Selective isolation of mycobacteria from soil: a statistical analysis approach

We compared four decontamination methods for the isolation of mycobacteria from soil specimens. Different media were used: L?wenstein-Jensen, Ogawa and various modified Ogawa media. Statistical analysis demonstrated that the best results (low contamination and high positivity rates) were obtained when the specimens were incubated in trypticase soy broth, treated with solutions containing malachite green and cycloheximide, then decontaminated with sodium hydroxide and inoculated onto Ogawa media. The lowest contamination rates were obtained with Ogawa medium containing 500 micrograms cycloheximide ml-1. The use of these techniques is proposed for the isolation of mycobacteria from heavily contaminated clinical specimens as well as from soil.     

Selective medium for the isolation and enumeration of Mycobacterium avium-intracellulare and M. scrofulaceum

A medium for the selective isolation and enumeration of Mycobacterium avium-intracellulare and M. scrofulaceum (MAIS) was developed, based upon the ability of these mycobacteria to utilize Tween 80 as sole carbon source and grow optimally at pH 5.5 on a simple mineral salts medium. Representative MAIS strains had higher efficiencies of plating on the Tween 80 medium compared with Middlebrook 7H10. It was shown that nonmycobacterial organisms in natural waters had lower efficiencies of plating on the Tween 80 medium and smaller colonies, thus allowing direct isolation and enumeration of the slowly growing mycobacteria without overgrowth.     

Direct Cord Reading Medium for Isolation of Mycobacteria

A study of growth and colony morphology of mycobacteria was performed on 7H10 medium and compared with the same medium to which WR 1339 was added. Collection strains of human, bovine, and atypical mycobacteria, in addition to 1,199 sputa, were planted on both media. The results showed that WR 1339 modifies the growth and colony morphology of human, bovine, atypical group I, and, to a lesser degree, atypical groups II, III, and IV mycobacteria. The great majority of human and bovine strains exhibit definite cords when examined at a magnification of 100 times. The colonies are larger when WR 1339 is added, especially if there is a small number of colonies. The addition of WR 1339 spreads the growth on the surface of the agar, producing a thin but larger colony as compared with 7H10 medium alone on which the colonies grow more vertically, thus producing thick but smaller colonies. WR 1339 spreads the growth, producing thin and transparent colonies where the cords are oriented in uniplane and easily visible directly on the isolation media. Half of the positive sputum cultures were easily identified at 12 days. The presence of typical cords permits a quick screening diagnosis of species directly on the isolation media and, in most instances, elimination of the possibility of atypical or contaminating colonies.     

Isolation of mycobacteria from acidic forest soil samples: comparison of culture methods

To evaluate which combination of decontamination method and medium is most reliable when examining acidic, organic forest soils for mycobacteria, three decontamination methods and five media supplemented with cycloheximide were compared. Before decontamination, the samples were incubated at 37°C for 5 h to allow germination of microbial spores. The recovery of mycobacteria was significantly influenced both by the method and by medium. Decontamination with NaOH or H₂SO₄ both combined with malachite green and cycloheximide yielded higher viable counts of mycobacteria than decontamination with NaOH followed by oxalic acid. Egg media at pH 5·5 resulted in lower mycobacterial counts than egg media at pH 6·5 or Mycobacteria 7H11 agar. The numbers of slopes totally free of contaminants revealed Mycobacteria 7H11 agar medium to be more prone to contamination than the four egg media tested. The highest counts of mycobacteria and a low rate of contamination were obtained when decontamination with NaOH-malachite green–cycloheximide was combined with culture on glycerol and cycloheximide supplemented egg medium at pH 6·5.     

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.     

