is not to create custom solutions applicable only to one instrument. Instead, we aim to develop methods that can be generalized across various aspects of photon science. In this respect, this work serves as a prototype for data-driven alignment procedures applicable not only to spectrometers but also to entire beamlines and detectors in general. Manual alignment procedure (1) At the outset, shed light on the CCD through a coarse alignment of the spectrometer's z -axis. (2) Precisely centre the light on the CCD by adjusting the spectrometer's y -axis. Utilize coarse tuning in 1 mm steps, followed by fine-tuning with 0.2 mm increments. (3) Optimize the x -position of the Mn wire while monitoring the counts on the CCD image. (4) Move the piezo crawler to the `DOUBLE HOLE' position (10.5 mm) – a double aperture. Adjust the spectrometer's z -axis and y -axis to locate an image of the holes. (5) Centre the image at the half height of the CCD using the spectrometer's y / z axes. (6) Verification step. Shift the spectrometer's z -axis by +1.7 mm and then by −1.7 mm. If, at d Z = −1.7 mm, another faint image of two holes (reflection) can be observed, this confirms that the initial `true' transmitted image of two holes existed. If no reflection image is seen, revert to the previous z -axis position and check if, at d Z = +1.7 mm, a brighter image of two holes can be seen. If so, the earlier image was a reflection, and you are now in the `true' two holes image position. Stay at this position and proceed to Step 7. (7) Set the piezo crawler to FILTER. Starting from the position of the `true' two holes, adjust the spectrometer by d Z  = −3.6 mm. You may now be near the final alignment. (8) Fine-tune the alignment of the spectrometer's y -axis. Straighten the stripe structure by moving the spectrometer's y -axis, adjusting by d Y = −0.3 mm. (9) Fine-tune the spectrometer's z -axis: decrease the Mn–O separation (move the image down) by increasing Z , and increase the Mn–O separation (move the image up) by decreasing Z . Use a step size of d Z = 0.2 mm, and for the final fine-tuning set d Z = 0.1 mm. Network architecture (i) Learning rate: 1 × 10 −3 (scheduler reduces rate by a factor of 0.5 every 100 epochs). (ii) Adam optimiser. (iii) MSELoss with reduction = `sum'. Acknowledgements We acknowledge the beam time (proposal 221-10888) as well as the support provided by the staff scientists at the UE52-SGM beamline of the BESSY II storage ring, Helmholtz-Zentrum Berlin für Materialien und Energie GmbH (Berlin, Germany). We gratefully acknowledge the assistance and support of the Joint Laboratory Artificial Intelligence Methods for Experiment Design (AIM-ED) between Helmholtz-Zentrum Berlin für Materialien und Energie GmbH and the University of Kassel. We are grateful to the other members of our machine learning group, Felix Möller and Gesa Goetzke, for their advice and consultation, and Junko Yano, Jan Kern and Vittal Yachandra for useful feedback regarding the manuscript. Open access funding enabled and organized by Projekt DEAL. Funding information The research was funded in the framework of the Röntgen-Ångström Cluster (RÅC, https://www.rontgen-angstrom.eu/ ) on the German side via the Bundesministerium für Bildung und Forschung (BMBF, contract No. 05K20CBA) and on the Swedish side from the Swedish Research Council (grant agreement No. 2019-06093). This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence , which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. Follow J. Synchrotron Rad. 276 COMMENTS Replica plating Replica plating. Replica plating is a microbiological technique in which one or more secondary Petri plates containing different solid ( agar -based) selective growth media (lacking nutrients or containing chemical growth inhibitors such as antibiotics) are inoculated with the same colonies of microorganisms from a primary plate (or master dish ... Replica Plating The replica plating experiment, devised by Joshua and Lederberg in 1952, is a simple process for screening large populations of microbial colonies on culture plates for genetic markers of interest. It has proved useful for discovery of rare auxotrophic mutations, detection and isolation of recombinant organisms after gene transfer procedures ... Replica Plating: Principle and Procedure J. Lederberg and E. Lederberg (1952) devised this procedure to demonstrate the spontane­ous nature of mutations. This method is used for detection of bio­chemical mutants, for the classification of fer­mentation reactions and for the determination of the spectra of antibiotic sensitivity. Phage sensitive strain of E.coli on nutrient agar plate is incubated until each cell […] 12.4: Replica plates accelerate the screening process Clare M. O'Connor. Boston College. GENTLY. Replica plating provides a rapid screening method for analyzing phenotypes. Colonies on a master plate are transferred to a sterile piece of velveteen. Copies of the mater plate are transferred. to additional selective or indicator media to monitor phenotypes under additional conditions. Aseptic Laboratory Techniques: Plating Methods 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. ... Replica-plate Procedure. Transferring cultures from ... Replica Plating Replica plating is an experimental technique that uses a printing-like transfer employing fabric with a pile (e.g., velveteen) to make multiple copies of an original culture plate with each microbial colony identified by its position on the culture plate. The copies can be made onto plates with different culture conditions that can select or identify variant strains derived from the original ... The Ledergberg Replica Plating Experiment explorebiology.orgJoshua Lederberg developed a "printing press" for bacteria called replica plating and he used it to distinguish between two models for evo... Replica Plating Technique Replica