Minimal Agar Plates Research Articles

Detection by replica plating of false revertant colonies induced in the Salmonella-mammalian microsome assay by hexavalent chromium.

The replica plating method as developed by Lederberg has been used to differentiate between "true" and "false" histidine-requiring revertant bacterial colonies which develop on minimal agar plates in the Ames test. Strains of S. typhimurium LT2, TA 100, when exposed to either sodium dichromate or the fumes from the welding of stainless steel, develop colonies whose apparent numbers are directly in proportion to the Cr(VI) content per plate in both cases, over a wide dose range. Replica impressions of the resulting colonies were transferred to Vogel Bonner minimal agar plates and incubated for 48 hr at 37 degrees C. It was then observed that considerable numbers of "false" revertant colonies were obtained at those Cr(VI) doses which resulted in a pronounced toxic effect, albeit with an acceptable level of the bacterial background lawn. No morphological distinction between "true" and "false" revertant colonies could be made. Although it would appear that at low doses (i.e., low toxicity) the true mutagenicity of stainless steel welding fumes can be completely accounted for by the presence of Cr(VI), the dose range over which the mutagenicity assay is reliable cannot be estimated from examination of the background lawn or from an estimate of the degree of survival of the treated cultures. Thus there is raised a serious question concerning the reliability of quantitative data published in bacterial mutagenicity testing where replica testing of the histidine requirement of the resulting "revertant" colonies is not routinely made. It is suggested that the replica technique can easily be developed as a simple and useful tool for the control of histidine requirement and ampicillin resistance in routine mutagenicity testing.(ABSTRACT TRUNCATED AT 250 WORDS)

Naturally occurring prototrophic strains of Salmonella typhi.

In a survey of the nutritional requirements of Salmonella typhi it was found that 3.2% of 560 recent clinical isolates were able to grow in a minimal medium consisting of phosphates, ammonium and magnesium sulfates, and glucose; the remainder required tryptophan. Both groups grew slowly and rather poorly in these media due to a deficient utilization of sulfur from sulfate. Addition of cysteine or sodium sulfide or thiosulfate promoted rapid and profuse growth. Minimal medium containing thiosulfate as a source of sulfur allowed for an easy and sharp differentiation of prototrophic variants needing none of the amino acids, and tryptophan auxotrophs. The prototrophic phenotype is not the result of the presence of rare prototrophic mutants, since these strains were able to develop in minimal medium from very small inocula (10(2)), all colonies were prototrophic in replica plating experiments, and the cultures gave comparable colony counts when seeded simultaneously in nutrient and on minimal agar plates.

Analysis of genetic recombination between two partially deleted lactose operons of Escherichia coli K-12

Genetic recombination between a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1) in Escherichia coli K-12 has been examined. Since the deletions were nonoverlapping, rare lactose-fermenting (Lac+) recombinants occurred and were detected qualitatively on lactose tetrazolium agar indicator plates as white papillae growing on the surface of red colonies or quantitively on lactose minimal agar plates. Formation of Lac+ recombinants required the recA, recB, and recC gene products. Indirect suppression of recB21 by sbcB15 led to an increase in the frequency of Lac+ recombinants over wild-type levels. recF143 did not appreciably alter the number of Lac+ progeny, whereas recL152 and sbcB15 strains yielded increased numbers of Lac+ recombinants. The nature and formation of Lac+ recombinants was also examined. Respreading analysis indicated that formation of recombinants occurred primarily as the cells entered early stationary phase on the surface of the minimal agar plates and that over 90% of the recombinants contained a phi80dIIlac+ prophage. Time-of-entry experiments suggested that the region of deoxyribonucleic acid between the two operons was not inverted as a result of the recombinational event.

Cell division in Escherichia coli BS-12 is hypersensitive to deoxyribonucleic acid damage by ultraviolet light

Escherichia coli BS-12 uvrA lon is hypersensitive to ultraviolet light. On minimal agar plates at densities in excess of about 10(7) bacteria per plate, as few as one or two photoreversible pyrimidine dimers in the entire genome are sufficient to cause inhibition of cell division. Most of the resulting filaments are unable to divide or form a viable colony. Inhibition of cell division appears to be a rapid consequence of replication of deoxyribonucleic acid containing a pyrimidine dimer. Photoreversibility of the inhibition of cell division persists indefinitely, indicating that the continued presence of the pyrimidine dimers (or the continued generation of daughter strand gaps) is necessary to maintain the division-inhibited state. In view of the kinetics for the production of filamentation by ultraviolet light and the extremely low average inducing fluence (0.03 J/m2), it is concluded that the initiating signal is not the same as that causing other inducible phenomena such as prophage induction or Weigle reactivation.

Glucose catabolite repression in Escherichia coli K12 mutants defective in methyl-alpha-d-glucoside transport.

1. Two spontaneous Escherichia coli K12 mutants resistant to glucose catabolite repression were isolated using minimal agar plates with methyl alpha-D-glucoside. Mutants grow well on glucose and mannitol. 2. Glucose does not inhibit the inducible enzyme synthesis in isolated mutants: mutant cell (in contrast to parent cells) produce high levels of beta-galactosidase and L-tryptophanase under the conditions of glucose catabolite repression. 3. The isolated mutants are negative in methyl-alpha-D-glucoside transport; glucose uptake is not severely damaged. But the mutants (named tgl, transport of glucose) retained the ability to phosphorylate methyl alpha-D-glucoside in vitro at the expense of phosphoenolpyruvate. 4. The tgl mutation is cotransduced with purB and pyrC markers, i.e. locates near 24 min of the E. coli chromosome map. 5. It is thought that E. coli cells possess two glucose transport systems. The first one is represented by the glucose-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system. The second glucose transport system (coded for tgl gene) functions as permease and possesses high affinity to methyl alpha-D-glucoside. The integrity of glucose permease determine the sensitivity of the cell to glucose catabolite repression.

