Replica Plating Technique Research Articles

Changes in cutaneous flora after wet occlusion

Aerobic flora from wet-occluded forearms of six volunteers was sampled the day before treatment, on the 3rd day when dressings were removed, and daily, when possible, for 8 days thereafter. Erythema was not present. All bacterial colonies appearing on appropriate dilution plates were identified with the aid of a replica-plating technique. Flora of each individual increased to over 10-4 colony-forming units/cm2 as a result of wet-occlusion, but counts rapidly fell by about 10-2 units once dressings were removed. Although similar types of bacteria were found on all subjects, the composition of each individual's flora during the recovery response appeared to be unique. Enterobacteriaceae were found on half the subjects with Enterobacter aerogenes being the most successful colonizer. Besides the expected presence of Baird-Parker Staphylococcus subgroup II, high numbers of subgroup IV and some colonies of subgroup III were also observed. Almost all cutaneous diphtheroids were lipophilic and lipolytic.

Effect of Experimental Dermatophyte Infection on Cutaneous Flora

The cutaneous aerobic bacterial flora was monitored during the course of experimental dermatiphyte (ringworm) infections on the forearms of 9 volunteers. Micrococcaceae were identified by the new Baird-Parker classification with the aid of a replica-plating technique. There were significant differences in total populations but not in kinds of flora compared with control (opposite) forearms. The proportion of penicillin-resistant microorganisms, however increased the infection, to a degree which varied as to individual. All coccal groups were affected; resistant flora diminished when the fungus was no longer DETECTED. Staphylococci were more frequently isolated than micrococci on the infected areas, but Staphylococcus aureus was not found. Trends toward hierarchies in persistance and quantities IV were observed among the flora, with Staphylococcus subgroups II and dominating the infected sites. On both areas almost all diphtheroids were nonfluorescent, lipophilic, and lipolytic. One subject consistently carried Bacillus firmus; another's normal microbiota contained Alcaligenes faecalis.

Improved rapid plate method for the isolation of bacteriophages from lysogenic bacteria.

A modification of a recently reported rapid plate method for the isolation of bacteriophages from lysogenic bacteria is described. The velveteen replica plate technique was used for inoculation of mitomycin C-induced colonies onto agar plates, and tetrazolium chloride was used to enhance detection of phage activity on replicated indicator plates.

Ultraviolet light sensitive mutants of Coprinus lagopus I. Isolation and characterization

4UV-sensitive mutants have been isolated from the wild type strain BC9/66 of Coprinus lagopus by following a new replica plating technique. Complementation and recombination studies between these mutants suggest 3 gene loci uvs1, uvs2 and uvs3, two of the mutants being allelic ( uvs3-1 and uvs3-2). The mutants uvs2, uvs3-1 and uvs3-2 show photoreactivation whereas the mutant uvs1 appears to be deficient in this respect. None of the mutants of the wild type showed significant recovery after dark holding.

Improved Rapid Plate Method for the Isolation of Bacteriophages from Lysogenic Bacteria

A modification of a recently reported rapid plate method for the isolation of bacteriophages from lysogenic bacteria is described. The velveteen replica plate technique was used for inoculation of mitomycin C-induced colonies onto agar plates, and tetrazolium chloride was used to enhance detection of phage activity on replicated indicator plates.

Isolation of sugar and antibiotic mutants in Chlorella vulgaris with a new replica plating technique

A new replica plating technique has been described to isolate physiological and biochemical mutants in the unicellular alga, Chlorella. In one operation, 115 different cultures can be inoculated. A pure culture of Chlorella was raised from a single cell which was treated with ethyl methanesulfonate at its LD 50 value to induce mutations. After a prolonged treatment with ethyl methanesulfonate (EMS), 0.87–39.4% mutants were isolated on media with different sugar and antibiotic contents. This technique can be effectively used for isolating physiological and biochemical mutants in other unicellular organisms and also for higher plant cells.

