An Unstable Strain of Aspergillus foetidus Segregating Proline Auxotrophs

Two basic colony types have been obtained through single conidial isolation from the Bode strain of Aspergillus foetidus as well as from mutants of this unstable strain. Type I is prototrophic whereas type II is an auxotroph requiring proline. When a type I strain is grown on complex medium it gradually becomes overwhelmed by type II sectors of growth. However, essentially pure cultures of type I can be maintained on minimal medium (lacking proline). The yield of glucoamylase from type II cultures is less than half that obtained with type I cultures. The instability of type I cultures when grown on complex medium can not be explained by heterokaryosis or the presence of virus-like particles found in the original Bode strain and its derivatives. The isolation of five stable prototrophic strains obtained as more rapidly growing sectors from type I subcultures grown on complex medium suggests that the instability most probably results from a duplicated chromosomal segment or other chromosomal aberration analogous to those described in A. nidulans.     

A new solid medium for the isolation and enumeration of Thiobacillus ferrooxidans and acidophilic heterotrophic bacteria

A solid medium (FeTSB) was developed for the simultaneous isolation and enumeration of the iron-oxidising bacterium Thiobacilluls ferrooxidans and acidophilic heterotrophic bacteria. The medium consisted of ferrous sulfate, tryptone soya broth and basal salts, solidified with agarose, although bacteriological agar could be substituted for some strains. The medium has been used to isolate bacteria from natural environments and also in laboratory studies of characterised strains. The factors which influence the success of colony growth on solid media are discussed.     

Improvements on colony morphology identification towards bacterial profiling

Colony morphology may be an indicator of phenotypic variation, this being an important adaptive process adopted by bacteria to overcome environmental stressors. Furthermore, alterations in colony traits may reflect increased virulence and antimicrobial resistance. Despite the potential relevance of using colony morphological traits, the influence of experimental conditions on colony morphogenesis has been scarcely studied in detail. This study aims to clearly and systematically demonstrate the impact of some variables, such as colony growth time, plate colony density, culture medium, planktonic or biofilm mode of growth and strain genetic background, on bacterial colony morphology features using two Pseudomonas aeruginosa strains. Results, based on 5-replicate experiments, demonstrated that all variables influenced colony morphogenesis and 18 different morphotypes were identified, showing different sizes, forms, colours, textures and margins. Colony growth time and composition of the medium were the variables that caused the highest impact on colony differentiation both derived from planktonic and biofilm cultures. Colony morphology characterization before 45 h of incubation was considered inadequate and TSA, a non-selective medium, provided more colony diversity in contrast to P. aeruginosa selective media. In conclusion, data obtained emphasized the need to perform comparisons between colony morphologies in equivalent experimental conditions to avoid misinterpretation of microbial diagnostics and biomedical studies. Since colony morphotyping showed to be a reliable method to evaluate phenotypic switching and also to infer about bacterial diversity in biofilms, these unambiguous comparisons between morphotypes may offer a quite valuable input to clinical diagnosis, aiding the decision-making towards the selection of the most suitable antibiotic and supportive treatments.     

Growth of Variants of Myxococcus virescens

Some observations on variant strains of Myxococcus virescens B2 with special emphasis on characteristics associated with the ability to grow in dispersion are reported. The isolated strains were divided into two major classes according to their mode of growth in shaken and static liquid cultures based on casitone and casamino acids media. Strains growing in dispersion were designated D⁺-strains and those growing in aggregates or as films, D^?-strains. Colony morphology, cell morphology, growth in liquid and on solid medium and morphogenesis were compared. The ability to grow in dispersion shown by D⁺-strains seemed to be associated with a smooth colony on casitone agar, inability to form typical fruiting bodies and a low linear growth rate of colonies on solid medium as compared with the D^?-strains. In contrast D^?-strains produced rough colonies on casitone agar, were able to fruit and evidently formed an adhesive slime in the form of fibrils extending from the cell surface. It is suggested that the observed differences depend on different envelopes of the cells in the two classes.     

Strategies for adaptation to antibiotics in wild type <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and in the strains with small colony phenotype

Emergence of ciprofloxacin stress-induced mutants in the cultures of a collection strain Pseudomonas aeruginosa ATCC 27853 and of two strains with a small colony phenotype, which were isolated from a swimming pool biofilm, was studied. In biofilm cultures of the small colony phenotype strains, which were already resistant to hypochlorite, prolonged incubation (up to 16 days) with sublethal ciprofloxacin concentrations was shown to result in emergence of the cells, which are resistant to the antibiotic and form colonies on media with rifampicin (100 μg/mL) and streptomycin (50 μg/mL). Under the same conditions, the mechanisms of temporary adaptation are switched on in the cells of strain ATCC 27853, which enabled its shortterm survival at an average level in liquid media and provided for colony formation on solid medium with ciprofloxacin (0.2 μg/mL). Only 20% of these colonies remained viable when transferred to a higher antibiotic concentration (2 μg/mL).     

