Optimization of procedures for isolation of mycobacteria from soil and water samples obtained in northern India

For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.     

Mycobacteria in water and loose deposits of drinking water distribution systems in Finland

Drinking water distribution systems were analyzed for viable counts of mycobacteria by sampling water from waterworks and in different parts of the systems. In addition, loose deposits collected during mechanical cleaning of the main pipelines were similarly analyzed. The study covered 16 systems at eight localities in Finland. In an experimental study, mycobacterial colonization of biofilms on polyvinyl chloride tubes in a system was studied. The isolation frequency of mycobacteria increased from 35% at the waterworks to 80% in the system, and the number of mycobacteria in the positive samples increased from 15 to 140 CFU/liter, respectively. Mycobacteria were isolated from all 11 deposits with an accumulation time of tens of years and from all 4 deposits which had accumulated during a 1-year follow-up time. The numbers of mycobacteria were high in both old and young deposits (medians, 1.8 x 10(5) and 3.9 x 10(5) CFU/g [dry weight], respectively). Both water and deposit samples yielded the highest numbers of mycobacteria in the systems using surface water and applying ozonation as an intermediate treatment or posttreatment. The number and growth of mycobacteria in system waters correlated strongly with the concentration of assimilable organic carbon in the water leaving the waterworks. The densities of mycobacteria in the developing biofilms were highest at the distal sites of the systems. Over 90% of the mycobacteria isolated from water and deposits belonged to Mycobacterium lentiflavum, M. tusciae, M. gordonae, and a previously unclassified group of mycobacteria. Our results indicate that drinking water systems may be a source for recently discovered new mycobacterial species.     

Inclusion of geothermal water in composition of nutrient medium for mycobacteria cultivation

New effective nutrient medium for isolation and cultivation of mycobacteria was developed. Time of their growth on the developed medium decreased by 3 to 11 days compared with control media (Finn II, Levenstein-Jensen). Rate of mycobacteria isolation increased by 20 to 40% compared with control media.     

Effect of bovine serum albumin on the isolation of boar spermatozoa and their fertility

A procedure for isolation of subpopulations of highly motile spermatozoa from ejaculates of several mammalian species was modified for use with boar semen. An initial trial was conducted to test the effectiveness of concentrations of 3 to 15% Bovine Serum Albumin (BSA) as an isolation medium. A concentration of 15% BSA resulted in isolation of a population of boar spermatozoa with 70% motility compared with an estimate of 53.5% determined after collection. Although there was a variable effect of isolation on the rate of forward movement, sperm cell concentration decreased (P<.05) as BSA level increased.To test the fertilizing capacity of isolated boar spermatozoa, 16 sows were randomly alloted to either a treated group bred with isolated spermatozoa (12% BSA) or control group bred with raw semen. Both the percent motile spermatozoa (64.1%) and the rate of forward movement (2.9) were increased (P<.01) after isolation in BSA compared with initial values obtained at collection (51.4% and 2.3). Farrowing rates (71.4% and 75% for treated and control groups, respectively) were not significantly different, and the sex ratio of the offspring was not altered by the procedure.     

Pollen Cryopreservation and Pollen Protoplast Isolation in Brassica carnpestris var.purpurea

The authors have investigated the factors affecting pollen cryopreservation in Brassica campestris var. purpurea, such as pollen development stages cryoprotectant and the process of freezing. A suitable procedure was established as follows :Pollen grains suspended in B5 medium containing 10X DMSO and 1SM sucrose were frozen by a three- –1℃/min – 1 ℃/min step method(0℃———→–10 ℃ ,standing for 15 min———→–40 ℃ , standing for 1 hr→liquid nitrogen)and later thawed in 40℃ water bath. During a period of 60, 90 days′preservation, the relative survival percentage of mature (at the day of anthesis)and nearly mature(2 days before anthesis, trinucleate stage)pollens maintained at ca. 91% that of young pollens(7-8 days before anthesis, late uninucleate stage to early binucleate stage)slightly declined from the original 91.6% to 84. 3%. Culture. experiment showed that the cryopreserved young pollen could be induced to cell division just as well as the fresh pollen. The method of isolating protoplasts from fresh mature pollen developed previously was improved and simplified. As a result, protoplasts were isolated more conveniently from mature pollen and young pollen for the first time. The protoplasts from cryopreserved mature and young pollen could be obtained as well with an isolation rate of 77.4% and 35.9% respectively. However, for isolation of protoplasts from preserved young pollen, an incubation in NLN medium at 35℃ after thawing was necessary.     

