Improved method of lipopolysaccharide isolation from gram-negative bacteria

A modification of the traditional method for lipoplysaccharide isolation from the cells of grammnegative bacteria was elaborated on the basis of extraction by the hot water solution of phenol (the method of Westfahl). To make the method simpler and to raise the yield of the product it was proposed to use the water-phenol extract without its division for plases. The nucleic acids are eliminated by precipitation from dialyzed extract at pH 3,2-3,4 achieved by addition of acetic acid. The comparative isolation of lipopolysaccharides by the classic and modified methods has confirmed the advantages of a new technique.     

A rapid method for the isolation of eicosapentaenoic acid-producing marine bacteria

Bacterial production of long chain polyunsaturated fatty acids (LC-PUFAs) is a promising biotechnological approach for the mass production of these valuable compounds, but extensive screening is currently needed to select a strain that meets industrial requirements.A method was developed for the rapid screening and isolation of eicosapentaenoic acid (EPA)-producing marine bacteria from mixed cultures using the dye 2,3,5-triphenyltetrazolium chloride (TTC). The method was first validated using two bacteria from the Shewanella genus, S. gelidimarina (known to contain EPA) and S. fidelis (known not to contain EPA), and subsequently applied to a range of bacterial samples collected from seven randomly selected New Zealand fish species.By incorporating TTC in both solid and liquid state fermentation treatments, a clear association between the reduction of TTC to the red-coloured triphenyl formazan (TF) and the presence of EPA within Gram-negative bacteria was confirmed. Incubation in 0.1% w/v TTC was optimal for colour response and cell growth in agar plates and liquid cultures. Bacteria that produce EPA reduced TTC to TF, but a number of non-EPA-producing bacteria also showed this capacity. By conducting a subsequent Gram staining, all EPA-producing strains were revealed to be G (−) rod bacteria while the non-producing ones were all G (+) cocci. The fatty acid methyl esters of the isolated bacteria that reduced TTC to TF were analysed using gas chromatography-mass spectrometry and the content of EPA was confirmed by gas chromatography.From a pool of 2.0 × 10⁸ CFU/ml, this method allowed the rapid isolation of 16 bacteria capable of producing EPA. This new approach significantly reduces the number of samples submitted for GC analysis and therefore the time, effort and cost of screening and isolating strains of EPA-producing marine bacteria.     

Paper replication method for isolation of radiation-sensitive mutants.

A filter paper replication system particularly useful for isolation of radiation-sensitive mutants of pigmented bacteria was devised. The fidelity of replication was high. Adhesion between a paper disk and a properly dried master plate provided adequate contact pressure. The replicas arising from this technique constitute a convenient apparatus for general application in isolation of clones sensitive to a discriminating treatment.     

Directed isolation of gram-negative asporogenous bacteria from natural substrates

A method for selective isolation of gramnegative nonsporulating bacteria from soil substrates was developed. The method consists of plating out the substrates on a glucose-yeast medium with addition of benzylpenicillin and nalidixic acid. The isolation frequency of gramnegative nonsporulating bacteria increased under such conditions from 9-16 (control) to 80-100 per cent. At the same time the isolation frequency of gram-positive bacteria decreased from 88.5 to 9.6 per cent.     

Rapid method for distinction of gram-negative from gram-positive bacteria

Summary A rapid method for distinction between gram-negative and grampositive bacteria by means of a 3% solution of potassium hydroxide is tested on 71 gram-positive and 55 gram-negative bacterial strains. The method proved reliable with one exception only, a Bacillus macerans strain. That strain was definately gram-negative on staining. Other Bacillus strains were proved gram-positive by the test, even those being gram-negative on staining.     

Paper replication method for isolation of radiation-sensitive mutants

A filter paper replication system particularly useful for isolation of radiation-sensitive mutants of pigmented bacteria was devised. The fidelity of replication was high. Adhesion between a paper disk and a properly dried master plate provided adequate contact pressure. The replicas arising from this technique constitute a convenient apparatus for general application in isolation of clones sensitive to a discriminating treatment.     

Simple screening method for isolation of penicillin acylase-producing bacteria.

A new screening method for bacteria capable of producing penicillin acylase is described. The method is based on the use of Serratia marcescens sensitive to 6-aminopenicillanic acid but comparatively resistant to benzylpenicillin. It is simple, quite specific, and requires no special equipment. It can also be used to screen for phenoxymethylpenicillin acylase activity. We also suggest an acidimetric method for rapid detection of cloned genes in genetic engineering studies of penicillin acylase.     

A rapid procedure for the isolation of plasmid DNA from environmental bacteria.

The INSTA-MINI-PREP method, a rapid protocol for plasmid DNA extraction, was originally developed to prepare plasmid DNA from 1 to 3 ml miniprep Escherichia coli cultures. Direct extraction of plasmid DNA is achieved by a two-phase solution which is separated by centrifugation in the presence of the INSTA-PREP gel barrier material. This method has been successfully tested on various environmental Salmonella strains, although it was not suitable for Pseudomonas aeruginosa and enterococci strains. The INSTA-MINI-PREP method is a new alternative procedure to screen plasmid contents of Salmonella and E. coli strains rapidly and easily.     