Mapping of sul, the suppressor of lon in Escherichia coli.

The suppressor sul, which is allele specific for the ultraviolet sensitivity gene lon, has been mapped by conjugation and transductional crosses in Escherichia coli K-12 and B/r. Previously, sul was reported to lie in the azi region of the Escherichia coli chromosome. Evidence is presented which positions sul close to and clockwise of fabA on the Escherichia coli map. Cotransductional frequencies of 31.3% were obtained between sul and pyrD, and frequencies of 82% were obtained between sul and fabA. Also, the mucoid phenotype of K-12 lon strains grown on minimal glucose agar plates at 37 C was not significantly effected in sul derivatives. No differences between the sul of Escherichia coli B/r and that of K-12 derivatives with regard to map location or effect on mucoid production were observed.

Suppression of initiation-negative strains of Escherichia coli by integration of the sex factor F.

Data are presented suggesting that the most critical factor determining whether an Hfr dnaAts strain can synthesize deoxyribonucleic acid and form colonies at temperatures that are nonpermissive for corresponding F- strains is neither the site of insertion of F nor the presence of additional mutations in the F particle or the chromosome; it is whether the particle is capable of autonomous replication at the temperature used. Consequently, suppression of the DnaA phenotype in Hfr strains occurs at 40 C but not, in most of them, at 42 C without the occurrence of additional mutations. The site of insertion of F may also be important since it is shown that in one Hfr dnaA strain partial suppression does occur at 42 C. In addition, it is shown that strains exhibiting suppression by integration of F at 40 C on minimal agar plates do not do so at this temperature on nutrient agar plates.

Characteristics of some single-step mutants to chloramphenicol resistance in Escherichia coli K12 and their interactions with R-factor genes.

Six one-step Chloramphenicol (Cm)-resistant mutants ofEscherichia coliK12 were graded for resistance to Cm, Tetracycline (Tc) and Puromyein (Pm) by streaking on minimal agar plates containing antibiotic. They fell into at least three distinct groups on the basis of their resistance patterns. One mutant showed increased sensitivity to Pm. Most of the mutants expressed their effect on resistance to Cm and Tc in the presence of R-factors carrying resistance genes for these antibiotics, but one mutant with a relatively high level of resistance to Cm had its resistance effect completely masked in the presence of R-mediated resistance. Similar cases were found among mutants selected for Cm-resistance in another strain of K12.

TRANSFORMATION OF BACILLUS LICHENIFORMIS.

Gwinn, Darrel D. (Oregon State University, Corvallis), and Curtis B. Thorne. Transformation of Bacillus licheniformis. J. Bacteriol. 87:519-526. 1964.-When a series of 28 auxotrophic mutants of Bacillus licheniformis were screened for transformation, only three of them, M28 (glycine(-)), M30 (uncharacterized), and M33 (purine(-)), produced a detectable number of transformants. The screening method consisted of spreading auxotrophic cells and deoxyribonucleic acid (DNA) from the prototrophic strain 9945A on minimal agar plates and observing the plates for development of prototrophic colonies. M28 transformed at a higher frequency than did the two other mutants, and it was studied in greater detail. Although up to 20% of the recipient cells spread on the plates in the presence of DNA gave rise to prototrophic colonies over a period of 72 hr, only about 10(-3)% of the cells produced transformants when they were incubated with DNA in liquid suspension for 1 hr. The most competent cultures of many tested were those grown on a shaker for 22 hr in a medium composed of nutrient broth, salts, and glycerol. When mutations resulting in requirements for histidine, leucine, serine, and trytophan were introduced singly into the glycine mutant, transformants for the leucine, serine, and histidine markers could be obtained at will, but transformants for the tryptophan marker were not detected even though all four of the double mutants could be transformed to glycine independence.

Radiation-Induced Reversion of Transition Mutants

X-irradiation increased the reversion frequency of two transition mutants of Escherichia coli, one of which (meth-) differed genetically from wild type by an adenine-thymine substitution of a guanine-cytosine base pair, the other (urg-2-) by a guanine-cytosine substitution of an adenine-thymine base pair. The lethal effects of the radiation were greatest when protein synthesis was prevented for a period immediately after irradiation. A period of 30 min at 37�0 was as effective as 22 hr. Irradiation of a saline suspension gave a lower survival of 0�2 times that of bacteria irradiated after being spread on minimal agar plates.

Development of resistance to streptomycin by Bact, lactis aerogenes (Aerobacter aerogenes). II. A comparison of mutants of different origins.

Streptomycin-resistant strains ofBact.lactis aerogeneshave been obtained by (a) direct selection in a minimal salt-glucose medium containing 1 unit/ml. streptomycin (strainRs1). (b) indirect selection in the absence of streptomycin with the replica plating technique (strainRrp). (c) irradiation with ultra-violet light (strainRuv). The rate at which colonies develop when these three strains respectively are inoculated on to minimal agar plates containing 1000 u/ml. streptomycin has been subjected to statistical comparison. The results suggest that a basic similarity exists between the strains. Experiments designed to investigate the possible specific induction of resistance by low concentrations of streptomycin in phosphate buffer suspensions of the organism yielded negative results.