The human tumor clonogenic assay as a model system in cell biology

The human tumor stem cell (clonogenic) assay (HTCA) is a soft agar system designed for growing fresh human tumor specimens in vitro. The assay has been extensively used in studies both of individual patients' response to chemotherapy and for screening new agents. The technical limitations of this assay have been extensively discussed. The use of this test as a model system to study fundamentals of tumor cell growth has not been stressed. The potentials and limitations of this assay for the study of the regulation of tumor growth are presented.     

Membrane Filter Method for the Isolation and Enumeration of Pseudomonas aeruginosa from Swimming Pools

A membrane filter technique using black membrane filters, MacConkey agar and fluorescence under ultraviolet (UV) light was investigated for the quantitative isolation of Pseudomonas aeruginosa from swimming pools. Three thousand four hundred forty-five samples were collected from public swimming pools and enumerated by this method over a 6-month period. Fluorescent cultures were isolated from 222 specimens. Seventy-seven of these fluorescent cultures were selected for biochemical screening, with 75 (97%) being verified as P. aeruginosa. To further assess the specificity and sensitivity of this UV screening technique, a comparative study was made of some morphological and biochemical characteristics of fluorescent pseudomonads obtained from different sources. The sensitivity of the method was unimpaired by either colony types or biochemical variations of P. aeruginosa. The failure of the other two fluorescent species, P. fluorescens and P. putida, to grow and/or fluoresce on MacConkey agar at 37 C illustrates the specificity of this technique. Further studies are needed to compare the viability of P. aeruginosa on MacConkey agar to that of efficacious nonselective media.     

Quantitative characterization of antagonistic activity of lactobacilli

In this work the method of serial dilutions of lactobacilli in two-layer agar was used. On the agar surface bacterial or fungal cultures were applied at different time intervals. A special quantitative characteristic was introduced. L. plantarum strain 8P-A3 was shown to have the maximum antagonistic activity. In great amounts L. casei and L. reuteri are capable to suppress the growth of bacteria and fungi. All lactobacilli under study produced a pronounced bactericidal effect on Pseudomonas, had different influence on the viability of Escherichia and staphylococci and exhibited fungistatic and fungicidal action only when inoculated at high concentrations.     

Sampling of solid spent agar media for gas chromatography of diffused acidic fermentation products

Abstract Sampling and processing of solid agar media for gas chromatographic analysis is described. Small pieces of agar are cut from a 'sterile' area of the plate leaving the actual bacterial growth undisturbed for further tests. It is shown that diffused fermentation acids are adequately represented in these samples to permit their use for phenotypic and taxonomic characterization of isolates. Because of the established qualitative and quantitative correspondence of fermentation patterns from agar-grown and from broth-grown bacteria, respectively, the method is offered as a rapid alternative for the sampling of spent culture broths. In addition, the sampling of spent agar plugs seems to hold promise for the study of defined mixed cultures and of mixed growth from primary plates.     

Quantitative, radial diffusion slide assay for staphylocoagulase.

A simple, quantitative radial diffusion assay for staphylocoagulase in culture fluids, using microscope slides coated with a thin layer of agar containing plasma and fibrinogen, was developed. No prior purification of the enzyme was needed, and only small quantities, 7 microliter, were required for each test. This method is particularly suitable for objectively comparing the relative amounts of coagulase produced by different cultures of Staphylococcus aureus.     

Membrane-assisted culture of fungal mycelium on agar plates for RNA extraction and pharmacological analyses

Fungal mycelium grown in liquid culture is easy to harvest for RNA extraction and gene expression analyses, but liquid cultures often develop rather heterogeneously. In contrast, growth of fungal mycelium on agar plates is highly reproducible. However, this biological material cannot be harvested easily for downstream analyses. This article describes a PVDF (polyvinylidene difluoride) membrane-assisted agar plate culture method that enables the harvest of mycelium grown on agar plates. This culture method leads to a strongly reduced variation in gene expression between biological replicates and requires less growth space as compared with liquid cultures.     