Development and diagnostic value evaluation of a nutrient medium for the yield and isolation of hemoculture

The effectiveness of the bacteriological study of the blood depends in many aspects on the quality of nutrient media. The currently approved nutrient media for the isolation and yield of hemocultures have low isolation rates and require prolonged time for the isolation of causative agents. A two-phase nutrient medium for the yield and isolation of hemoculture has been developed. This medium has been found to have advantages over the control "double medium" in sensitivity, yield effect, growth rate and isolation rate with respect to the causative agents of bacteriemia and sepsis. The medium is standard and ready for use. The above-mentioned advantages of the medium increase the quality of the bacteriological diagnosis of bacteriemia and sepsis.     

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 %).     

A comparison of thiosulphate-citrate-bile salts-sucrose (TCBS) agar and thiosulphate-chloride-iodide (TCI) agar for the isolation of Vibrio species from estuarine environments

AIMS: The purpose of this study was to compare a recently described medium, thiosulphate-chloride-iodide (TCI), for the isolation of estuarine vibrios with thiosulphate-citrate-bile salts-sucrose (TCBS). METHODS: A total of 492 colonies which developed on these media from estuarine water samples taken monthly over a 10-month period were examined. RESULTS: A much larger number of colonies developed on TCBS than TCI, and minimal taxonomic criteria indicated that a higher percentage (61%) of TCBS colonies could be identified as Vibrio spp. when compared with TCI (46%). SIGNIFICANCE: This study suggests that TCBS is a superior medium when compared with TCI for the isolation of Vibrio spp. from estuarine waters. Because of the public health risk presented by V. vulnificus, V. parahaemolyticus, V. cholerae and other vibrios, the selection of the most appropriate medium for their isolation is extremely important.     

分枝杆菌全合成琼脂培养基的研究

本文报道了一种适合常见致病分枝杆菌生长的全合成固体琼脂培养基体系。它为实验室研究分枝杆菌的营养生理、生化特性、遗传变异、药理和耐药机制提供了一种新工具,也为研制更加满意的分枝杆菌培养基创造了条件。试验过的大多数非典型分枝杆菌,在全合成琼脂培养基上比在罗氏培养基上生长快,生长量少于或等于罗氏培养基。标准牛型株的细胞群体中,仅有少量细胞能在全合成琼脂培养基上生长。鸟型．胞内和偶发等分枝杆菌标准株在全合成琼脂培养基上产生两种不同的菌落。     

Comparison of methods for processing drinking water samples for the isolation of Mycobacterium avium and Mycobacterium intracellulare

Several protocols for isolation of mycobacteria from water exist, but there is no established standard method. This study compared methods of processing potable water samples for the isolation of Mycobacterium avium and Mycobacterium intracellulare using spiked sterilized water and tap water decontaminated using 0.005% cetylpyridinium chloride (CPC). Samples were concentrated by centrifugation or filtration and inoculated onto Middlebrook 7H10 and 7H11 plates and Lowenstein-Jensen slants and into mycobacterial growth indicator tubes with or without polymyxin, azlocillin, nalidixic acid, trimethoprim, and amphotericin B. The solid media were incubated at 32 degrees C, at 35 degrees C, and at 35 degrees C with CO(2) and read weekly. The results suggest that filtration of water for the isolation of mycobacteria is a more sensitive method for concentration than centrifugation. The addition of sodium thiosulfate may not be necessary and may reduce the yield. Middlebrook M7H10 and 7H11 were equally sensitive culture media. CPC decontamination, while effective for reducing growth of contaminants, also significantly reduces mycobacterial numbers. There was no difference at 3 weeks between the different incubation temperatures.     

