Diversity of the growth patterns of Bacillus subtilis colonies on agar plates

Abstract A Bacillus subtilis strain showed a variety of colony growth patterns on agar plates. The bacterium grew to a fractal colony through the diffusion-limited aggregation process, a round colony reminiscent of the Eden model, a colony with a straight and densely branched structure similar to the dence branching, morphology, a colony spreading without any openings, and a colony with concentric rings, on plates with various agar and nutrient concentrations. The microstructures of these colonies were also characteristic and dynamic. The patterns of these bacterial colonies were thought to grow in relation to the diffusion of nutrient in the agar plate.     

Adaptive divergence in experimental populations of Pseudomonas fluorescens. I. Genetic and phenotypic bases of wrinkly spreader fitness

A central feature of all adaptive radiations is morphological divergence, but the phenotypic innovations that are responsible are rarely known. When selected in a spatially structured environment, populations of the bacterium Pseudomonas fluorescens rapidly diverge. Among the divergent morphs is a mutant type termed "wrinkly spreader" (WS) that colonizes a new niche through the formation of self-supporting biofilms. Loci contributing to the primary phenotypic innovation were sought by screening a WS transposon library for niche-defective (WS(-)) mutants. Detailed analysis of one group of mutants revealed an operon of 10 genes encoding enzymes necessary to produce a cellulose-like polymer (CLP). WS genotypes overproduce CLP and overproduction of the polymer is necessary for the distinctive morphology of WS colonies; it is also required for biofilm formation and to maximize fitness in spatially structured microcosms, but overproduction of CLP alone is not sufficient to cause WS. A working model predicts that modification of cell cycle control of CLP production is an important determinant of the phenotypic innovation. Analysis of >30 kb of DNA encoding traits required for expression of the WS phenotype, including a regulatory locus, has not revealed the mutational causes, indicating a complex genotype-phenotype map.     

Strain improvement of Penicillium janthinellum NCIM 1171 for increased cellulase production

The strain of Penicillium janthinellum NCIM 1171 was subjected to mutation involving treatment of Ethyl Methyl Sulfonate (EMS) for 24h followed by UV-irradiation for 3min. Successive mutants showed enhanced cellulase production (EMS-UV-8), clearance zone on Avicel containing plate (SM2) and rapid growth on Walseth cellulose agar plates containing 0.2% 2-deoxy-d-glucose (SM3). These mutants were transferred to Walseth cellulose plates containing higher concentration (1.5%) of 2-deoxy-d-glucose (SM4) in which only five mutants showed clearance zone on SM4. All these mutants showed approximately two-fold increase in activity of both FPase and CMCase in shake flask culture when grown on basal medium containing CP-123 (1%) and wheat bran (2.5%). The enzyme preparations from these mutants were used to hydrolyze Avicel. Higher hydrolysis yields of Avicel were obtained with enzyme preparations of EU1. This is the first report on the isolation and selection of mutants based on hydrolysis of Avicel, which is the most crystalline substrate.     

Automated counting of bacterial colony forming units on agar plates

Manual counting of bacterial colony forming units (CFUs) on agar plates is laborious and error-prone. We therefore implemented a colony counting system with a novel segmentation algorithm to discriminate bacterial colonies from blood and other agar plates.A colony counter hardware was designed and a novel segmentation algorithm was written in MATLAB. In brief, pre-processing with Top-Hat-filtering to obtain a uniform background was followed by the segmentation step, during which the colony images were extracted from the blood agar and individual colonies were separated. A Bayes classifier was then applied to count the final number of bacterial colonies as some of the colonies could still be concatenated to form larger groups. To assess accuracy and performance of the colony counter, we tested automated colony counting of different agar plates with known CFU numbers of S. pneumoniae, P. aeruginosa and M. catarrhalis and showed excellent performance.     

A simple and effective plating method to screen polycyclic aromatic hydrocarbon-degrading bacteria under various redox conditions

Agar plates with a polycyclic aromatic hydrocarbon (PAH) layer have been used to screen for microorganisms that degrade PAHs, leaving clear zones around colonies; however, there are several problems with previous methods such as undesired contamination in the fume hood and difficulty in controlling the amount of PAH on the plates. In this study, we developed a modified screening method to address the drawbacks encountered with previous screening methods. A uniform white layer of PAHs was generated by spreading PAHs dissolved in volatile solvents over a surface of solidified agar medium, followed by the evaporation of the solvents. An inoculation was then performed by spreading a molten agar medium containing microbial samples over the solidified agar medium with a PAH layer. Subsequently, the white PAH layer migrated to the surface of the molten agar medium. This essential modification enabled us not only to solve problems of the previous screening methods but also to prepare an agar plate with a PAH layer without a complicated experimental scheme in the anaerobic chamber. After solidification of the molten agar medium and incubation of the plates, clear zones were successfully detected around colonies with aerobic and anaerobic PAH-degrading microbial cultures.     