plating is a microbiological technique in which one or more secondary Petri plates containing different solid (agar-based) selective growth media (la... How to Replica Patch Plate Dr. Patrick demonstrates the common microbiology lab technique of replica patch plating. She first does an over view of the equipment used. Then she picks an... PDF Replica Plating to Identify Mixed E. coli Populations Prepare Replica Plates (10 minutes) Attach a replica-plating grid to the bottom of an LB/amp plate and to the bot-tom of an LB/kan plate. Use a permanent marker to label each plate with your name and the date. Replica plate a sample of cells from one colony on the L LB/amp plate onto the fresh LB/amp and LB/kan plates. Replica_plating In molecular biology and microbiology, replica plating is a technique in which one or more secondary Petri plates containing different solid (agar-based) selective growth media (lacking nutrients or containing chemical growth inhibitors such as antibiotics) are inoculated with the same colonies of microorganisms from a primary plate (or master dish), reproducing the original spatial pattern of ... Chapter 17 Replica Plating of Animal Cells Lederberg-Style Replica Plating Indirect selection by classic replica plating is the technique of choice for isolation of temperature-sensitive strains and nutrient auxotrophs (Table I). To create authentic replica plates, a 1 : 8 colony replica prepared on filter paper at 37°C or a I : 16 replica prepared at 33°C is placed cell side-down in ... PDF How to do replica plating. David Perkins Background test plate to show that inoculation has been effective. Orientation on the master and raplica plates must be ... Littlewood, R. K., and K. D. Munkres. 1972. Simple and reliable method for replica plating Neurospora crassa. J. Bacteriol. 110: 1017-1021. Maling, B. 1960. Replica plating and rapid ascus collection of Neurospora. J. Gen. Microbiol ... Ame's Test, Replica plate technique and Fluctuation test Replica plate technique: This technique was devised by Lederberg and Lederberg (1952). The replica plating technique was used to verify the spontaneous origin of bacterial resistance. By this procedure samples from all the colonies on a plate may be transferred simultaneously to another plate by means of a velveteen covered stamp pad. Colonies picked up from first plate this plate is known as ... Replica plating Definition and Examples replica plating (Science: technique) technique for testing the genetic characteristics of bacterial colonies. A dilute suspension of bacteria is first spread, in a petri dish, on agar containing a medium expected to support the growth of all bacteria, the master plate. Each bacterial cell in the suspension is expected to give rise to a colony. A sterile velvet pad, the same size as the petri ... Aseptic Laboratory Techniques: Plating Methods 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. ... Replica-plate technique used to transfer cells from ... A laboratory class experiment illustrating basic principles of DNA a SB plate and 100 ~1 on a SBA plate. Do the same with the cell suspension corresponding to the "-" tube. Incubate plates 12- 16 h at 37°C and record results. Identification of trunsformunt phenotypes by replica plating in selective media (screening) The following procedure allows Replica Plating and Indirect Selection of Bacterial Mutants Luria SE, Delbrück M. Mutations of Bacteria from Virus Sensitivity to Virus Resistance. Genetics. 1943 Nov;28(6):491-511. [ PMC free article] [ PubMed] [ Google Scholar] NOVICK A, SZILARD L. Experiments with the Chemostat on spontaneous mutations of bacteria. Proc Natl Acad Sci U S A. 1950 Dec;36(12):708-719. Unveiling The Mystery: What Is Replica Plating? Replica plating is a fundamental technique in scientific research that plays a crucial role in understanding the behavior and characteristics of microorganisms. By replicating bacterial colonies onto different media, researchers can study their growth patterns, genetic traits, and responses to various environmental conditions. This article will provide an overview of replica plating, its ... Isolation and Identification of Auxotrophic and Drug Resistant Mutants This technique isolates both nutritional mutants and antibiotic resistant mutants. Their actual experiment concerned with replicating master plates of sensitive cells to two or more plates containing either streptomycin or T1 phage. Replica plating allows the observation of microbes under a series of growth conditions. (PDF) Aseptic Laboratory Techniques: Plating Methods 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 ... Isolation of antibiotic resistance mutant by replica plating benazeer fathima. This document describes the replica plating technique used to isolate antibiotic resistance mutants from a bacterial population. The technique involves spreading bacteria onto a "master plate" containing nutrients. A velveteen cloth is then used to transfer the bacterial colonies onto a "replica plate" containing the same ... The Replica Plate Method for Screening Antibiotic-Producing Organisms The Replica Plate Method for Screening Antibiotic-Producing Organisms 1. H. A. Lechevalier and C. T. Corke. ... LEDERBERG J, LEDERBERG EM. Replica plating and indirect selection of bacterial mutants. J Bacteriol. 1952 Mar; 63 (3):399-406. [PMC free article] [Google Scholar] Stansly PG. A Bacterial Spray Apparatus Useful in Searching for ... (IUCr) Automated spectrometer alignment via machine learning Achieving precise alignment of the reflection zone plate with respect to the sample and detector typically demands approximately one hour of skilled operation. The alignment process involves a meticulous nine-point procedure. Initially, a `grid-search' method is applied to locate two reference markers. essay homework paper phd project resume review speech thesis