Cultural and Biochemical Characteristics of Mycobacterium Paratuberculosis Isolated from Goats in Norway

In Norway a variant of Mycobacterium paratuberculosis occurs which causes disease in goats but very seldom in sheep and cattle. Cultural and biochemical characteristics of this variant are investigated by comparing different pre-treatment methods and culture media for primary isolation and by subjecting a number of strains to different enzymatic and biochemical tests. Decontamination of materials with 5% oxalic acid and 0.1% benzalkonium chloride and culture on Dubos, Finleyson’s and Herrold’s medium was tested. The investigations showed that the combination oxalic acid decontamination/Dubos’ medium is most suitable for isolation of the goat-pathogenic variant. The morphology of the colonies was also most easily studied after culture on Dubos’ medium from material pre-treated with oxalic acid. The biochemical tests were found to be poorly suitable for the identification of M. paratuberculosis and for its differentiation from other mycobacteria. Mycobactin dependence for growth seems not to be absolute as a few goat strains produced growth on Dubos’ medium without mycobactin. However, growth was in all cases far better in the presence of mycobactin.     

Mycobacteria from leprous tissue of an armadillo cultivated on a hyaluronic acid based medium.

Mycobacteria were isolated from pooled leprous tissues of an armadillo. The suspensions of acid fast bacilli obtained were inoculated into a culture medium composed of umbilical cord extract, supplemented with yeast extract powder and glycerol with sheep serum added. Incubation temperature was 34 degrees C. An abundant growth of mycobacteria was observed in the primo culture in four weeks. The culture was easily sub-cultured on the homologous media. The primo culture did not grow on Lo?wenstein medium. The identity of the cultures of mycobacteria obtained is not yet established. The same strain of mycobacteria was cultured in media inoculated with suspensions of M. leprae decontaminated with sodium hydroxide-citrate solution. We confirm the findings of Skinsnes et al. (1975) that mycobacteria from human and animal leprous tissue can be cultured repeatedly on a hyaluronic based medium.     

Development of novel Alicyclobacillus spp. isolation medium

AIM: To develop a new isolation medium with higher recovery rates of Alicyclobacillus spp. METHODS AND RESULTS: SK agar was developed with optimized incubation temperature, pH, acidulant, Tween 80 concentration and divalent cation addition. Results indicate that detection of Alicyclobacillus spp. by SK agar was significantly higher (P > 0.05) than those obtained by K agar, orange serum agar, and potato dextrose agar. CONCLUSIONS: Current media used for Alicyclobacillus spp. isolation still resulted in high numbers of false negative products. The sensitivity of SK agar to Alicyclobacillus spp. allows detection of low numbers of Alicyclobacillus spp. and also provides a more higher isolation results compared with currently used media. SIGNIFICANCE AND IMPACT OF THE STUDY: SK agar will be useful to the fruit juice industry to obtain more accurate numbers of contaminant Alicyclobacillus spp. With this media, false negative samples can be reduced, and the likelihood of exported products being rejected can be greatly reduced.     

Enhancement of mycobacterial growth in middlebrook 7H12 medium by polyoxyethylene stearate

Studies were carried out to enhance mycobacterial growth in 7H12 (BACTEC 12A) radiometric medium. Out of 10 different additives investigated, addition of 100 mcg polyoxy-ethylene stearate (POES)/ml enhanced growth of many mycobacteria, especially ofMycobacterium tuberculosis andM. bovis (TB), with no adverse effects.Evaluation of the growth-enhancing effect of POES in 7H12 medium during primary isolation of 117 mycobacterial cultures from clinical specimens indicated significant time saving due to the rapid growth of mycobacteria, especially of TB. The growth index (GI) in 7H12 medium increased much more rapidly in the POES-containing medium, with smears for acid-fast bacilli available sooner owing to higher biomass. This enhanced growth also allowed earlier differentiation of TB from other mycobacteria by the BACTEC NAP test and earlier initiation of indirect drug susceptibility tests, with results available approximately 2–3 days sooner.     