A method for the isolation of asporogenic mutants of Bacillus thuringiensis.

Asporogenic and oligosporogenic mutants of Bacillus thuringiensis can be isolated by streaking the bacteria onto a solid medium, incubating at 42 degrees C, and picking small, raised areas of growth which appear on the streaks after 48 h.     

Simple and rapid method for the isolation of Aspergillus niger mutants with enhanced cellulase and xylanase activity

Summary An agar plate isolation technique was developed for screening the mutants of Aspergillus niger. Fungal growth in solid media containing carboxymethylcellulose(CMC) was selected within 2–3 days. The mutants selected by the new technique showed a remarkable enhancement of CMCase, FPase, -glucosidase, xylanase, and -xylosidase activities. These results clearly show that the new isolation technique is a practical method for the selection of better strains.     

Plaque color method for rapid isolation of novel recA mutants of Escherichia coli K-12: new classes of protease-constitutive recA mutants

As a prerequisite to mutational analysis of functional sites on the RecA protein of Escherichia coli, a method was developed for rapid isolation of recA mutants with altered RecA protease function. The method involves plating mutagenized lambda recA+ cI ind on strains deleted for recA and containing, as indicators of RecA protease activity, Mu d(Ap lac) fusions in RecA-inducible genes. The lambda recA phages were recognized by their altered plaque colors, and the RecA protease activity of the lambda recA mutant lysogens was measured by expression of beta-galactosidase from dinD::lac. One class of recA mutants had constitutive protease activity and was designated Prtc; in these cells the RecA protein was always in the protease form without the usual need for DNA damage to activate it. Some Prtc mutants were recombinase negative and were designated Prtc Rec-. Another class of 65 recA mutants isolated as being protease defective were all also recombinase defective. Unlike the original temperature-dependent Prtc Rec+ mutant (recA441), the new Prtc Rec+ mutants showed constitutive protease activity at any growth temperature, with some having considerably greater activity than the recA441 strain. Study of these strong Prtc Rec+ mutants revealed a new SOS phenomenon, increased permeability to drugs. Use of this new SOS phenomenon as an index of protease strength clearly distinguished 5 Prtc mutants as the strongest among 150. These five strongest Prtc mutants showed the greatest increase in spontaneous mutation frequency and were not inhibited by cytidine plus guanosine, which inhibited the constitutive protease activity of the recA441 strain and of all the other new Prtc mutants. Strong Prtc Rec+ mutants were more UV resistant than recA+ strains and showed indications of having RecA proteins whose specific activity of recombinase function was higher than that of wild-type RecA. A Prt+ Rec- mutant with an anomalous response to effectors is described.     

An improved method for rapid isolation of plasmid DNA from wild-type Gram-negative bacteria for plasmid restriction profile analysis

An improved method for the isolation of large and small plasmids from wild-type Gram-negative bacteria has been developed. The protocol combines the lysis and purification procedures of two popular plasmid isolation methods, and produces DNA sufficiently pure for restriction enzyme digestion in less than three hours.     

Simple and rapid method for disruption of bacteria for protein studies.

A simple and rapid method was developed for the extraction of proteins from both pathogenic and nonpathogenic bacteria. The method involves the treatment of cells with acetone followed by sodium dodecyl sulfate extraction of cellular proteins. Polyacrylamide gel electrophoresis revealed that the protein composition of extracts made by this method was comparable to that of extracts made by established methods, namely, sonication and agitation with beads. This technique has been successfully applied to the extraction of proteins from a wide variety of bacteria, including pathogens.     

A rapid column chromatographic method for the isolation of catechol-type siderophores.

The ester groups of glycerophospholipids in tissue extracts can be cleaved in less than 10 min at room temperature if the lipids are extracted with hexane-isopropanol and the filtrate is treated with methanolic NaOH. The resulting mixture can be treated with aqueous Na-sulfate containing sulfuric acid and partitioned to remove the inorganic reagents and hydrophilic ester degradation products. When the procedure is applied to brain lipid extracts, the addition of alkali produces a second, lower phase that contains much of the hydroxycerebroside, virtually all of the sulfatide in the extract, and small amounts of other lipids. The sulfatide can be isolated from the lower phase by neutralizing it with HCl in aqueous methanol, and partitioning with chloroform to remove nonlipid components. The lower phase is evaporated to dryness and treated with periodic acid to convert the cerebroside to a less polar product. The lipids recovered from the reaction mixture are then fractionated with a Florisil column, which yields highly purified sulfatide. Starting with 300 g of pig brain one can obtain about 1.1 g of sulfatide in 4 working days, using conventional, compact equipment. Since the precipitation step is almost complete, and the procedure can be scaled down to very low levels, the method has promise for quantitation methods and isotopic studies of sulfatide metabolism.