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.     

Culture media for differential isolation of Lactobacillus casei Shirota from oral samples

This study aimed to develop a solid culture medium for differential isolation of the probiotic strain Lactobacillus casei Shirota (LcS) and for selective cultivation of lactobacilli present in oral samples. Type strains of lactobacilli and isolates from commercial probiotic products were inoculated onto modified de Man Rogosa Sharpe agar (termed 'LcS Select'), containing bromophenol blue pH indicator, vancomycin and reducing agent L-cysteine hydrochloride for differential colony morphology development. L. casei Shirota cultured on the novel medium produced distinctive colony morphologies, different from other lactobacilli tested. LcS-characteristic colonies were recovered on LcS Select medium from samples of saliva and tongue plaque following a four-week probiotic intervention study. The viable count of presumptive LcS colonies correlated with those isolated on a non-commercial lactitol-LBS-vancomycin agar (LLV) developed for a selective isolation of LcS from faeces. The novel LcS Select medium proved suitable for differential isolation of the probiotic strain L. casei Shirota from oral samples containing mixed microbial populations. It can also be used for selective growth of vancomycin-resistant lactobacilli. There are few available culture media that are sufficiently selective to enable isolation of probiotic strains from mixed populations. LcS Select medium provides a cheaper, yet effective tool in this context.     

Characterization of colony morphology variants isolated from Streptococcus pneumoniae biofilms

In this study, we report the isolation of colony morphology variants from Streptococcus pneumoniae serotype 3 biofilms. The colony variants differed in colony size (large, medium, and small) and their mucoid appearance on blood agar. The small nonmucoid variant (SCV) emerged during the initial attachment stage of S. pneumoniae biofilm formation and dominated over the course of biofilm growth. Mucoid variants appeared at later biofilm developmental stages. The reduction in colony size/mucoidy correlated with a decrease in capsule production and an increase in initial attachment. The large mucoid variant formed flat unstructured biofilms, failed to aggregate in liquid culture, and adhered poorly to solid surfaces. In contrast, SCVs autoaggregated in liquid culture, hyperadhered to solid surfaces, and formed biofilms with significant three-dimensional structure, mainly in the form of microcolonies. The variants showed similar antibiotic resistance/susceptibility based on a modified Kirby-Bauer test and when grown as biofilms. However, antimicrobial treatment of S. pneumoniae biofilms altered the colony variant's distribution and mainly affected the most interior areas of biofilm microcolonies. To further explore the nature of the variants, the capsule biosynthetic operon (cps3DSUM) was explored in greater detail. The genetic analysis indicated that the emergence of nonmucoid variants was due to a deletion comprising cps3DSU as well as additional genes upstream of the cps3 operon. Overall, our findings suggest that in vitro biofilm formation of S. pneumoniae serotype 3 coincides with the emergence of colony variants with distinct genotypic and phenotypic characteristics.     

Growth ofThiobacillus ferrooxidans on solid media containing heterotrophic bacteria

Summary the study of the chemolithotrophic bacteriumThiobacillus ferrooxidans, an organism important in mining and metallurgical applications, has always been complicated by the inability to cultivate and enumerate many wild strains on solid agar. Here, we have described a plate-count procedure that uses an acidophilic heterotrophic bacterium to scavenge organic material from normal agar, permitting colony formation of the autotroph without visible evidence of growth of the heterotroph. This technique could enable anyT. ferroxidans isolate, including those that are agar-sensitive, to grow and develop colonies on solid medium.     

Growth characteristics, cytopathic effect in cell culture, and virulence in mice of 36 type strains belonging to 19 different Acanthamoeba spp

A total of 36 strains belonging to 19 different species of Acanthamoeba were compared for temperature tolerance, ability to grow in an axenic medium, cytopathic effect in Vero cell culture, and virulence in mice. Pathogenic strains appeared to belong to different species, whereas pathogenic and nonpathogenic strains occurred in one species. Although growth at high temperatures and readiness to grow axenically indicated a potential for pathogenicity, each such strain had to be tested in cell cultures or laboratory mice to determine whether or not it was virulent. This study was not intended to differentiate Acanthamoeba spp., but to provide methods to be used for the specific isolation and identification of pathogenic Acanthamoeba strains.     

