Comparison of four different culture media for isolation and growth of type II and type I/III Mycobacterium avium subsp. paratuberculosis strains isolated from cattle and goats

Culture is considered the definitive technique for Johne's disease diagnosis, and it is essential for later applications of certain molecular typing techniques. In this study, we have tested four solid media (Herrold's egg yolk medium [HEYM] with sodium pyruvate and mycobactin [HEYMm-SP], HEYM with mycobactin and without sodium pyruvate [HEYMm], Middlebrook 7H11 with mycobactin [Mm], and L?wenstein-Jensen with mycobactin [LJm]) for isolation of Mycobacterium avium subsp. paratuberculosis strains in 319 tissue samples from cattle herds and goat flocks. We have shown that each of the two main groups of M. avium subsp. paratuberculosis (type II and type I/III) has different requirements for growth in the culture media studied. The recommended solid media for isolation of type I/III strains are LJm and Mm, since the combination of both media allowed the recovery of all these strains. The most widespread culture medium, HEYM, is not suitable for the isolation of this group of M. avium subsp. paratuberculosis strains. Regarding the type II strains, HEYMm-SP was the medium where more strains were isolated, but the other three media are also needed in order to recover all type II strains. The incubation period is also related to the strain type. In conclusion, because the type of strain cannot be known in advance of culture, coupled with the fact that cattle and goats can be infected with both groups of strains, we recommend the use of the four solid media and the prolongation of the incubation period to more than 6 months to detect paratuberculous herds/flocks and to determine the true prevalence of the infection.     

Isolation of Mycobacterium Paratuberculosis from Sheep and Cattle in Iceland

Isolation of Mycobacterium paratuberculosis from sheep and cattle in Iceland. Acta vet. scand. 1979, 20, 191–199. — Culture experiments concerning the Icelandic variant of Mycobacterium paratuberculosis are described. Various decontaminating agents and culture media were employed and the colonial morphology of freshly isolated strains on different media described. The growth rate and culture requirements are compared with those of the Norwegian goat-pathogenic variant of M. paratuberculosis. For primary isolation modified Herrold’s medium gave the best results. However, on all the various culture media used, the growth of the Icelandic variant was much more sporadic than that of the Norwegian goatpathogenic variant. It is concluded that bacteriological culture is not useful for the diagnosis of Johne’s disease caused by the Icelandic variant of M. paratuberculosis.     

Comparison of Four Different Culture Media for Isolation and Growth of Type II and Type I/III Mycobacterium avium subsp. paratuberculosis Strains Isolated from Cattle and Goats

Culture is considered the definitive technique for Johne's disease diagnosis, and it is essential for later applications of certain molecular typing techniques. In this study, we have tested four solid media (Herrold's egg yolk medium [HEYM] with sodium pyruvate and mycobactin [HEYMm-SP], HEYM with mycobactin and without sodium pyruvate [HEYMm], Middlebrook 7H11 with mycobactin [Mm], and Löwenstein-Jensen with mycobactin [LJm]) for isolation of Mycobacterium avium subsp. paratuberculosis strains in 319 tissue samples from cattle herds and goat flocks. We have shown that each of the two main groups of M. avium subsp. paratuberculosis (type II and type I/III) has different requirements for growth in the culture media studied. The recommended solid media for isolation of type I/III strains are LJm and Mm, since the combination of both media allowed the recovery of all these strains. The most widespread culture medium, HEYM, is not suitable for the isolation of this group of M. avium subsp. paratuberculosis strains. Regarding the type II strains, HEYMm-SP was the medium where more strains were isolated, but the other three media are also needed in order to recover all type II strains. The incubation period is also related to the strain type. In conclusion, because the type of strain cannot be known in advance of culture, coupled with the fact that cattle and goats can be infected with both groups of strains, we recommend the use of the four solid media and the prolongation of the incubation period to more than 6 months to detect paratuberculous herds/flocks and to determine the true prevalence of the infection.     

Growth and Metabolic Characteristics of Mycobacterium paratuberculosis

The cultural characteristics of newly isolated strains of Mycobacterium paratuberculosis on a variety of media were studied. The mycobactin dependence ascribed to this species of Mycobacterium was found to be easily circumvented by incorporation of 1% ferric ammonium citrate in serum or egg yolk media, but the earliest and most abundant growth occurred on serum medium that contained mycobactin and sodium pyruvate. In the presence of ferric ammonium citrate, respiration did not decrease during 5 days; but in the presence of all other agents studied, respiration decreased after the first day.     

