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2 .7.1.1 Curing Experiments

Samples (1.0 ml) of overnight cultures (50 ml) grown on defined medium (Section 2.1.1) with succinate (0.5g carbon S,"^) as the carbon source, and ethidium bromide at concentrations of 10, 20, 40, 60, 80 and 100 pgml ^ were serially diluted into Tris-sulphate buffer, (0.02M), pH 7.0. Aliquots (0.1 ml) were spread onto Petri dishes with either succinate or 2MCPA as the carbon source (Section 2.1.2) and incubated at 30°C for 24 h and 48 h respectively. After incubation colony counts were determined and the values on succinate and 2IICPA plates for each concentration of ethidium bromide compared.

Colonies (100) from the succinate containing plates were transferred, using sterile tooth-picks onto succinate and 2MCPA plates to examine for the ability or otherwise to grow on 2MCPA, which was further investigated

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by inoculating a number of these organisms into succinate-defined medium (Section 2.1.1) incubating overnight, and transferring a sample (25 ml) into fresh medium (25 ml) containing 2MCPA, incubating for 24 h and assaying for free chloride ions as previously described (Section 2.3.1). A control of the parental organism was used to ensure sufficient time had elapsed for chloride release to be measured.

Cultures of the isolates incapable of growth on 2MCPA were screened for the presence of plasmid DNA by the method described (Section 2.6.2) using a plasmid containing strain of Pseudomonas

sp. strain E4 as a control.

2.7 . 1 . 2 Rate o f re v e rsio n to p aren tal phenotype

The rate of reversion of the ethidium bromide treated cells, characterised by absence of dehalogenase activities back to the parental phenotype, characterised by the presence of dehalogenase activities, was investigated.

Cultures cured of dehalogenase activity were repeatedly cultured on succinate-defined medium (Section 2.1.1) and aliquots (0.1 ml) spread onto 2MCPA-containing plates (Section 2.1.2) at Intervals.

After incubation for 48 h the plates were studied for the presence of large colonial growth. This experiment was repeated three months after the original work on cured strains of Pseudomonas sp. strain E4. 2.7.2 Mating Experiments

Further evidence for the plasmid association of dehalogenase activity in the soil isolates was sought by attempting to transfer the plasmids from the soil Isolates to organisms possessing no dehalogenase activity, and selecting for such transconjugants.

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Two approaches were used, firstly attempts were made to transfer the plasmid in Pseudomonas sp. strain E4 back into a cured strain of

this isolate strain E41, and secondly, mating experiments between the soil isolates and other Pseudomonas species were set up.

2.7.2.1 Selection of antibiotic resistant strains used as recipients in mating experiments

Two selection procedures were used, though the second proved to be the more efficient. The first involved culturing the required organism on succinate-defined medium (Section 2.1.1) or on nutrient broth (13g Nutrient broth i,"1) overnight, and spreading a sample (0.1 ml) onto succinate plates (Section 2.1.2) or nutrient agar plates

(2% wv"1) supplemented with an antibiotic (50 to 100 ugml’1) and incubating at 30°C until colonial growth was observed. The colonies were transferred onto media containing higher concentrations of the antibiotic and onto media with a second antibiotic at the lower concentration. The resistant organisms were finally transferred to media containing two antibiotics at the required concentrations.: streptomycin (Sm), 750 ugml"1; kanarnycin (Km), 100 or 200 ugml"1 and chloramphenicol (Cm) 100 u g m l .

The second approach was used to isolate strains of Pseudomonas aeruginosa strain PA01162 and P. putida strain KT 2440 (kindly supplied

by Prof. K. Timmis, Max-Planck Institute, Berlin) which were resistant either to Km (200 ugml’1) and Cm (100 ugml’1) or Km (200 ugml"1) and ampicillin (Ap) (50 ugml’1) respectively.

Both strains were inoculated into nutrient broth and incubated at 30°C overnight. Samples (1.0 ml) were transferred into fresh nutrient

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broth and incubated for 2 to 3 h to initiate growth, after which the antibiotics were added at half the required concentrations. After overnight incubation samples (0.1 ml) were spread onto nutrient agar plates containing both pairs of antibiotics at the required concentrations and incubated for 48 h at 30°C. The resistant organisms were cultured and maintained on fresh antibiotic containing media.