A novel method for the isolation of mycobacterial DNA

Abstract DNA was isolated from mycobacteria by a simplified procedure. Cells were suspended in 6 M guanidinium chloride, the suspension was cooled to −70 °C, then incubated at 65 °C for 10 min, cooled in ice, deproteinized by chloroform and DNA was recovered from the supernatant. The procedure was used to obtain DNA from several mycobacteria (1 × 10⁹) or more cells) including Mycobacterium neoaurum M. fortuitum M. phlei and M. smegmatis . Each of the species was shown to have two ribosomal RNA operons per genome, and preliminary evidence was obtained which suggests that one of these operons is homologous with one of the operons of M. smegmatis .     

Rapid enumeration of respiratory active mycobacteria with fluorescent double staining

A new method for rapid enumeration of physiologically active mycobacteria was developed by acid-fast bacilli staining (Auramine O) following formazan reduction. Results can be obtained within 90 min by the optimized procedure, while more than one week is required for the widely used culture-dependent approach.     

Prostacyclin and prostaglandin E2 secretions by bovine pulmonary microvessel endothelial cells are altered by changes in culture conditions

The isolation and culture of pulmonary microvascular endothelial (MVE) cells from bovine lungs were established. Primary and early passaged cultures grew best in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% equine plasma-derived serum, bovine retinal growth extract (1%), and heparin (90 micrograms/ml) on gelatin coated plates. A second tissue culture procedure was prepared in which the isolation technique was the same except the culture medium consisted of DMEM supplemented with 10% plasma-derived serum. Either growth medium produced homogeneous, long term, serial cultures for up to 16 passages. MVE cells were characterized in part based on their morphology by light and electron microscopy and positive reaction to Factor VIII-related antigen and uptake of 1,1'-dioctacecyl-1,3,3,3'3-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein (Dil-Ac-LDL). MVE cells were also positive for angiotensin-converting enzyme (ACE) activity and the presence of ACE was localized on the cells by indirect immunofluorescence. MVE cells maintained in the presence of heparin and growth factor principally synthesized prostaglandin (PG) E2 (1512 +/- 159 pg/mg protein at 15 min) and smaller amounts of prostacyclin (PGI2) and thromboxane (Tx) A2 (316 +/- 43 and 588 +/- 105 pg/mg protein/15 min respectively) as measured by radioimmunoassay. However, prostanoid release was not elevated from basal levels upon incubation with arachidonic acid, bradykinin, or ionophore A23187. In contrast, MVE cells cultured without heparin and growth factor secreted more PGI2 than PGE2 (862 +/- 84 and 89 +/- 12 respectively). Incubation with arachidonic acid, bradykinin, or ionophore A23187 induced significant increases in PGI2 and PGE2 production (P less than 0.01). Pulmonary artery endothelial (PAE) cell cultures used as a control for comparison predominantly synthesized PGI2. These findings suggest that in vitro the vessel source and culture conditions may qualitatively and quantitatively affect the pattern and levels of prostanoid synthesized and secreted.     

Direct Comparison of the N-Acetyl-L-Cysteine-Sodium Hydroxide and the Trisodium Phosphate Digestion Methods for the Culture of Mycobacteria

Comparison of sputum digestion procedures utilizing acetylcysteine-sodium hydroxide (AC) and trisodium phosphate for the isolation and culture of mycobacteria indicated that the AC procedure provides faster growth, thus permitting the earlier detection of positives. Also, in untreated patients and those in whom treatment has been recently instituted, the number of positives was slightly larger with the use of the AC procedure. However, of greater interest was finding that the AC procedure provided a larger quantity of positive cultures with sputum from subjects who had been intensively treated with antimicrobials and other drugs. Contamination was higher with the AC procedure, but when the sputa were diluted 1:10 this interference due to contamination was decreased.     