Numerical taxonomy of mycobactin-dependent mycobacteria, emended description of Mycobacterium avium, and description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. nov., and Mycobacterium avium subsp. silvaticum subsp. nov

We performed a numerical taxonomy analysis of 38 Mycobacterium paratuberculosis and related mycobacterial strains, including wood pigeon mycobacteria; this analysis was based on 22 tests, which were selected for their potential discriminative value from a total of 51 tests studied and produced four well-defined clusters. Cluster 1 contained the M. paratuberculosis strains, including two strains isolated from Crohn's disease patients; cluster 2 contained Mycobacterium avium and Mycobacterium intracellulare reference strains; cluster 3 consisted of the wood pigeon mycobacteria; and the only strain in cluster 4 was M. paratuberculosis 316F, which is used for antigen and vaccine production. Strains in cluster 1 were mycobactin dependent even when they were subcultured, whereas strains in cluster 3 were unable to grow on egg medium and their growth was stimulated by pH 5.5. Growth stimulation by pyruvate, resistance to D-cycloserine (50 micrograms/ml), and alkaline phosphatase activity also were characteristics that were useful for discriminating between clusters 1 and 3. The results of previous DNA-DNA hybridization studies have demonstrated that M. avium Chester 1901, M. paratuberculosis Bergey et al. 1923, and the wood pigeon mycobacteria belong to a single genomic species, and we propose that the name of this species should be M. avium. On the basis of the results of previous genomic analyses based on restriction fragment length, the results of polymorphism studies, and DNA patterns determined by field inversion gel electrophoresis as well as the results of our phenotypic study, we propose that the species should be divided into subspecies which correspond to pathogenicity and host range characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)     

An Improved Medium for Culture of Mycoracterium Paratuberculosis from Bovine Faeces

Löwenstein-Jensen medium with mycobactin, cyclo-heximide, penicillin, and chloramphenicol, and enriched with sodium pyruvate, was compared with an ordinary L.-J. medium with mycobactin. Faeces samples from cattle experimentally infected with M. paratuberculosis were cultured on both media. The improved medium gave 11 % more positive cultures and 90 % more colonies. Of the positive cultures 97 % showed detectable growth after 8 weeks of incubation, and the contamination rate was reduced to 0.4 %. By culture of faeces samples from naturally infected cattle the improved medium identified 23.2 % more infected animals than the basic medium, mostly due to a reduction of the contamination rate to about 3 %.     

Inactivation of Mycobacterium paratuberculosis in cows' milk at pasteurization temperatures.

The thermal inactivation of 11 strains of Mycobacterium paratuberculosis at pasteurization temperatures was investigated. Cows' milk inoculated with M. paratuberculosis at two levels (10(7) and 10(4) CFU/ml) was pasteurized in the laboratory by (i) a standard holder method (63.5 degrees C for 30 min) and (ii) a high-temperature, short-time (HTST) method (71.7 degrees C for 15 s). Additional heating times of 5, 10, 15, 20, and 40 min at 63.5 degrees C were included to enable the construction of a thermal death curve for the organism. Viability after pasteurization was assessed by culture on Herrold's egg yolk medium containing mycobactin J (HEYM) and in BACTEC Middlebrook 12B radiometric medium supplemented with mycobactin J and sterile egg yolk emulsion. Confirmation of acid-fast survivors of pasteurization as viable M. paratuberculosis cells was achieved by subculture on HEYM to indicate viability coupled with PCR using M. paratuberculosis-specific 1S900 primers. When milk was initially inoculated with 10(6) to 10(7) CFU of M. paratuberculosis per ml, M. paratuberculosis cells were isolated from 27 of 28 (96%) and 29 of 34 (85%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. Correspondingly, when 10(3) to 10(4) CFU of M. paratuberculosis per ml of milk were present before heat treatment, M. paratuberculosis cells were isolated from 14 of 28 (50%) and 19 of 33 (58%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. The thermal death curve for M. paratuberculosis was concave in shape, exhibiting a rapid initial death rate followed by significant "tailing." Results indicate that when large numbers of M. paratuberculosis cells are present in milk, the organism may not be completely inactivated by heat treatments simulating holder and HTST pasteurization under laboratory conditions.     