A replica plating technique for the study of plaque-forming centers

Mouse spleen cells were cultured on Millipore filters supported on the surface of Eagle's agarose medium. The secretory products of individual plaque-forming centers (PFC) revealed after diffusion into the agarose, were studied over an extended period of time by transferring the Millipore filters to fresh agarose surfaces repeatedly in a manner analogous to the replica plating technique employed with bacteria.

Rapid mapping of conditional and auxotrophic mutations in Escherichia coli K-12.

The approximate genetic map locations of auxotrophic and conditional lethal mutations of Escherichia coli can be rapidly determined with replica plating techniques. A set of patches of 15 streptomycin-sensitive (Str(S)) Hfr strains with points of origin distributed around the map is replica plated onto a recombinant-selective plate with a lawn of Str(R) cells which carry an unmapped mutation. The map interval defined by the Hfr points of origin which are closest to the mutant locus is seen by the presence or absence of heavy patches of recombinants produced by transfer of early wild-type genes from the Hfrs. An alternative method is to replicate patches of different mutant strains (100 per plate) onto Hfr lawns; in this case more than 1,000 different mutants can be mapped in a single experiment in a few days. In this way, many types of mutations with similar phenotypes can be grouped as to approximate location on the genetic map. For ordering mutations within groups, the same replica plating methods can be used to cross F-prime derivatives of mutants with other mutants of the same group. Relative merits of these and other mapping methods of E. coli are discussed.

Chapter 5: A Replica Plating Method of Cultured Mammalian Cells

This chapter discusses a replica plating method of cultured mammalian cells. The chapter describes the technical procedure for replica plating and illustrates an example of the application of the technique to the characterization of mutants of cultured mammalian cells. The replica plating method described is useful for the replica plating of various cultured mammalian cell lines in vitro . Genetic and biochemical studies of single somatic mammalian cells in vitro were developed by Puck and his co-worker who introduced the colony-forming technique as used in the field of microbial genetics. The lack of a replica plating technique, as used in the field of microbial genetics, for cultured mammalian cells has delayed more detailed genetic analysis in somatic mammalian cells. This procedure isuseful for the isolation of useful mutants, for further purification of a mutant cell line, and for the investigation of mutagenesis at the somatic cell level similar to that at bacterial and fungal levels.

A rapid method for complementation testing of Ultraviolet-Sensitive (UVS) mutants of Streptomyces coelicolor

Seventy-one uvs mutations were isolated and tested for complementation with mutations representing the known closely linked uvsA, C and D loci by a rapid replicaplating technique involving “ultra-fertile” crosses. Most of the mutations were successfully classified by this technique. Of the 71 mutations, 23 were assiggned to uvsA, 15 to uvsC and 28 to uvsD. Four strains may have carried mutations in more than one of the three loci, or conceivably polar mutations. The remaining mutation occurred outside the uvsA,C,D cluster.

A technique for replica platingCoprinus lagopus, a filamentous fungus

SUMMARYThe problem of replica plating filamentous fungi such asCoprinus lagopusis overcome by inducing micro-colonies with sodium deoxycholate and using ‘Velcro’, a hooked material, to replace velveteen in the standard replica plating technique. ‘Velcro’ is advantageous in that it has a regular pattern of closely spaced hooks which transfer small inocula from the colonies on the master plate.

Ultraviolet-sensitive mutants of Streptomyces coelicolor I. Phenotypic characterisation

23 UV-sensitive ( uvs) mutants were isolated by a replica-plating technique after mutagenesis by UV or nitrosoguanidine. Phenotypic classification of the uvs mutations (and genetic analysis described in the accompanying paper) have defined 6 loci, uvsA-F. Mutations in uvsA-E result in an increased UV-sensitivity of strains carrying them; mutations in uvsF, on the other hand, confer no increased sensitivity alone but enhance the sensitivity of strains carrying uvsA, C or D mutations. None of the UV-sensitive mutants is X-ray-sensitive. Mutations in none of the uvs genes have a major effect on recombination frequencies between pairs of strains, both carrying the same mutation. Wild-type strains of S. coelicolor were found to be heterogeneous in respect of photoreactivability.

