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.     

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.     

A high-throughput ultrasonic spraying inoculation method promotes colony cultivation of rare microbial species

Current method for obtaining microbial colonies still relies on traditional dilution and spreading plate (DSP) procedures, which is labor-intensive, skill-dependent, low-throughput and inevitably causing dilution-to-extinction of rare microorganisms. Herein, we proposed a novel ultrasonic spraying inoculation (USI) method that disperses microbial suspensions into millions of aerosols containing single cells, which lately be deposited freely on a gel plate to achieve high-throughput culturing of colonies. Compared with DSP, USI significantly increased both distributing uniformity and throughput of the colonies on agar plates, improving the minimal colony-forming abundance of rare Escherichia coli mixed in a lake sample from 1% to 0.01%. Applying this novel USI to a lake sample, 16 cellulose-degrading colonies were screened out among 4766 colonies on an enlarged 150-mm-diameter LB plate. Meanwhile, they could only be occasionally observed when using commonly used DSP procedures. 16S rRNA sequencing further showed that USI increased colony-forming species from 11 (by DSP) to 23, including seven completely undetectable microorganisms in DSP-reared communities. In addition to avoidance of dilution-to-extinction, operation-friendly USI efficiently inoculated microbial samples on the agar plate in a high-throughput and single-cell form, which eliminated masking or out-competition from other species in associated groups, thereby improving rare species cultivability.     

Improved protocols for the isolation and in-situ cryopreservation of cell colonies

Stable transfection and cloning of cells often require physical separation of cell colonies. In order to conveniently isolate cell clones from petri dishes, we developed a protocol starting with a soft agar overlay of cells. This reduces the risk of cell diffusion between different colonies. Cells from individual colonies are mechanically removed, incubated with trypsin, and cell suspensions are seeded onto parallel microtiter plates. The cell clones on one microtiter plate can be cryopreserved in situ using the protocol described here which was tested for a variety of cell lines. Replica plates can be used for screening and further expansion of interesting clones. If screening can also be performed in situ, e.g., by immunocytochemistry, immunofluorescence, or the polymerase chain reaction, it is possible to perform most steps necessary in cell cloning experiments on microtiter plates.     

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.     

Genetic transformation system in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

A wild-type strain of Methanobacterium thermoautotrophicum Marburg was transformed by DNA from strains resistant to 5-fluorouracil. Recipient cells were grown without selection on gellan gum (GELRITE) plates with DNA. Drug-resistant cells were recovered by replica plating the resulting colonies onto drug plates. Transformation required high-molecular-weight DNA with appropriate markers and was not observed on agar or in liquid media under a variety of conditions.     

Analysis of recombinant protein expression by MALDI-TOF mass spectrometry of bacterial colonies

E. coli expressing soluble recombinant HIV antigens were analyzed directly by MALDI-TOF mass spectrometry (MS) from bacterial colonies picked from agar plates. An HIV envelope (ENV) antigen construct, penvA, was expressed in E. coli by transformation of the plasmid pPL/penvA-M. The plasmid was co-transformed into E. coli DH5 alpha cells with an equal quantity of the plasmid pKRR826, the parent vector without the penvA insert, and plated at medium density on L-agar plus ampicillin plates. A total of 24 colonies from four agar plates (six colonies per plate) were picked and transferred into 50% acetonitrile--0.1% trifluoroacetic acid aliquots for analysis by MALDI-TOF MS. The MS analysis detected 10 of 24 colonies expressing the recombinant protein; one colony expressed a mutant penvA protein; eleven of 24 colonies showed ions only from E. coli; and two of 24 colonies showed no detectable proteins. When E. coli transformed only with plasmid pPL/penvA-M were examined, all (10 of 10) colonies showed the penv insert by the MALDI-TOF MS method. The method is fast (less than 1.5 h for 24 colonies) and allows identification of colonies expressing intact or mutant proteins directly from culture plates without sample purification.     

