Colony formation of mammalian cells on agar plates and its application to Lederberg's replica plating

In this paper we describe a new technique of cloning by use of agar plates and its application to replica plating. It was found that most cell lines form colonies on the surface of solid agar, although the plating efficiency and size of colony is dependent on specimens and concentrations of agar and agarose used. When 0.5% Noble-agar was used as substrate, plating efficiencies were obtained comparable to those of conventional cloning techniques in liquid medium and of agar suspension cultures. In some cases, including the primary culture of Yoshida sarcoma, the efficiency of plating was apparently higher than that obtained by the already established procedures. In an experiment with a series of BHK-21 cells, it was found that virally transformed cells could form colonies on agar plate, whereas untransformed and reverted cells could not divide, suggesting that agar plate culture, as well as agar suspension culture, can be used for a selective assay of transformation.Two methods of replica plating were employed. Method I is that devised by Lederberg in which colonies on the master plate are imprinted on pile fabrics and then transferred to the replica plates. With FM3A cells, the fidelity of replica plating was around 95%. Method II is inoculation of clones by applying a glass rod to the replica plates on which positions of inocula were identified by a grid. Fidelity of replica plating of FM3A, L5178Y and YSC cells was 99.7, 100 and 100% respectively.     

一种改进的平板影印工具及其应用

[目的]建立一种简便高效的平板影印工具,克服传统影印工具的缺陷.[方法]将大量竹签按照一定方法捆扎成与平皿内盖直径相近的捆,将单根牙签转移菌落的效果进行扩大,从而实现菌落的大规模转移.[结果]本实验室使用上述工具成功地进行了平板影印和传代,并进行转化子大规模筛选.[结论]与传统工具相比,该影印工具更为经济简便,而且效果清晰,为微生物平板筛选提供新的方法和思路.国家发明专利申请号:2007100291331.     

Replica plating of colonies from Listeria-selective agars to blood agar to improve the isolation of Listeria monocytogenes from foods

Bacterial colonies from Listeria-selective agars were replica plated to sheep blood agar to screen for beta-hemolysis. By using the replica plating method to test for the beta-hemolytic characteristic of all the colonies growing on Listeria-selective agars instead of picking 3 to 10 suspected colonies for further testing, we recovered Listeria monocytogenes from 59 of 142 Listeria-selective agar plates which contained colonies of hemolytic and nonhemolytic Listeria species and were negative when tested by conventional colony picks.     

Replica plating of colonies from Listeria-selective agars to blood agar to improve the isolation of Listeria monocytogenes from foods.

Bacterial colonies from Listeria-selective agars were replica plated to sheep blood agar to screen for beta-hemolysis. By using the replica plating method to test for the beta-hemolytic characteristic of all the colonies growing on Listeria-selective agars instead of picking 3 to 10 suspected colonies for further testing, we recovered Listeria monocytogenes from 59 of 142 Listeria-selective agar plates which contained colonies of hemolytic and nonhemolytic Listeria species and were negative when tested by conventional colony picks.     

Direct spectrophotometric observation of an acyl-enzyme intermediate in elastase catalysis.

A technique is presented that is useful for selecting conditional-lethal mutants of mycoplasma cells and viruses. The method is based on growing mycoplasma on Millipore filters. Mutants can be isolated directly from filters seeded with mycoplasma. The filters can be transferred from condition to condition, acting as its own “master” and “replica” template. Virus mutants from the non-lytic Mycoplasma Group L1 and L2 viruses can also be picked from filters seeded with infected cells. This method is analogous to classical “replica plating” which is not a practical technique for mycoplasmas.     

A Replica Plating Method for Plant Cells

A replica plating method is described for plant cells growing in Petri dishes. The method involved a uniform application of plant cells (Morinda citrifolia L.) by spraying cells evenly on agar plates containing 60% conditioned medium. Subsequently the cells were allowed to grow through a nylon net. The net was removed from the master plate and placed upside down on replica plates. Cells from colonies adhering to the threads of the net were thus transferred to the replica plate and yielded colonies that, after a growth period of about 10–20 days, corresponded in position to the colonies on the master plate. An 80% transfer of colonies from the master plate to the copy plate was possible.     

A simple method for cloning and replica plating of mammalian cells using multicellular spheroids.

V 79/4 Chinese hamster cells or HeLa cells grow in Eagle's MEM supplemented with 25 microgram/ml dextran sulphate to form clonal multicellular spheroids. These cell clones, consisting of 5-10(2) cells, are easy to separate, to transfer from one culture vessel into another and grow as normal monolayer colonies on Dederon cloth circles after subculture in Eagle's MEM without dextran sulphate. A simple replica technique is described by which 500 clones can be transfered onto at least 3 replica cloth circles, 10 cm in diameter, with a replica plating efficiency of approximately 100%.     

Replica Plating Method for Estimating Phenanthrene-Utilizing and Phenanthrene-Cometabolizing Microorganisms

A replica plating method was developed for detecting and enumerating phenanthrene-degrading microorganisms. The method is designed to discriminate between aquatic organisms that utilize phenanthrene as the sole carbon and energy source and organisms that cometabolize phenanthrene. The method was used to demonstrate that phenanthrene utilizers and phenanthrene cometabolizers coexist in estuarine sediments.     