Genetic analysis of simian virus 40: II. Comparison of seven dilution end-point titration microassays

Mammalian cells have been successfully cultured in volumes of 0.002 to 0.012 ml. Using these microcultures and an indirect immunofluorescent staining procedure, we have developed a dilution end-point titration micromethod for use with simian virus 40. This titration procedure is faster, as accurate, and less susceptible to contaminating infection than the presently available plaque assay method. The primary advantage of the technique, however, is that it should allow an investigator to carry out extensive physiologic and genetic analyses of viruses and cells on a modest budget and without technical assistance.The kinetics of SV40 tumor antigen and virion antigen synthesis at various temperatures, and the propagation of single virions were studied in microcultures and found to be similar to those determined by conventional methods.A replica-plating technique for virus has been developed that allows one person to screen more than 1000 single virus clones per week.

Genetic analysis of simian virus 40: I. Description of microtitration and replica-plating techniques for virus

Mammalian cells have been successfully cultured in volumes of 0.002 to 0.012 ml. Using these microcultures and an indirect immunofluorescent staining procedure, we have developed a dilution end-point titration micromethod for use with simian virus 40. This titration procedure is faster, as accurate, and less susceptible to contaminating infection than the presently available plaque assay method. The primary advantage of the technique, however, is that it should allow an investigator to carry out extensive physiologic and genetic analyses of viruses and cells on a modest budget and without technical assistance. The kinetics of SV40 tumor antigen and virion antigen synthesis at various temperatures, and the propagation of single virions were studied in microcultures and found to be similar to those determined by conventional methods. A replica-plating technique for virus has been developed that allows one person to screen more than 1000 single virus clones per week.

A rapid and efficient replica plating technique

SUMMARYA new type of apparatus for replica plating is described, which reduces operator error and fatigue. Several replica plates can be made immediately, thus eliminating the need for a master plate in many cases.

Aerobic Heterotrophic Bacterial Populations of Sewage and Activated Sludge

The replica-plating technique and Lochhead's nutritional method were combined in exploratory experiments to test their feasibility as useful means for characterizing the aerobic heterotrophic flora of activated sludge and to minimize the burdensome process of isolation, purification, and testing of isolates. In the test run, the method was about 86% reliable at the 0.05 level of significance. About 40% of the total number of bacteria able to grow on an aqueous extract of activated sludge did not grow on media containing glucose, amino acids, growth factors, and inorganic salts. The requirement for activated sludge extract suggested the existence of a requirement for unidentified nutrients contained in the activated sludge extract.

Differentiation of cutaneous anaerobic bacteria. The use of the replica plating technique

Differentiation of cutaneous anaerobic bacteria. The use of the replica plating technique

Apparatus for rapid replica plating in rhizosphere studies.