Effect of selective media on loss of congo red binding inShigella flexneri

Summary The effect of growth ofShigella flexneri on various selective media on retention of congo red (CR) binding ability was determined to evaluate the effectiveness of isolation techniques regarding maintenance of the virulence plasmid. WhenS. flexneri was surface-plated onto selective agars and the resulting colonies replica plated onto CR plates, no white colonies indicative of loss of virulence were found despite repeated trials. However, whenS. flexneri was grown in liquid media (agar was removed from agar-containing media by centrifugation), white colonies were found upon plating onto CR plates. Most common selective media for shigellae produced fewer than 5–10 white colonies/1000 red colonies. However, growth in broth prepared from violet red bile agar, desoxycholate citrate agar, and SS agar gave more than 100 white colonies/1000 red colonies. Loss of CR binding was demonstrated whenS. flexneri was grown in broth containing tergitol 7, sodium dodecyl sulfate, bile salts #3, crystal violet, eosin y or methylen blue. However, concentrations of selective agents that led to loss of CR binding were much higher than those used in selective media. Results indicate that under usual conditions of isolation ofS. flexneri from food and clinical specimens, CR binding appears to be a relatively stable character with most selective media; however, use of violet red bile agar, desoxycholate citrate agar, and SS agar may lead to substantial loss of congo red binding indicating that the isolates may not be virulent.     

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.     

Microimmunofluorescence using Terasaki plates and direct plate freezing method--rapid and reliable screening system of hybridomas

Microimmunofluorescence using Terasaki plates and a direct plate freezing method were combined for effective screening of hybridoma supernatants. The microplates, in which the fused cells (myeloma and spleen cells) were cultured and hybridoma colonies were growing, were frozen after harvest of supernatants and saved at -80 C for several weeks without affecting antibody production ability of hybridomas. Microimmunofluorescence was performed in Terasaki plates on which target cells were attached by poly-L-lysine and glutaraldehyde or by short time culture of the cells in Terasaki plates. The direct plate freezing method prevented initial hybridoma cells from changes or disappearance of antibody productions during screening of hybridoma supernatants; the microimmunofluorescence staining method permits fast and detailed estimation of specificity of antibodies of hybridomas by saving time and minimal consumption of supernatant for checking. The combination of these two methods is a powerful tool for obtaining desired monoclonal antibodies.     

Application of Replica Plating and Computer Analysis for Rapid Identification of Bacteria in Some Foods: I. Identification Scheme

A method was devised and tested for a quantitative identification of microbial flora in foods. The colonies developing on the initial isolation plates were picked with sterile toothpicks and inoculated on a master plate in prearranged spacing and order. The growth on the master plates was then replicated on a series of solid-agar plates containing differential or selective agents. The characteristic growth and physiological responses of microbial isolates to penicillin, tylosin, vancomycin, streptomycin, chloramphenicol, neomycin, colistin, and to S S Agar, Staphylococcus Medium No. 110, and Potato Dextrose Agar were recorded, together with Gram reaction and cell morphology. This information was then fed into an IBM 1410 digital computer which grouped and analyzed each isolate into 10 microbial genera, or groups, according to the identification key. The identification scheme was established by use of reference culture studies and from the literature. This system was used to analyze the microbial flora in dover sole (Microstomus pacificus) and ground beef. The method described in this article enables one to examine large numbers of microbial isolates with simplicity.     

Quantification of the ease of removal of bacteria from surfaces

This paper describes a technique which reproducibly quantifies the ease of removal of microorganisms from surfaces. Tiles (22 mm×22 mm) of various materials were colonised withStaphylococcus epidermidis NCTC 11047,Escherichia coli K12 HB101 orPseudomonas aeruginosa PaWH, by submersion, for various times (2 min–48 h), in inoculated Tryptone Soya broth (37°C). Colonised tiles were blotted onto a Tryptone Soya agar plate for 1 min and the process was repeated through a succession of agar plates. The final plate contained tetrazolium salts (0.05% w/v) and was incubatedin situ with the tile. Tetrazolium plates indicated that very few organisms remained on the tiles after 15 successive blots. In all instances, the number of recovered colonies per plate decreased exponentially with plate succession number, according to the relationship, CFU-A.10^–kN, where CFU is the number of colonies transferred,k is the removal exponent, A is the intercept and N is the plate succession number. Removal exponents differed significantly between organisms (P>0.95), depended on the nature of the test surface, and decreased as the inital attachment and colonisation time was increased from 2 min–48 h. Intercept values (A) but not the gradients were dependent upon the initial numbers of bacteria in suspension. These data indicate that the gradients derived from counting recoverable viable cells from successive blots of test tiles onto agar is a measure of the strength of attachment of the organisms to the surface.     