The resistant strains were used as recipients in mating experiments and were selected for by the double resistances each possessed.

2 . 7 . 2 . 2 Method f o r membrane mating experim ents

Overnight cultures (50 ml) (Section 2.1.1) of donor and recipient organisms were grown under selective conditions. For donor cultures the selective pressure was the presence of the halogenated substrated as the sole carbon source for the soil isolates and the presence of Km (100 ugml’^) in a nutrient broth culture medium for P. aeruginosa

PA08R which carried the resistance factor R68-45 (supplied by B.W. Holloway, Monash, Australia). The recipient organisms were cultured in nutrient broth with the two relevant antibiotics present at half the final concentration used to maintain the resistant organisms.

The recipient organisms were washed and resuspended in Tris-HCl buffer (20 mM), pH 7.0 by centrifugation at 5,000g for 10 min. Given volumes, to ensure more recipient than donor cells were present, of the donor and recipient cultures were mixed in a Gallenkamp filter and filtered onto 0.45p membranes (Sartorius Instruments Ltd., Sutton Surrey). The filters were transferred aseptically onto nutrient agar and incubated overnight at 30°C. Samples (1 ml) of the two parental cultures were serially diluted in Tris-HCl buffer (20 mM), pH 7.0 and

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broth and incubated for 2 to 3 h to initiate growth, after which the antibiotics were added at half the required concentrations. After overnight incubation samples (0.1 ml) were spread onto nutrient agar plates containing both pairs of antibiotics at the required concentrations and incubated for 48 h at 30°C. The resistant organisms were cultured and maintained on fresh antibiotic containing media.

The resistant strains were used as recipients in mating experiments and were selected for by the double resistances each possessed.

2. 7.2.2 Method fo r membrane mating experiments

Overnight cultures (50 ml) (Section 2.1.1) of donor and recipient organisms were grown under selective conditions. For donor cultures the selective pressure was the presence of the halogenated substrated as the sole carbon source for the soil isolates and the presence of Km (100 ygml"1) in a nutrient broth culture medium for P. aeruginosa

PA08R which carried the resistance factor R68-45 (supplied by B.W. Holloway, Monash, Australia). The recipient organisms were cultured in nutrient broth with the two relevant antibiotics present at half the final concentration used to maintain the resistant organisms.

The recipient organisms were washed and resuspended in Tris-HCl buffer (20 mM), pH 7.0 by centrifugation at 5,000g for 10 min. Given volumes, to ensure more recipient than donor cells were present, of the donor and recipient cultures were mixed in a Gallenkamp filter and filtered onto 0.45p membranes (Sartorius Instruments Ltd., Sutton Surrey). The filters were transferred aseptically onto nutrient agar and incubated overnight at 30°C. Samples (1 ml) of the two parental cultures were serially diluted in Tris-HCl buffer (20 mM), pH 7.0 and

spread onto succinate-defined medium Tetri dishes (Section 2.1.2) to ascertain the numbers of organisms used in the mating experiment. Colony counts were made after 24 to 48 h incubation.

After overnight incubation the bacteria on the membranes were suspended in sterile Tris-HCl buffer (20 mM), pH 7.0 (3 ml), by whirlimixing, and samples (0.1 ml) spread onto the selection plates and incubated at 30°C for 7 to 14 d. These plates contained 2MCPA as the carbon source, which selected against the recipients, and the two antibiotics to which the recipients were resistant, to select against the donor organisms. Any bacteria capable of growth on those media could only be transconjugants.

Such organisms were purified by dilution streaking onto fresh selection plates, after which they were transferred onto Kings' A plates (King e t a l . , 1954), if the recipient was a P. aeruginosa

strain, and screened for plasmids as described in Section 2.6.2.

2 .7 .2 ,3 . Membrane mating experim ents

The membrane mating experiments attempted are suranarised in Table 2.1.