Mycobacterial infection in farmed turbot Scophthalmus maximus

Mycobacteriosis (piscine tuberculosis) has been reported to affect a wide range of freshwater and marine fish species; however, this is the first report describing mycobacterial infections in turbot Scophthalmus maximus. High numbers of granulomas were initially observed in the organs of moribund farmed turbot. Bacteriological analysis of organs with granulomas led to the isolation of Mycobacterium marinum. Further analysis, to determine the prevalence of the infection in the farm and to identify its source, showed the occurrence of a dual infection by M. marinum and M. chelonae. The presence of Nocardia sp. in some of the fish infected with mycobacteria was also detected. The presence of granulomas in internal organs of apparently healthy fish indicated a high prevalence of the disease, a conclusion that was supported by isolating mycobacteria from all fish with or without granulomas. The infection was probably responsible for the mortality observed (approximately 2% mo(-1)), as most of the recently dead fish presented high numbers of granulomas and isolation of mycobacteria was possible from all of the fish. The isolation of M. marinum from the inlet water suggested this as the most plausible source for the infection occurring in the farm.     

Quantitation of lysophosphatidylcholine molecular species in rat cardiac tissue.

We have developed a rapid and sensitive procedure for isolation and measurement of 1-acyllysophosphatidylcholine (LPC) species in rat myocardial tissue. Tissues were spiked with heptadecanoyl-LPC internal standard and extracted with chloroform/methanol. The chloroform phase was dried, resuspended in chloroform/propan-2-ol (2/1, v/v), and applied to an aminopropyl-bonded phase (Bond Elut) column. Following stepwise elution with several solvent mixtures, the LPC fraction (ethyl acetate/methanol, 4/6, v/v) was separated by HPLC with direct quantitation of palmitoyl-LPC (P-LPC), oleoyl-LPC (O-LPC), and stearoyl-LPC (S-LPC), using an evaporative light scattering mass detector. Calibration curves were generated for each individual LPC species. Recoveries of added [14C]LPC and of heptadecanoyl-LPC internal standard after extraction and chromatography were 85.8 +/- 1.9% (mean +/- SE, N = 10) and 83.4 +/- 1.8% (N = 15), respectively. This assay showed satisfactory sensitivity, reproducibility, and accuracy for measurement of LPC species in rat myocardial tissue. The major molecular species of LPC in rat myocardium were found to be P-LPC and S-LPC, which were two- to sixfold as abundant as O-LPC. In isolated, crystalloid-perfused rat hearts the time of perfusion was found to significantly influence the content of P-LPC (0 min, 252 +/- 10; 15 min, 178 +/- 10, P less than 0.001, compared with 0 min; 40 min, 131 +/- 4, P less than 0.001; and 70 min, 129 +/- 4, P less than 0.001; nmol/g dry weight), but not the content of O-LPC and S-LPC. The method will be useful for studying the participation of LPC species in physiology, pathophysiology, and therapeutics.     