Growth characteristics, cytopathic effect in cell culture, and virulence in mice of 36 type strains belonging to 19 different Acanthamoeba spp.

A total of 36 strains belonging to 19 different species of Acanthamoeba were compared for temperature tolerance, ability to grow in an axenic medium, cytopathic effect in Vero cell culture, and virulence in mice. Pathogenic strains appeared to belong to different species, whereas pathogenic and nonpathogenic strains occurred in one species. Although growth at high temperatures and readiness to grow axenically indicated a potential for pathogenicity, each such strain had to be tested in cell cultures or laboratory mice to determine whether or not it was virulent. This study was not intended to differentiate Acanthamoeba spp., but to provide methods to be used for the specific isolation and identification of pathogenic Acanthamoeba strains.     

Variation of colony morphology and chromosomal rearrangement in Candida tropicalis pK233

UV irradiation treatment of the asexual yeast Candida tropicalis gave rise to morphological mutants exhibiting at least four different types of abnormal colonies on glucose-containing solid medium. These mutants were named according to their colony morphologies: 'doughnut', 'frilly', 'echinoid' and 'walnut' mutants. The doughnut mutant produced a wrinkled colony with a hollow in its central region that was rich in filamentous pseudohyphal cells. With increased incubation time, the colony gradually changed to a reticulate shape. The parent strain, which normally produced smooth colonies, gave similar colonies to those of the doughnut mutant when grown in medium containing oleic acid as carbon source. Both the frilly and the walnut mutants produced pseudohyphal cells in a similar fashion to the doughnut mutant. The echinoid mutant produced an echinulate colony morphology with aerial hyphae and contained true hyphal cells as well as pseudohyphal ones. Pulsed-field gel electrophoresis showed that the echinoid and frilly mutants had different karyotypes from that of their parent strain, suggesting the occurrence of chromosomal rearrangements associated with these morphological mutations.     

Biological properties of promastigotes of Leishmania having undergone lyophilization]

Materials are presented of a comparative study of morphological and cultural properties and virulence of liophylized cultures of Leishmania tropica major after their rehydration or after a long passage on the NNN medium. Results of investigations of two initial strains and three substrains obtained after the recovery of liophylized cultures have shown that liophylization does not reduce essentially the initial strain virulence. The strains undergone liophylization and passed repeatedly on the NNN medium have a tendency to a quicker virulence reduction rate as compared to initial ones. The culture of promastigotes recultivated after liophylization does not differ from the initial one in the ranges of 1-7 passages in its morphology, mobility and ability to grow on the NNN medium.     

New medium for isolating iron-oxidizing and heterotrophic acidophilic bacteria from acid mine drainage.

A new solid medium is described for growing iron and heterotrophic bacteria from acid mine drainage (AMD). Examination of AMD from five states revealed several kinds of colonies of iron-oxidizing bacteria: (i) smooth, (ii) smooth with secondary growth sectors or branching, (iii) star-shaped, (iv) radiating lobe, and (v) flat-rough. All AMD samples yielded whitish colonies that could not use ferrous iron, sulfur, or hydrogen, nor could they grow on nutrient agar, brain heart infusion agar, or Trypticase soy agar. Glucose and sucrose supported growth if the sugar-salts medium was at pH 3.0. The new iron medium has several advantages over others: (i) easy preparation, (ii) rapid growth, (iii) larger colonies, (iv) differentiation of colony morphology, and (v) detection of a new group of heterotrophic acidophilic bacteria.     

表型与亲本不易区分的茯苓同核体菌株新类型

茯苓Wolfiporia hoelen是我国传统中药材之一,也是一种食药兼用的大型真菌,目前已规模化栽培,但由于其交配系统一直不明确,影响了种质改良。前期我们发现了茯苓的同核体,明确了茯苓的交配系统和生活史,并建立了以培养特性和分子标记区分同核体的方法,但未明确是否适用于茯苓种群的不同个体。在对多个菌株的研究中,发现了同核体表型与亲本不易区分的茯苓菌株。本研究主要以来自日本的菌株775 (NBRC 30628)为亲本,对其同核体菌株进行收集鉴定,并对同核体菌株的培养特性、交配现象和杂交等进行了研究。此类菌株的同核体菌株可通过与亲本对峙培养进行鉴定,但菌丝生长、菌落形态和吃料速度等与亲本没有显著差异,不同交配型的同核体之间交配时没有明显的交配现象。rpb2杂合位点标记可以用于鉴定该类型同核体菌株,且能验证是否交配。该类型同核体与之前发现的同核体类型之间可以进行杂交,杂交菌株可与两亲本都产生拮抗现象。该发现补充了之前建立的茯苓同核体鉴定方法,加深了对茯苓物种群体的了解,同时丰富了茯苓的育种资源。     

Isolation and characterization of ethanol tolerant yeast strains

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains.     