Demonstration of Aminopeptidase Activities Secreted by Amsonia tabernaemontana Walt. Cells

Using synthetic substrates, an uncomplicated and sensitive procedure for the determination of extracellular aminopeptidase was developed. The studied enzyme produced by the tested plant material (calli, cell suspension culture and roots of Amsonia tabernaemontana Walt. seedlings) hydrolyzed the substrates β‐naphthylamides (βNA) and 4‐(phenylazo) phenylamides (PAP‐amide) of the amino acids to β‐naphthylamine and 4‐(phenylazo) aniline, respectively, and amino acid. The β‐naphthylamides of the amino acids were applied for the identification of extracellular aminopeptidase, whereas the 4‐(phenylazo) phenylamides of the amino acids were used for the determination of intra‐ and extracellular aminopeptidase activity. By simultaneous azocoupling of β‐naphthol with Fast Garnet GBC salt on agar plates a corresponding brown‐red hardly water‐soluble azo‐dye was produced. The evaluation of dyed zones allowed the extracellular aminopeptidase activity to be assessed. No coloration of the agar medium was observed without inoculum, with heat‐inactivated cells (10 min at 100 °C) or in medium inoculated without substrate. On the agar plates with substrate and sterile Amsonia seedlings, changes in coloration were observed indicating a release of aminopeptidase from the roots during germination. The results show a 91.0 % intracellular and 9.0 % extracellular distribution of aminopeptidase activity, when a cell suspension culture of A. tabernaemontana Walt. as the plant material was used. The agar plate method described permits the rapid, uncomplicated and specific detection of plant producers of extracellular aminopeptidase, which could be particularly useful in future inhibitory and/or biotechnological studies.     

TWO DISTINCT COLONIAL MORPHOTYPES OF AMPHORA COFFEAEFORMIS (BACILLARIOPHYCEAE) CULTURED ON SOLID MEDIA1

When cultured on different types of solid media, the marine-fouling diatom Amphora coffeaeformis (Ag.) Kütz. consistently formed two distinct colonial morphotypes named tight and fuzzy. Tight colonies were comprised mainly of small, morphologically distorted, nonmotile cells, whereas morphologically normal and highly motile cells formed the fuzzy colonies. Cells from tight colonies were less adherent to glass, grew more slowly in liquid media, and had a slightly decreased viability on plates with copper than cells from fuzzy colonies. Whereas the protein profiles of the two types of cells were nearly identical in polyacrylamide gels stained with Coomassie blue, cells from tight colonies produced a significantly lower amount of a protease-resistant, low M_r polysaccharide or glycoconjugate as detected in silver-stained gels. The frequency of appearance of the fuzzy and tight morphotypes was not influenced by the mode of nutrition or the type of substratum to which the algal cells adhered. However, certain formulations of solid medium and the presence of growth-inhibitory concentrations of copper in agar plates favored the formation of tight colonies. Due to their frequencies and patterns of appearance, it was clear that the two naturally formed morphotypes were not the consequence of spontaneous mutations, genetic rearrangement, or selection of stable natural variants, and we have hypothesized that they were linked to a normal physiological behavior. The tight colonial morphotype was used as a valuable marker to screen for true motility/adhesion mutants within an ultraviolet-mutagenized population of A. coffeaeformis. Seven mutants were isolated that were non-motile on agar plates, poorly adherent to glass, and distinguished from naturally formed cells from tight colonies by their inability to form fuzzy colonies upon subculture on solid media.     

Chromium(VI) and apparent phenotypic reversion in Salmonella TA100

After treatment with potassium chromate at concentrations causing ultramicroscopic cellular lesions, a significant proportion (up to 75%) of TA100 colonies fail to replicate on fresh minimal plates containing biotin. This suggests that chromium(VI) may not always induce his- reversion to his+ in Salmonella TA100. The terms 'false' or phenotypic reversion have been used to distinguish such instances from 'true' or genotypic reversion, where progeny his+ cells readily grow on biotin replica plates. Results of the present study indicate that the majority of chromate-exposed colonies, initially scored as his-, are identifiable as his+ after 24 h culture on nutrient agar. Moreover, chromate exerts a cytostatic effect on TA100 since early colony development is suppressed at high chromate concentrations. A gradual chemical reduction of chromium(VI) ions by normal media compounds is probably responsible for the re-emergence of colony growth during prolonged incubation of test plates. Thus, temporary growth inhibition at high chromate concentration appears to be responsible for most of the non-replicating colonies detected in mutagenicity assays of chromium(VI).     