Isolation, identification, and structural analysis of the mycobactins of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, and Mycobacterium paratuberculosis.

Methods were devised to purify the cell-associated, iron-binding compounds known as mycobactins from the closely related species Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (i.e., the MAIS complex of organisms). The mycobactins from these three species showed a structure that is common to the mycobactins from all the mycobacteria examined to date. However, these mycobactins were unique in that they had more than one alkyl chain. The M. scrofulaceum mycobactins differed from other MAIS mycobactins by a shift in the position of the double bond in the R1 alkyl chain. Traces of other mycobactin types were observed in ethanol extracts of the three species, and examination of the chromatographic properties of these mycobactins showed that each species produced five mycobactin types. Each mycobactin could be subdivided further by the length of its R1 alkyl chain. No differences in the production of these novel mycobactin were observed among species. Mycobactins from three strains of Mycobacterium paratuberculosis and two wood pigeon strains of Mycobacterium avium which had lost their original growth requirements for mycobactin after repeated subculturing in laboratory growth media were examined by thin-layer chromatography and high-pressure liquid chromatography. Each organism produced a mycobactin with similar chromatographic properties to those synthesized by MAIS organisms. M. paratuberculosis NADC 18 produced at least two components in our laboratory, and nuclear magnetic resonance analysis of the major component showed this mycobactin to be identical to that produced by M. intracellulare M12. However, a sample of mycobactin J isolated by Merkal and McCullough (Curr. Microbiol. 7:333-335, 1982) from M. paratuberculosis NADC 18 was different from our isolates and appeared to correspond to a minor mycobactin component we had seen by thin-layer chromatography. No reason for this difference could be evinced. Our findings indicate that there is a close taxonomic relationship between M. paratuberculosis and the MAIS complex.     

Estimation of Mycobacterium avium subsp. paratuberculosis growth parameters: strain characterization and comparison of methods

The growth rate of Mycobacterium avium subsp. paratuberculosis was assessed by different methods in 7H9 medium supplemented with OADC (oleic acid, albumin, dextrose, catalase), Tween 80, and mycobactin J. Generation times and maximum specific growth rates were determined by wet weight, turbidometric measurement, viable count, and quantitative PCR (ParaTB-Kuanti; F57 gene) for 8 M. avium subsp. paratuberculosis strains (K10, 2E, 316F, 81, 445, 764, 22G, and OVICAP 49). Strain-to-strain differences were observed in growth curves and calculated parameters. The quantification methods gave different results for each strain at specific time points. Generation times ranged from an average of 1.4 days for viable count and qPCR to approximately 10 days for wet weight and turbidometry. The wet-weight, turbidometry, and ParaTB-Kuanti qPCR methods correlated best with each other. Generally, viability has been assessed by viable count as a reference method; however, due to M. avium subsp. paratuberculosis clumping problems and the presence of noncultivable M. avium subsp. paratuberculosis cells, we conclude that qPCR of a single-copy gene may be used reliably for rapid estimation of M. avium subsp. paratuberculosis bacterial numbers in a sample.     

Variation in resistance of Mycobacterium paratuberculosis to acid environments as a function of culture medium

Acid resistance of Mycobacterium paratuberculosis was examined as a function of growth conditions (i.e., in vitro growth medium and pH). M. paratuberculosis was cultured in either fatty acid-containing medium (7H9-OADC) or glycerol-containing medium (WR-GD or 7H9-GD) at two culture pHs (pHs 6.0 and 6.8). Organisms produced in these six medium and pH conditions were then tested for resistance to acetate buffer at pHs 3, 4, 5, and 6 at 20 degrees C. A radiometric culture method (BACTEC) was used to quantify viable M. paratuberculosis cell data at various acid exposure times, and D values (decimal reduction times, or the times required to kill a 1-log(10) concentration of bacteria) were determined. Soluble proteins of M. paratuberculosis grown under all six conditions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify proteins that may be associated with acid resistance or susceptibility. The culture medium affected growth rate and morphology: thin floating sheets of cells were observed in 7H9-OADC versus confluent, thick, waxy, and wrinkled pellicles in WR-GD. Culture medium pH affected growth rate (which was highest at pH 6.0), but it had little or no effect on D values for M. paratuberculosis at any test pH. When grown in 7H9-OADC, M. paratuberculosis was more acid resistant at all test pHs (higher D values) than when grown in WR-GD. Glycerol appeared to be the culture medium component most responsible for lower levels of M. paratuberculosis acid resistance. When glycerol was substituted for OADC in the 7H9 medium, D values were significantly lower than those of 7H9-OADC-grown M. paratuberculosis and were approximately the same as those for M. paratuberculosis grown in WR-GD medium. Comparison of the SDS-PAGE protein profiles for M. paratuberculosis cultures grown in 7H9-OADC, WR-GD, or 7H9-GD medium revealed that increased expression of 34.2- and 14.0-kDa proteins was associated with higher levels of acid resistance of M. paratuberculosis grown in 7H9-OADC medium and that 56.6- and 41.3-kDa proteins were associated with lower levels of acid resistance. This is the first report showing that in vitro culture conditions significantly affect growth characteristics, acid resistance, and protein expression of M. paratuberculosis, and the results emphasize the importance of culture conditions for in vitro susceptibility studies.     