An autoradiographic screening test for mycoplasmal contamination of mammalian cell cultures

Summary Autoradiographic detection of incorporation of tritiated thymidine into the cytoplasm of cultured mammalian cells has been evaluated as a test of contamination of the cultures by cell-associated microorganisms, which usually are mycoplasmas. Criteria which indicate the presence of cell-associated mycoplasmas have been established, and the reliability of the standardized autoradiographic method has been assessed by testing the same cultures by two colony formation methods of mycoplasmal detection. The autoradiographic method demonstrated cell-associated microorganisms in all cultures from which characteristic colonies were grown on mycoplasma agar. The autoradiographic method did not produce false positive results, and the outcome of this test was evident in 3 days as opposed to 7 to 14 days by agar culture methods. Some applications of the autoradiographic method are shown, and it is suggested that this method be employed for routine surveillance for mycoplasmal contamination in laboratories where facilities for frequent agar culture tests are not easily available. This research was supported by U.S. Public Health Service Grants CA 12351-02 and CA 12334-01 from the National Cancer Institute.     

Development of an improved selective agar medium for isolation of Yersinia pestis

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.     

Use of ristomycin diffused into the culture medium for separation of meningococci from nasopharyngeal mucus

The method for enhancing the growth of meningococci after the inoculation of the specimens of nasopharyngeal mucus is proposed. The method is based on the diffusion of ristomycin into agar from ristomycin-impregnated filter paper strips. During the study of 694 specimens of nasopharyngeal mucus the above-mentioned method allowed isolation of 108 meningococcal cultures, while in serum agar only 21 cultures were isolated. The effectiveness coefficient of this method was found to be 80.6%, its effectiveness index being 5.1. The method facilitated the isolation of meningococci and in some cases reduced the time of analysis by 1 day.     

Differential media for quantitative recovery of waterborne Aeromonas hydrophila.

Because of the ubiquity of Aeromonas spp., their prevalence in drinking water, and the increasing number of reports on Aeromonas sp.-related infections, a standard method for routine and quantitative recovery had to be defined. On the basis of a survey of 10 media for recovery analysis and subsequent differentiation assays in mixed cultures, we conclude that ampicillin-dextrin agar performed the best for the recovery of Aeromonas spp. in drinking water and the differentiation by simple criteria of that genus from other common waterborne bacteria.     

Stick-plaque test--an economic method of quantitative determination of viruses]

An economic method for quantitative assay of viruses is presented. In this "canule stick-plaque test" (German abbreviation SPT) samples of viruses, geometrically diluted and taken up by a canule, are inoculated by sticking into monolayer cell cultures overlayed with agar medium. A plaquelike CPE detectable by neutral red staining develops in the area of the inoculation. The frequency of this CPE formation depends on the concentration of viruses in the inoculated dilution. This dose-response allows calculation of the ID50. In this way it is possible to carry out titration involving 6 dilutions and 10 inoculations per dilution using 3 common Petri dishes (6 cm in diameter), only. The sensitivity, accuracy, and reproductibility of this method are described and discussed.     

Rapid method for detecting thermostable nuclease in staphylococci

A rapid test for the detection of staphylococcal thermostable nuclease (TNase) is described. The procedure consists of heat inactivation of solid cultures of staphylococci and microslide agar diffusion technique in toluidine blue agar containing deoxyribonucleic acid. Using this method the results are obtained about 1 d sooner than with the conventional method. Translated by I. Miler     

A preparative suspension culture system permitting quantitation of anchorage-independent growth by direct radiolabeling of cellular DNA

We have developed a hybrid methylcellulose/agar suspension culture system which permits long-term colony formation of transformed mesenchymal cells. In contrast to traditional agar suspensions, our system allows for recovery of cells and direct biochemical analysis of anchorage-independent growth. The ability to readily radiolabel cellular macromolecules in these preparative cultures permits a quantitative and objective analysis of colony formation by incorporation of [3H]thymidine into newly synthesized DNA.     

Development of an Improved Selective Agar Medium for Isolation of Yersinia pestis

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.     

A Simple Method for Staining Fungal Substrate Hyphae in Agar Media

Morphogenetic processes often occur in fungal cultures in agar medium. These processes are difficult to study by light microscopy because the hyphae or other structures fail to have sufficient contrast for detailed study and photography. To overcome this difficulty, we developed a method to stain hyphae inside the agar without affecting the medium itself.     

Clear Media for the Recovery of Pasteurella tularensis

Peptic digests of blood and hemoglobin were investigated as substitutes for the blood used in the preparation of glucose-cysteine-blood agar plating medium for the recovery of the virulent Schu strain of Pasteurella tularensis. Digest media so prepared were found to be satisfactory for the quantitative recovery of freshly grown cells but not for cells stored longer than several days. The addition of appropriate quantities of human plasma, bovine sera, or soluble starch rendered the digest media appropriate for use with stored cultures. The peptic digest-plasma (PDP) and peptic digest-starch (PDS) media were evaluated and found satisfactory for the quantitative recovery of P. tularensis Schu from freshly prepared and stored cultures, and from aerosols produced therefrom. With cultures stored longer than 6 weeks, the starch modification (PDS) was not as satisfactory as, and the plasma variation (PDP) was better than, glucose-cysteine-blood agar (GCBA) for the recovery of the organisms. PDP was superior to either GCBA or PDS medium for the recovery of the weakly virulent Jap 4 and Niieg-blue strains of P. tularensis.     