Fitness of isogenic colony morphology variants of Pseudomonas aeruginosa in murine airway infection

Chronic lung infections with Pseudomonas aeruginosa are associated with the diversification of the persisting clone into niche specialists and morphotypes, a phenomenon called 'dissociative behaviour'. To explore the potential of P. aeruginosa to change its morphotype by single step loss-of-function mutagenesis, a signature-tagged mini-Tn5 plasposon library of the cystic fibrosis airway isolate TBCF10839 was screened for colony morphology variants under nine different conditions in vitro. Transposon insertion into 1% of the genome changed colony morphology into eight discernable morphotypes. Half of the 55 targets encode features of primary or secondary metabolism whereby quinolone production was frequently affected. In the other half the transposon had inserted into genes of the functional categories transport, regulation or motility/chemotaxis. To mimic dissociative behaviour of isogenic strains in lungs, pools of 25 colony morphology variants were tested for competitive fitness in an acute murine airway infection model. Six of the 55 mutants either grew better or worse in vivo than in vitro, respectively. Metabolic proficiency of the colony morphology variant was a key determinant for survival in murine airways. The most common morphotype of self-destructive autolysis did unexpectedly not impair fitness. Transposon insertions into homologous genes of strain PAO1 did not reproduce the TBCF10839 mutant morphotypes for 16 of 19 examined loci pointing to an important role of the genetic background on colony morphology. Depending on the chosen P. aeruginosa strain, functional genome scans will explore other areas of the evolutionary landscape. Based on our discordant findings of mutant phenotypes in P. aeruginosa strains PAO1, PA14 and TBCF10839, we conclude that the current focus on few reference strains may miss modes of niche adaptation and dissociative behaviour that are relevant for the microevolution of complex traits in the wild.     

Single-tube colony PCR for DNA amplification and transformant screening of oleaginous microalgae

Recently, several colony PCR methods have been developed to simplify DNA isolation procedure and facilitate PCR-based colony screening efforts in microalgae. A main drawback of current protocols is that cell collection, disruption, and genomic DNA extraction are required preceding the PCR step, making the colony PCR process laborious and costly. In the present study, we have developed a novel procedure that eliminates any steps of DNA extraction and allows the colony screening to be performed in a single PCR tube: algal cells (as low as 5,000) from agar plates or liquid cultures were directly transferred into a PCR tube containing 2× PCR buffer and boiled for 5–10 min depending on different algal strains, followed by addition of other PCR components (dNTPs, primers, and polymerase) and then subjected to conventional PCR reaction. The procedure documented here worked well not only for the model alga Chlamydomonas reinhardtii, but also for the thick-walled oleaginous strains such as Chlorella, Haematococcus, Nannochloropsis, and Scenedesmus with its efficacy independent on amplicon sizes and primer pairs. In addition, screening of Chlorella zofingiensis transformants was achieved using this method. Collectively, our single-tube colony PCR is a much simpler and more cost-effective procedure as compared to those previously reported and has broad applications including gene cloning, strain determination, and high-throughput screening of algae colonies and transformants for biomass and biofuel production.     

Isolation and characterization of heterotrophic,aerobic bacteria from oil storage caverns in northern Germany

Aerobic heterotrophic bacteria were enriched and isolated from three oil storage caverns of the German national oil reserve at different distances from the oil/brine interface. Microscopically no bacteria were found in the original samples, but colony counts showed more than 100 colony-forming units (cfu)/ml in two samples, whereas 0 to 4 cfu/ml were found in the other samples. Enrichments using defined mineral salts medium or complex medium revealed culturable organisms in all samples. All colony types were isolated and further separation of organisms during isolation was completed microscopically. Enrichments in media containing complex organic compounds led to higher numbers of isolates in samples near the oil/brine interface than enrichments with oil as the sole source of carbon. Micro-organisms that could utilize oil as the sole source of carbon were isolated from all enrichment cultures. Identification of the isolates revealedBacillus strains in all samples and coryneform bacteria in the samples from cavern 123.