Effect of turbulent-flow pasteurization on survival of Mycobacterium avium subsp. paratuberculosis added to raw milk

A pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) was used to study the heat sensitivity of Mycobacterium avium subsp. paratuberculosis added to raw milk. The ATCC 19698 type strain, ATCC 43015 (Linda, human isolate), and three bovine isolates were heated in raw whole milk for 15 s at 63, 66, 69, and 72 degrees C in duplicate trials. No strains survived at 72 degrees C for 15 s; and only one strain survived at 69 degrees C. Means of pooled D values (decimal reduction times) at 63 and 66 degrees C were 15.0 +/- 2.8 s (95% confidence interval) and 5.9 +/- 0.7 s (95% confidence interval), respectively. The mean extrapolated D72 degrees C was <2.03 s. This was equivalent to a >7 log10 kill at 72 degrees C for 15 s (95% confidence interval). The mean Z value (degrees required for the decimal reduction time to traverse one log cycle) was 8.6 degrees C. These five strains showed similar survival whether recovery was on Herrold's egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). Milk was inoculated with fresh fecal material from a high-level fecal shedder with clinical Johne's disease. After heating at 72 degrees C for 15 s, the minimum M. avium subsp. paratuberculosis kill was >4 log10. Properly maintained and operated equipment should ensure the absence of viable M. avium subsp. paratuberculosis in retail milk and other pasteurized dairy products. An additional safeguard is the widespread commercial practice of pasteurizing 1.5 to 2 degrees above 72 degrees C.     

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis

The efficacy of high-temperature, short-time (HTST) pasteurization (72 °C/15 s) when low numbers (≤ 10³ cfu ml ⁻¹ ) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10³ cfu ml⁻¹, 10² cfu ml⁻¹, 10 cfu ml⁻¹, and 10 cfu 50 ml⁻¹) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14·8% and 10% of HTST-pasteurized milk samples at the 10³ and 10² cfu ml⁻¹ inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3·7% and 6·7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis . No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml⁻¹ or 10 cfu 50 ml⁻¹.     

Use of a methylene blue azide medium for isolation of enterococci

A methylene blue azide medium (MBA), developed by Schaedler, Dubos, and Costello to isolate enterococci from the gastrointestinal tract of animals, was evaluated. This was done by comparing the isolation of enterococci from feces and saliva on the medium. Fifty-two catalase-negative, gram-positive cocci from human feces isolated from MBA were classified as enterococci. All strains grew in S F, 6.5% NaCl, and streptomycin broths, and all fermented mannitol. The isolates were provisionally subdivided into Streptococcus faecalis and S. faecium groups. S. faecalis-like strains fermented glycerol and pyruvate aerobically and produced acid in Snyder's medium (initial pH, 4.8). The S. faecium group fermented raffinose. Among all strains, several tests were variable. These included growth at 45 C, in 0.1% tellurite and in methylene blue milk. Three methods were employed to isolate and identify enterococci from the oral cavity. Direct streaking of MBA with saliva failed to produce any growth on the medium. Two other methods, with the use of various selective broths to promote the recovery of oral enterococci, failed to produce any bacteria capable of growing on MBA. The MBA-isolated fecal strains and oral viridans streptococci were generally indistinguishable on Mitis-Salivarius and K F agars. In experiments with fecal material, no gram-negative bacilli were found among the isolates selected. The MBA medium was judged as a high selectivity-low yield medium, and may provide a means of separating fecal and nonfecal enterococci.     