Green fluorescent protein as a new expression marker in mycobacteria

This study describes the use and the advantages of the green fluorescent protein (GFP) as a reporter molecule for mycobacteria. The gfp gene from Aequorea victoria was placed under the control of the hsp60 promoter in the shuttle vector pGFM-11. The gfp expression in the recombinant Mycobacterium smegmatis and BCG was readily detected on agar plates by the development of an intense green fluorescence upon irradiation with long-wave u.v. light. In mycobacteria containing a pGFM-11 derivative that lacks the hsp60 promoter, no fluorescence was observed. However, this plasmid was successfully used as a promoter-probe vector to identify BCG promoters. The fluorescence emission of GFP in mycobacteria harbouring pGFM-11 and grown in liquid media could be quantified by spectrofluorimetry. This allowed for easy assessment of drug susceptibility. As GFP does not require the addition of substrates or co-factors, the green fluorescent bacilli could be directly observed within infected macrophages using fluorescence and laser confocal microscopy, or in tissue sections of infected mice. Finally, infected cells or free-living recombinant mycobacteria could also be analysed by flow cytometry. The GFP thus appears to be a convenient reporter for mycobacteria, allowing tracing of recombinant mycobacteria, isolation of promoters with interesting properties, in vivo drug testing and the development of new diagnostic tools.     

Isolation of motile aeromonads from foods of animal origin

The purpose of this work was to select an available medium for Aeromonads isolation as well as to point out the Aeromonads presence in different foodstuffs of animal origin. Out of 15 tested media for Aeromonas isolation, two media, were selected: DFS as control medium and AGOS medium, both giving superposable results. 404 samples of foodstuffs of animal origin were examined; Aeromonads testing was performed qualitatively and quantitatively. 142 samples with a positivity percent of 51.40% were qualitatively tested. 262 samples with a positivity percent of 48.85% were quantitatively tested.     

Diagnostic effectiveness of nutrient media for the bacteriological detection of enterobacteria

The effectiveness of new nutrient media intended for the differential isolation of pathogenic and opportunistic enterobacteria (SS agar), as well as for the preliminary identification of enterobacteria, was determined on a wide range the strains of the family Enterobacteriaceae. The comparative evaluation of SS agar and Ploskirev's bactoagar was carried out. The newly developed medium was shown to have advantage in its differentiating and inhibiting properties. The clinical trials of the media, carried out in this study, revealed high detection rate and exceedingly exact differentiation of lactose-negative lactose-positive enterobacteria, as well as 100% coincidence of the results obtained on the nutrient medium for the preliminary identification of enterobacteria and on laboratory-made control media.     

"Tween egg medium" for isolating mycobacteria from sputum specimens.

'Tween egg medium,' in which Tween 80 was used as a carbon source, showed a higher rate of positive cultures than Ogawa egg medium in isolation of mycobacteria from sputum specimens.     

Inhibition of Fungal Growth in the Cultural Isolation of Mycobacteria

Antifungal antibiotics were compared to determine their usefulness in primary mycobacterial cultures. Amphotericin B was found to be more effective in preventing fungal growth from bovine fecal specimens than were cycloheximide, nystatin, and tetracycline. Amphotericin B did not affect the growth rate of the following Mycobacterium species: M. avium, M. bovis, M. intracellulare, M. paratuberculosis, M. phlei, or M. tuberculosis, but it inhibited the growth of M. fortuitum. There was no observable effect on numbers of colonies of M. paratuberculosis on primary isolation from fecal specimens. It is recommended that, for the primary isolation of pathogenic mycobacteria from specimens likely to contain fungi, the inoculum should be pretreated with benzalkonium chloride, followed by mixing with amphotericin B or inoculation onto media containing amphotericin B.     

Arginine medium for the isolation and primary identification of Pseudomonas aeruginosa in environmental objects

A culture medium for the isolation and primary identification of P. aeruginosa has been developed. The medium contains L-arginine and ions of K, Na, Mg, C and P; it has also an overlay of plain agar with 0.6% of 2,3,5-triphenyltetrazolium chloride added. The comparison of arginine medium with other media proposed for the isolation of P. aeruginosa from various objects in the environment has demonstrated the advantage of this highly selective medium permitting the primary identification of up to 95% of characteristic colonies.