Rhizosphere investigations are restricted by the sheer number of cultures that must be handled in the multitude of different tubed media needed to characterize each isolate. A current rhizosphere study called for the use of 25 different diagnostic media with each of hundreds of isolates, a burdensome and laborious problem. Moreover, the replica plating technique of J. Lederberg and E. M. Lederberg (J. Bacteriol. 63:399, 1952) was unsatisfactory, because colonies were smeared when transferred by use of a velveteen pad after five or six plates were replicated from the master. Replication of 25 plates, each containing differential media, therefore required five masters. Because a more satisfactory method was needed, a replicator was devised adapting certain features of the replicating apparatus of G. Stotzky (Can. J. Microbiol. 11:629, 1965) and of C. Quadling and R. R. Colwell (Can. J. Microbiol. 10:87, 1964). A grid of inoculating needles was attached to a press (Fig. 1) to serve as a replicating inoculator. The replicator consists of three main parts. The press is a bottle capper, modified to fit into a wooden frame (Fig. 1A). The replicating part (Fig. 1B) is cut to the size of a petri dish from 16-gauge galvanized sheet metal. Brass nails (1.3 cm long) were inserted through and soldered in small holes drilled through the metal, 1.3 cm apart. The third part (Fig. 1C) consists of a sliding platform supporting two wood stands. One functions as a culture tube rack, the tubes being spaced 1.3 cm apart to match position of the brass nails. The other stand holds the petri dish into which the nails carrying inocula are to be imprinted. To prevent contamination, a sterile petri-dish lid covers the array of culture tubes when not in replicating position. In operation, 1-ml culture tubes containing suspensions of isolates obtained by standard methods are slid to the right under the metal replicator which is then depressed, each brass nail serving as an inoculation needle. The culture tubes are then covered with the protective petri lid, and the stand holding the dish to be inoculated is then slid to the left under the replicator. Depression of the replicator imprints the agar medium with an inoculum of each culture. Replication of 25 cultures occurs simultaneously; the process may be repeated as often as desired. The metal replicator may be autoclaved or flamed in alcohol when cultures are changed.

Apparatus for rapid replica plating in rhizosphere studies.

Rhizosphere investigations are restricted by the sheer number of cultures that must be handled in the multitude of different tubed media needed to characterize each isolate. A current rhizosphere study called for the use of 25 different diagnostic media with each of hundreds of isolates, a burdensome and laborious problem. Moreover, the replica plating technique of J. Lederberg and E. M. Lederberg (J. Bacteriol. 63:399, 1952) was unsatisfactory, because colonies were smeared when transferred by use of a velveteen pad after five or six plates were replicated from the master. Replication of 25 plates, each containing differential media, therefore required five masters. Because a more satisfactory method was needed, a replicator was devised adapting certain features of the replicating apparatus of G. Stotzky (Can. J. Microbiol. 11:629, 1965) and of C. Quadling and R. R. Colwell (Can. J. Microbiol. 10:87, 1964). A grid of inoculating needles was attached to a press (Fig. 1) to serve as a replicating inoculator. The replicator consists of three main parts. The press is a bottle capper, modified to fit into a wooden frame (Fig. 1A). The replicating part (Fig. 1B) is cut to the size of a petri dish from 16-gauge galvanized sheet metal. Brass nails (1.3 cm long) were inserted through and soldered in small holes drilled through the metal, 1.3 cm apart. The third part (Fig. 1C) consists of a sliding platform supporting two wood stands. One functions as a culture tube rack, the tubes being spaced 1.3 cm apart to match position of the brass nails. The other stand holds the petri dish into which the nails carrying inocula are to be imprinted. To prevent contamination, a sterile petri-dish lid covers the array of culture tubes when not in replicating position. In operation, 1-ml culture tubes containing suspensions of isolates obtained by standard methods are slid to the right under the metal replicator which is then depressed, each brass nail serving as an inoculation needle. The culture tubes are then covered with the protective petri lid, and the stand holding the dish to be inoculated is then slid to the left under the replicator. Depression of the replicator imprints the agar medium with an inoculum of each culture. Replication of 25 cultures occurs simultaneously; the process may be repeated as often as desired. The metal replicator may be autoclaved or flamed in alcohol when cultures are changed.

Replica plating technique for studying microbial interactions in soil.

A replica plating method was developed to study ecology of microorganisms in soil. Precise placement of inocula and amendments at desired loci in sterile soil contained in petri plates were accomplished with a template. Subsequent growth and distribution of individual species, even when part of a mixed population, was measured by periodic transfer with an easily constructed replicator to agar plates of differing nutritional composition or containing selective inhibitors. The method is rapid and reproducible, and permits the study of many variables and interactions in a single soil plate; it can also be used with non-sterile soil and other suitable microbial habitats.