Chemical and biological properties of mycobactins isolated from various mycobacteria

Nine different strains of mycobacteria grown on media deficient in iron all produced mycobactins. Most strains produced one mycobactin in great preponderance. Mycobacteria from clearly distinct taxonomic groups gave mycobactins differing in the structure of their nuclei. One group of taxonomically related mycobacteria produced mycobactins having the same nucleus but with different distributions of side chains within the homologous mixtures. Simple methods are described for identifying mycobactins on a small scale; these may be of value in classifying mycobacteria. Structures are proposed for mycobactin A from Mycobacterium aurum, mycobactin R from M. terrae, mycobactin F, produced together with mycobactin H by M. fortuitum, and mycobactins M and N from M. marinum. The first three of these differ from known mycobactins in details of substitution and configuration of asymmetric centres in the nucleus. Mycobactins M and N are substantially different, having only small acyl groups (acetyl and propionyl respectively) at the hydroxamic acid centre of the mycobactic acid moiety. Both are homologous mixtures having long-chain saturated 3-hydroxy-2-methyl acid fragments in the cobactin moiety. All mycobactins so far isolated promote almost maximal growth of M. johnei at 30ng./ml. in liquid medium. The activity of some mycobactins extends to much lower concentrations, mycobactin S showing significant growth promotion at 0.3ng./ml. Mycobactin M or N in combination with mycobactins having a long side chain in the mycobactic acid moiety exerts a mutually antagonistic effect on the growth of M. johnei, the mixture giving less growth than either mycobactin separately. Mycobactin M also decreases the growth of M. kansasii and M. tuberculosis on liquid media. These antagonistic effects are probably caused by a lengthening of the lag phase.     

Analysis of Antigens in Mycobacterium Paratuberculosis

Analysis of antigens in Mycobacterium paratuberculosis. Acta vet. scand. 1979, 20, 200–215. — Using crossed immunoelectrophoresis (GIE) and crossed line immunoelectrophoresis (GLIE), antigens from different strains and variants of Mycobacterium paratuberculosis were compared, and cross-reactions between 1 of these strains and Mycobacterium avium and BGG studied. In each of 4 bovine laboratory strains of M. paratuberculosis examined, altogether 44 different antigens were demonstrated. This is the largest number of antigens in M. paratuberculosis which has been described so far. No important difference in the antigenic structure of the strains was found. The 4 laboratory strains are being used routinely in the production of vaccine against Johne’s disease in Norway and Iceland. One of the aims of the present work was to investigate the antigenic relationship between these strains and the goat-pathogenic Norwegian and the Icelandic variant of M. paratuberculosis. Out of 44 different antigens demonstrated in the laboratory strains, 39 and 31 gave cross-reactions against the Norwegian and the Icelandic variant, respectively. This is in accordance with practical experience, as the results of vaccination against Johne’s disease, performed in Norway for many years, are very good. Twenty-seven and 24 cross-reacting antigens between M. paratuberculosis and strains of M. avium and BGG, respectively, were observed. This finding agrees with clinical observations. Another aim of the investigation was to identify species-specific antigens as regards M. paratuberculosis. One antigen showed a marked cross-reaction between the strains of M. paratuberculosis examined, but did not react with antisera against M. avium and BGG. Some other antigens showed partial specificity. The results obtained stress the complicated antigenic situation in mycobacteria which is of decisive significance as regards the diagnosis and classification of mycobacterial infections.     

A new mycobactin,mycobactin J,fromMycobacterium paratuberculosis

This paper reports the production, purification, and biological assay of a new mycobactin fromMycobacterium paratuberculosis. This new mycobactin, designated mycobactin J, is produced by a strain ofM. paratuberculosis adapted to growth on synthetic medium without exogenous mycobactin. Mycobactin J reduces the incubation period ofM. paratuberculosis in complex medium by 3 weeks when compared with medium containing mycobactin P, and a higher proportion of the organisms, in inocula from infected tissues or fecal specimens, produce colonies. Also, some strains ofM. paratuberculosis will grow on medium containing mycobactin J that will not grow on medium containing mycobactin P.     