New screening test to determine the acceptability of 0.45-micron membrane filters for analysis of water

During routine membrane filter (MF) quality control testing, irregularities such as partial or complete inhibition of microbial growth at grid lines, abnormal spreading of colonies, growth in or along the grid lines, nonwetting areas, poor colony sheen and metallic sheen on the MF surface with mEndo agar, brittleness, decreased recovery, and severe wrinkling were observed with several lots of filters. To study these effects and to develop a more sensitive screening test for MF quality, we compared five different MFs with various types and degrees of defects by using five stock coliform cultures and five different media. Results showed that the Enterobacter aerogenes-tryptic soy agar test system detected more MF defects than any other combination did and was superior to the Escherichia coli-mFC agar American Society for Testing and Materials method for grid line inhibition. Filtered natural samples grown on the same media showed the same effects as were observed with the pure cultures. Poor colony sheen and sheen on the MF surface were best detected with Enterobacter aerogenes on mEndo agar. The use of tryptic soy agar and mEndo agar with this organism permitted the maximum detection of MF irregularities. Of the 142 MF lots tested by this method, 30% were acceptable, 10% were marginally acceptable, and 61% were unacceptable. This method provides a valuable screening test for determining the acceptability of 0.45-microm-pore-size MFs used for coliform and heterotroph analysis and may also be useful in conjunction with other methods.     

New screening test to determine the acceptability of 0.45-micron membrane filters for analysis of water.

During routine membrane filter (MF) quality control testing, irregularities such as partial or complete inhibition of microbial growth at grid lines, abnormal spreading of colonies, growth in or along the grid lines, nonwetting areas, poor colony sheen and metallic sheen on the MF surface with mEndo agar, brittleness, decreased recovery, and severe wrinkling were observed with several lots of filters. To study these effects and to develop a more sensitive screening test for MF quality, we compared five different MFs with various types and degrees of defects by using five stock coliform cultures and five different media. Results showed that the Enterobacter aerogenes-tryptic soy agar test system detected more MF defects than any other combination did and was superior to the Escherichia coli-mFC agar American Society for Testing and Materials method for grid line inhibition. Filtered natural samples grown on the same media showed the same effects as were observed with the pure cultures. Poor colony sheen and sheen on the MF surface were best detected with Enterobacter aerogenes on mEndo agar. The use of tryptic soy agar and mEndo agar with this organism permitted the maximum detection of MF irregularities. Of the 142 MF lots tested by this method, 30% were acceptable, 10% were marginally acceptable, and 61% were unacceptable. This method provides a valuable screening test for determining the acceptability of 0.45-microm-pore-size MFs used for coliform and heterotroph analysis and may also be useful in conjunction with other methods.     

Permanent mounts of fungus cultures grown on media optimal for production of characteristic structures

Summary 1. Dermatophyte cultures in plastic flasks were compared on four different media for total growth, sporulation, and pigment production. Czapek's agar favored more rapid and more abundant sporulation in most cultures than Sabouraud's dextrose agar, potato dextrose agar, or wort agar.2. A simple technique for embedding agar island cultures of fungi is described. These plastic-embedded cultures form permanent mounts useful for study of microscopic morphology.3. A relatively durable slant culture in a similar plastic flask is suggested as a companion for study of gross morphologic features of the cultures.     

The quantitative and useful expression of the hardness of agar plate medium for mycoplasmas and bacteria

A method for quantitative expression of the hardness of agar plate medium was studied. As the method for expressing the hardness by using real values of the load which an agar plate medium could sustain for a certain length of time was found to be inaccurate, we proposed a method to express the hardness by utilizing the frequency with which various loads were sustained for a given period of time and the obtained value is referred to as 'gel solidity' (GS). The GS value within a certain range was found to be statistically useful because it linearly reflected the changes in variables in experimental conditions in respect to agar, such as agar concentration, thickness of the agar layer and the temperature of the environment, and especially because it can provide a quantitative as well as reproducible value for the hardness of agar plate medium. On the other hand, GS was little, if at all, affected by variables unrelated to agar.