Effect of some biocides on non-tuberculous mycobacteria

The incidence of disease and nosocomial infections produced by non tuberculosis mycobacteria (NTM) has increased in immunocompetent patients, but also and more frequently, in immunosuppressed patients. Several studies have disclosed that mycobacteria are more resistant to biocides than non-sporulating bacteria; in addition, some species are particularly resistant. The biocide action of sodium hypochloride and glutaraldehyde on Mycobacterium aviumintracellulare, Mycobacterium gordonae, Mycobacterium fortuitum and Mycobacterium chelonae was studied, using a modified Kelsey Maurer test. For the different species, both the minimal inhibitory concentration (MIC) and the minimal action time were determined. Effectiveness of sodium hypochloride and glutaraldehyde against the different mycobacterial species varied. The same was true for different isolates of the same species. Sodium hypochloride effective MIC and exposure time (killing of 99.99% of all NTM) were 0.2% and 5 minutes, respectively. In order to achieve 100% killing, 0.5% MIC and 15 minute exposure were needed. In the case of glutaraldehyde, 99.99% of the bacteria were killed with 1% MIC and a 15 minute exposure. An effectiveness of 100% was achieved with a 2% MIC of glutaraldehyde and a 15 minute exposure. Sodium hypochloride and glutaraldehyde are effective biocides for mycobacteria. The first biocide is cheap and effective at low concentrations, but its corrosive and oxidant nature makes it impossible for use in hospitals or with laboratory equipment. Glutaraldehyde (neither corrosive nor oxidant) is a safe alternative for disinfection of this type of equipment. However, it is important to bear in mind that these pathogens may develop resistance to biocides.     

A novel small antifungal peptide from Bacillus strain B-TL2 isolated from tobacco stems

A novel small antifungal peptide produced by a Bacillus strain B-TL2 isolated from tobacco stems was purified. The purification procedure consisted of ammonium sulfate precipitation, cation exchange chromatography on CM-Sepharose Fast Flow column and reverse-phase HPLC on SOURCE 5RPC column. After the final isolation step, one peptide with antifungal activity, designated as BTL, was obtained. The molecular mass of the purified BTL was determined as 2500 Da and 2237.7 Da by SDS-PAGE and matrix-assisted laser desorption/ionization time of flight mass spectrometry, respectively. The N-amino acid sequence of BTL was determined to be NH(2)-KQQLATEAESAGPIL, which shows relatively low identity to other antimicrobial peptides from bacteria. The peptide exhibited strong inhibitory activity against mycelial growth of Bipolaris maydis, Alternaria brassicae, Aspergillus niger, Cercospora personata. The purified BTL displayed thermostability, almost retaining 100% activity at 100 degrees C for 15 min.     

Comparison of the conventional culture,the manual fluorescent MGIT system and the automated fluorescent MGIT 960 culture system for the detection of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> ssp. <Emphasis Type="Italic">avium</Emphasis> in tissues of naturally infected hens

Different methods for the detection of Mycobacterium avium ssp. avium (MAA) in naturally infected hens were compared. They included the conventional culture method (solid Herrold’s and Stonebrink media and liquid Sula medium) and newly developed liquid culture systems, the manual mycobacteria growth indicator tube (M-MGIT) and the fully automated BACTEC MGIT 960 system (A-MGIT). 152 tissues originating from 15 naturally infected hens have been processed. The overall detection rates (percentage of positive cultures from the number of positive cultures determined by all the methods together) were 60, 70 and 76 % for the conventional media, M-MGIT and A-MGIT systems, respectively, the mean time of mycobacteria detection being 32.6, 17.6 and 14.6 d, respectively. The lowest contamination rate (2.0 %) was found in A-MGIT compared with M-MGIT (4.6 %) and conventional media (10.4 %).     

Comparison of NaOH-N-acetyl cysteine and sulfuric acid decontamination methods for recovery of mycobacteria from clinical specimens

We compared the NaOH-N-acetyl cysteine (NaOH-NALC) and the sulfuric acid decontamination procedure in the detection of mycobacteria using the Mycobacteria Growth Indicator Tube (MGIT). In total 219 sputum specimens were collected from 142 Zambian patients and subjected to mycobacterial culture. One half of the specimen was decontaminated with NaOH-NALC and the other half was decontaminated with sulfuric acid. From the 438 samples a total of 261 (60%) cultures yielded growth of mycobacteria, consisting of 22 different species. The sulfuric acid method was more successful than the NaOH-NALC method in recovering mycobacteria in MGITs (146 versus 115 respectively, p = 0.001). Of the 146 positive mycobacterial cultures recovered after sulfuric acid decontamination 28 were Mycobacterium tuberculosis, 84 nontuberculous mycobacteria (NTM) and 34 acid fast bacterial isolates which could not be identified to the species level. The 115 mycobacteria recovered by the NaOH-NALC method consisted of 34 M. tuberculosis strains, 55 NTM and 26 acid fast bacteria that could not be identified. The most frequently isolated NTM were Mycobacterium lentiflavum and Mycobacterium intracellulare. Comparing the two decontamination methods the recovery of NTM in the sulfuric acid group was significant higher than in the NaOH-NALC group (p = 0.001). In contrast, no significant difference was found for the recovery of M. tuberculosis. These results show that the decontamination method used affects the recovery of nontuberculous mycobacteria in particular.     