Influence of metal ions on the formation of mycobactin and salicylic acid in Mycobacterium smegmatis grown in static culture

Mycobacterium smegmatis was grown on trace-metal-free medium in static culture. Throughout the growth phase, the concentration of mycobactin increased continuously, reaching a maximum of about 30 to 40 mug of mycobactin/mg of cell dry weight after 6 days; the concentration of salicylic acid remained approximately constant at 1 to 2 mug of salicylic acid/mug of cell dry weight. Fe(2+) (or Fe(3+)), Zn(2+), Mn(2+), and Mg(2+) were all essential to a maximum formation of mycobactin. Optimum concentrations required were: Fe(2+), about 1.8 mum; Mn(2+) and Zn(2+), about 0.5 mum; and Mg(2+), at least 0.17 mm. Higher levels of Fe(2+) (9 to 90 mum) and Zn(2+) (2 to 7 mum) repressed mycobactin to about half the maximum value. No other cation or anion apparently is required for mycobactin biosynthesis. Salicylic acid concentration increased about fourfold when iron was omitted from the medium, but this is not as great as the increase reported previously for this strain of M. smegmatis. Mycobactin formation in another strain of M. smegmatis, NCIB 8548, showed similar dependencies on Fe(2+), Zn(2+), and Mn(2+). Maximum accumulation of mycobactin with this strain was 85 mug of mycobactin/mg of dry cell weight, under iron-deficient (1.8 mum Fe(2+)) conditions.     

水貂粪便中双歧杆菌的分离与鉴定

目的运用TPY培养基,从健康水貂的粪便中分离培养、筛选出2个肠道菌株。方法细菌培养、菌落形态观察、染色镜检、分离纯化、生化试验和药敏试验。结果分离培养出的2株菌株为双歧杆菌,其中1株为长双歧杆菌,另1株为青春双歧杆菌;双歧杆菌对氯霉素极其敏感,对阿米卡星耐药。结论本实验为毛皮特种经济动物微生态制剂的研究工作奠定了基础。     

Selection of methods of detecting lactose-fermenting Salmonella strains and evaluating their usefulness for diagnostic studies

Salmonella rods of subspecies I, lactose-fermenting were first isolated in Poland in 1980. They were isolated from a plus sample taken from a brain abscess of a child. Next strains were isolated from faeces of newborn and hospitalized children. Growth characteristic of colonies of lactose-fermenting Salmonella strains on selective-differentiating media (Mac Conkey's Levine, SS, So?tys) recommended for inoculation of clinical material resembled Escherichia coli. So far these type of colonies were omitted in diagnostic examinations. Lactose-fermenting variants showed on Bismuth sulfate agar "Difco" (WB) typical for Salmonella growth pattern. They grew on this medium after 48 hr of incubation in a form of black, medium sized colonies, with some metallic brilliance and characteristic blackening of the medium undercolonies. Precise knowledge of biochemical properties of lactose-fermenting Salmonella allows to supplement so far used diagnostic scheme with additional tests permitting differentiation of lactose-fermenting variants of Salmonella from the other members of Enterobacteriaceae family. Taking into consideration biochemical variants in diagnostic procedure i.e. lactose-fermenting Salmonella, allowedns to isolate in the years 1983-1985 lactose-positive strains in 1305 out of 2773 (47%) individuals positive for S. agona. In 1987, 246 persons (28.3%) out of 869 with lactose-fermenting Salmonella of various serotypes were simultaneously infected with lactose-negative variant. Lactose-fermenting strains of Salmonella belonged most frequently to the following genera: S. agona, S. enteritidis, S. oranienburg, S. typhimurium, and S. goldcoast. It was found that the modified diagnostic procedure makes possible the isolation and the identification of lactose-positive varians of Salmonella.     

Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii.

An isolate of Penicillium bilaii previously reported to solubilize mineral phosphates and enhance plant uptake of phosphate was studied. Using agar media with calcium phosphate and the pH indicator alizarin red S, the influence of the medium composition on phosphate solubility and medium acidification was recorded. The major acidic metabolites produced by P. bilaii in a sucrose nitrate liquid medium were found to be oxalic acid and citric acid. Citric acid production was promoted under nitrogen-limited conditions, while oxalic acid production was promoted under carbon-limited conditions. Citric acid was produced in both growth and stationary phases, but oxalic acid production occurred only in stationary phase. When submerged cultures which normally produce acid were induced to sporulate, the culture medium shifted toward alkaline rather than acid reaction with growth.