Mitochondrial genome structure of rice suspension culture from cytoplasmic male-sterile line (A-58CMS): reappraisal of the master circle

Summary The mitochondrial DNA (mtDNA) from the cultured cells of a cytoplasmic male-sterile line (A-58CMS) of rice (Oryza sativa) was cloned and its physical map was constructed. There was structural alteration on the mitochondrial genome during the cell culture. Detailed restriction analysis of cosmid clones having mtDNA fragments suggested either that the master genome has a 100-kb duplication (the genome size becomes 450 kb) or that a master circle is not present in the genome (the net structural complexity becomes 350 kb). The physical map of plant mitochondrial genomes thus far reported is illustrated in a single circle, namely a master circle. However, no circular DNA molecule corresponding to a master circle has yet been proved. In the present report, representation of plant mitochondrial genomes and a possibility for mitochondrial genome without a master circle are discussed.     

Non‐host plant essential oil volatiles with potential for a ‘push‐pull’ strategy to control the tea green leafhopper,Empoasca vitis

The tea green leafhopper, Empoasca vitis Göthe (Hemiptera: Cicadellidae), is an economically important pest of tea crops, Camellia sinensis (L.) O. Kuntze (Theaceae), in China. The use of non‐host plant essential oils for manipulation of E. vitis was investigated for potential incorporation into a ‘push‐pull’ control strategy for this pest. The effectiveness of 14 plant essential oils in repelling E. vitis was investigated in laboratory assays. Rosemary oil, geranium oil, lavender oil, cinnamon oil, and basil oil repelled leafhoppers in a Y‐shaped olfactometer. We also compared the efficacy of these five plant essential oils to repel E. vitis in the presence of a host plant volatile‐based leafhopper attractant, (Z)‐3‐hexenyl acetate, in a tea plantation. In the treatment combination, four plates (north, south, east, and west) treated with an essential oil surrounded a central sticky plate treated with (Z)‐3‐hexenyl acetate. Fewer E. vitis were found on the plates treated with rosemary oil (12.5% reduction) than on the four water‐sprayed control treatment plates surrounding a central plate with (Z)‐3‐hexenyl acetate. We compared the distribution of E. vitis on the plates, and the relative numbers of E. vitis on each plate were compared with similar plates in the control treatment. When four plates treated with rosemary oil surrounded a central (Z)‐3‐hexenyl acetate‐treated plate, the distribution of E. vitis on the different plates changed significantly compared with that of the control. Relatively fewer E. vitis were found on the east (13.0% reduction) rosemary oil‐treated plates and more E. vitis (11.3% increase) were found on the central attractant‐treated plate. Our findings indicate that rosemary oil is a promising leafhopper repellent that should be tested further in a ‘push‐pull’ strategy for control of E. vitis.     

An easy method for screening and isolating rod mutants of<Emphasis Type="Italic"> Bacillus subtilis</Emphasis>

A convenient and rapid method for screening and identifying rod mutants of Bacillus subtilis is described. At the restrictive temperature (45 °C), all rod mutants of B. subtilis screened lost their ability to sporulate. The morphology and colour of mutant colonies grown on sporulation agar plates differed from those of rod⁺ cells, which were able to sporulate even at elevated temperature. These characteristics provide an alternative approach for the identification of rod mutants in B. subtilis culture by streaking the cells onto a minimal glucose agar plate and incubating at the restrictive temperature. After 30 h of incubation at this temperature, rod mutants are easily identified. This method will facilitate the screening and isolation of rod mutants of B. subtilis.     

High frequency plant regeneration from rice protoplasts by novel nurse culture methods

Summary Novel nurse culture methods have been developed for plant regeneration from protoplasts of rice (Oryza sativa). The nurse culture methods use the agarose-bead type culture in combination with actively growing nurse cells that are either in the liquid part of the culture or inside a culture plate insert placed in the centre of the dish. Protoplasts isolated from either primary seed calluses or suspension cultures of various callus origins, divided and formed colonies with a frequency of up to 10% depending on the protoplast source and the genotype. The presence of nurse cells was absolutely required for the induction of protoplast division. Plants were regenerated from protoplast-derived calluses of five tested cultivars with a frequency of 17%–50%. Close examination of the plant regeneration process suggested that plants are regenerated through somatic embryogenesis from protoplast-derived calluses. Over 300 protoplast-derived plants were transferred to either pots or the field and are being examined for karyotypic stability and various plant phenotypes.