A novel procedure for the isolation of glycolipids from Bifidobacterium adolescentis 94 BIM using supercritical carbon dioxide

This study describes a novel isolation procedure for major glycolipids from Bifidobacterium adolescentis 94 BIM. The procedure consists of the use of supercritical carbon dioxide (scCO(2)) with hydro-methanolic solution as co-solvent. The major glycolipids were isolated using the following operating conditions: pressure, 30 MPa, co-solvent concentration, 10% (9:1, methanol/water, v/v), CO(2) flow rate, 5 g/min, extraction time and temperature, 2h and 55 degrees C, respectively. The reference glycolipids sample was prepared by classical organic solvent extraction followed by chromatographic purification. All isolates were characterized by TLC and the major glycolipids additionally by enzyme linked immunosorbent (ELISA). Sixty milligrams of glycolipids with similar immunoreactivity as the reference glycolipids were isolated from 1g of freeze-dried biomass (6% of yield).     

Comparison of some decontamination methods and growth media for isolation of mycobacteria from northern brook waters

Two decontamination methods and five media were compared for the isolation of mycobacteria from brook waters of different physical, chemical and bacteriological characteristics. The decontaminants used were: 0.7 mol 1^-1 NaOH followed by 50 g 1^-1 oxalic acid and 0.9 mol 1^-1 H₂SO₄ combined with 0.5 g 1^-1 cycloheximide. The media compared were: Mycobacteria 7H11 agar with OADC enrichment (pH 6.6), glycerol egg (pH 6.5 and 5.5), and pyruvate egg (pH 6.5 and 5.5). All media contained cycloheximide, 0.5 g 1^-1. The NaOH—oxalic acid method generally resulted in lower contamination and higher isolation of mycobacteria than the H₂SO₄-cycloheximide method. With the NaOH—oxalic acid method, all five media were equal in positivity rates but contamination was a problem on Mycobacteria 7H11 agar. Of the four egg media tested, the highest positivity rate (92% of the samples) was obtained on the pyruvate modification (pH 6.5), and the highest mean colony count of mycobacteria (900 cfu 1^-1) on the glycerol modification (pH 6.5). Characteristics of water and sampling site had similar effects on the isolation frequencies of mycobacteria obtained by different combinations.     

A method for simultaneous isolation and cryopreservation of bovine lymphocytes

A technique for the simultaneous isolation and cryopreservation of bovine lymphocytes was presented. Whole blood was slowly diluted 1:2 with RPMI-Hepes containing 15% Me₂SO to yield final concentrations of 50% whole blood and 7.5% Me₂SO. Aliquots were cooled to at least ?80 °C at a rate of 2.5 °C/min and subsequently immersed in liquid nitrogen for storage. Samples were thawed rapidly by agitation in a 37 °C water bath and diluted rapidly with warm RPMI-Hepes. After centrifugation, the lymphocyte pellet was washed and suspended in medium for cell identification and lymphocyte stimulation assays. Few red blood cells, granulocytes, and monocytes survived this freezing and thawing procedure. Recovery of lymphocytes was 69–75%, as compared to a recovery of 58–68% using isolation on density gradients. Only small differences in the numbers of lymphocytes that (i) form spontaneous rosettes with sheep red blood cells and (ii) bind anti-IgG were found between the two isolation procedures. Cell proliferation in response to PPD-B or PHA and the enhancement of amino acid transport in response to PPD-B were the same for lymphocytes isolated by both methods.