A Simple Method For Plating and Cloning Ciliates and Other Protozoa

SYNOPSIS. A simple method is described for plating and cloning ciliates and other protozoa, based on a principle differing from that traditionally used for plating and cloning bacteria and other microorganisms. This procedure, referred to as the silicone-oil-plating-procedure (SOPP), involves vortexing small volumes of culture medium containing protozoa with larger volumes of a non-toxic silicone oil and plating the resulting unstable emulsion in small plastic petri plates. Discrete microdroplets of culture medium form containing protozoa entrapped and immobilized between the hydrophobic surfaces of the plastic petri dish and the oil. Protozoa, isolated by this method grow, divide, and multiply to form clones. the procedure may be used for plating and cloning protozoa in bacterized and axenic culture. Variations of the basic method may be applied to isolating protozoa from the wild, washing protozoa to remove microorganisms, screening for potential mutants, and for replica plating.     

Identification of Thiobacilli by Replica Plating on Thallous Sulfide Paper

Colonies of Thiobacillus thioparus and T. thiooxidans grown on thiosulfate medium can be identified by replica plating on thallous sulfide paper.     

Insertion of plasmids into the chromosome of Streptomyces griseofuscus

The results demonstrate a general method for randomly integrating plasmids into the genome by a single crossover between a cloned DNA fragment and homologous DNA in the chromosome. The integrated plasmid is flanked by directly repeated copies of the cloned homologous DNA sequence. Two protocols, "replica plating" and "liquid transfer," yielded strains with integrated plasmids.     

Quantifying antimicrobial resistance at veal calf farms

This study was performed to determine a sampling strategy to quantify the prevalence of antimicrobial resistance on veal calf farms, based on the variation in antimicrobial resistance within and between calves on five farms. Faecal samples from 50 healthy calves (10 calves/farm) were collected. From each individual sample and one pooled faecal sample per farm, 90 selected Escherichia coli isolates were tested for their resistance against 25 mg/L amoxicillin, 25 mg/L tetracycline, 0.5 mg/L cefotaxime, 0.125 mg/L ciprofloxacin and 8/152 mg/L trimethoprim/sulfamethoxazole (tmp/s) by replica plating. From each faecal sample another 10 selected E. coli isolates were tested for their resistance by broth microdilution as a reference. Logistic regression analysis was performed to compare the odds of testing an isolate resistant between both test methods (replica plating vs. broth microdilution) and to evaluate the effect of pooling faecal samples. Bootstrap analysis was used to investigate the precision of the estimated prevalence of resistance to each antimicrobial obtained by several simulated sampling strategies. Replica plating showed similar odds of E. coli isolates tested resistant compared to broth microdilution, except for ciprofloxacin (OR 0.29, p≤0.05). Pooled samples showed in general lower odds of an isolate being resistant compared to individual samples, although these differences were not significant. Bootstrap analysis showed that within each antimicrobial the various compositions of a pooled sample provided consistent estimates for the mean proportion of resistant isolates. Sampling strategies should be based on the variation in resistance among isolates within faecal samples and between faecal samples, which may vary by antimicrobial. In our study, the optimal sampling strategy from the perspective of precision of the estimated levels of resistance and practicality consists of a pooled faecal sample from 20 individual animals, of which 90 isolates are tested for their susceptibility by replica plating.     

A comparison of two plating techniques to estimate plasmid stability of a prolonged chemostat culture

Summary A comparison of two plating techniques to estimate the segregational stability ofEscherichia coli RR1 harboring plasmid pBR322 in a chemostat was studied. No significant differences were observed between the spread and replica plating techniques in the beginning of the experiments. However, a noticeable discrepancy between these two methods appeared after approximately 100 hours. This inconsistency can be shown to be statistically significant.     

Analysis of seawaters for the recovery of culturable Vibrio parahaemolyticus and some other vibrios

We investigated the recovery of dormant and injured cells along with the normally culturable cells of Vibrio species with special emphasis on V. parahaemolyticus using both selective and non-selective media at moderate (20 C) and standard (37 C) culture temperatures from a bay water environment. Culture temperatures (20 or 37 C) did not affect the recovery of V. parahaemolyticus but did for other vibrios. We observed similar seasonality of V parahaemolyticus as in most other environmental studies. V. parahaemolyticus and other Vibrio species were recovered in higher numbers by a replica plating method compared to most probable number (MPN) and direct TCBS (thiosulfate citrate bile-salt sucrose) agar counts. Even with the replica plating method, however, vibrios number goes down to a minimum level and V. parahaemolyticus was undetectable during the cool temperature period of the year, although total bacterial cells and CFU on nutrient agar (with 2% NaCl) did not vary so much during the study period.     

Improved method for detection of glycosidases in bacterial colonies

An assay has been developed to detect bacterial glycosidases in colonies grown on the surface of agar plates. Advantages of this technique over previously described methods include elimination of the need for replica plating, better visualization of chromagenic reaction products, and a simple permeabilization step to enable better penetration by chromagenic substrates.     

Improved method for detection of glycosidases in bacterial colonies.

An assay has been developed to detect bacterial glycosidases in colonies grown on the surface of agar plates. Advantages of this technique over previously described methods include elimination of the need for replica plating, better visualization of chromagenic reaction products, and a simple permeabilization step to enable better penetration by chromagenic substrates.     

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.     

Direct detection of yeast mutants with reduced viability on plates by erythrosine B staining.

In this paper we report a rapid method to screen yeast mutants exhibiting reduced viability directly on plates. This method avoids the need for replica plating and is based on the addition of the vital dye erythrosine B in nutrient medium. After 2 or 3 days of culture, colonies containing a large proportion of dead cells show a pink or a dark pink color whereas normal colonies are practically white.     

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.