Prescreening bacterial colonies for bioactive molecules with Janus plates, a SBS standard double-faced microbial culturing system

Despite the availability of many culture-based antibiotic screening methods, the lack of sensitive automated methods to identify functional molecules directly from microbial cells still limits the search for new biologically active compounds. The effectiveness of antibiotic detection is influenced by the solubility of the assayed compounds, indicator strain sensitivity, culture media and assay configuration. We describe a qualitative high throughput screening system for detecting cell-perturbing molecules from bacterial colonies employing two opposed agar layers sequentially formed in prototype Society for Biomolecular Screening (SBS) plates, named Janus plates. Direct assay of microbial colonies against target organisms in opposed agar layers overcomes some of the limitations of agar overlay methods. The system enables the rapid detection of extracellular cell-perturbing molecules, e.g., antibiotics, excreted directly from environmental isolates. The source bacterial colonies remain separate from the target organism. The growth layer is prepared and grown independently, so environmental strains can be grown for longer intervals, at temperatures and in media that favor their growth and metabolite expression, while the assay layer with pathogens, usually requiring nutrient-rich medium and elevated temperatures, are added later. Colonies to be tested can be precisely arrayed on the first agar surface, thus avoiding dispersion and disturbance of potential antibiotic-producing colonies by overlaying agar with the target strain. The rectangular SBS configuration facilitates factorial replication of dense microbial colony arrays for testing with multiple assays and assay conditions employing robotic colony pickers and pin tools. Opposed agar layers only slightly reduced the effectiveness for detecting growth inhibition from pure antibiotics compared to single-layer agar diffusion assays. The Janus plate enabled an automation-assisted workflow where a lone operator can effectively identify and accumulate bioactive soil bacterial strains within a few weeks. We also envisage the method’s utility for functional prescreening colonies of clones from genomic and metagenomic libraries or improved strains originating from mutagenized cells.     

Colony spreading in Staphylococcus aureus

Wild-type Staphylococcus aureus rapidly expands on the surface of soft agar plates. The rates of expansion and the shapes of the resultant giant colonies were distinct for different strains of laboratory stocks and clinical isolates. The colony spreading abilities did not correlate with the biofilm-forming abilities in these strains. Insertional disruption of the dltABCD operon, which functions at the step of D-alanine addition to teichoic acids, and of the tagO gene, which is responsible for the synthesis of wall teichoic acids, decreased the colony spreading ability. The results indicate that wall teichoic acids and D-alanylation of teichoic acids are required for colony spreading.     

Colony formation of mammalian cells on agar plates and its application to Lederberg's replica plating

In this paper we describe a new technique of cloning by use of agar plates and its application to replica plating. It was found that most cell lines form colonies on the surface of solid agar, although the plating efficiency and size of colony is dependent on specimens and concentrations of agar and agarose used. When 0.5% Noble-agar was used as substrate, plating efficiencies were obtained comparable to those of conventional cloning techniques in liquid medium and of agar suspension cultures. In some cases, including the primary culture of Yoshida sarcoma, the efficiency of plating was apparently higher than that obtained by the already established procedures. In an experiment with a series of BHK-21 cells, it was found that virally transformed cells could form colonies on agar plate, whereas untransformed and reverted cells could not divide, suggesting that agar plate culture, as well as agar suspension culture, can be used for a selective assay of transformation.Two methods of replica plating were employed. Method I is that devised by Lederberg in which colonies on the master plate are imprinted on pile fabrics and then transferred to the replica plates. With FM3A cells, the fidelity of replica plating was around 95%. Method II is inoculation of clones by applying a glass rod to the replica plates on which positions of inocula were identified by a grid. Fidelity of replica plating of FM3A, L5178Y and YSC cells was 99.7, 100 and 100% respectively.     

Improved Rapid Plate Method for the Isolation of Bacteriophages from Lysogenic Bacteria

A modification of a recently reported rapid plate method for the isolation of bacteriophages from lysogenic bacteria is described. The velveteen replica plate technique was used for inoculation of mitomycin C-induced colonies onto agar plates, and tetrazolium chloride was used to enhance detection of phage activity on replicated indicator plates.     

Hypermotility in Clostridium perfringens Strain SM101 Is Due to Spontaneous Mutations in Genes Linked to Cell Division

Clostridium perfringens is a Gram-positive anaerobic pathogen of humans and animals. Although they lack flagella, C. perfringens bacteria can still migrate across surfaces using a type of gliding motility that involves the formation of filaments of bacteria lined up in an end-to-end conformation. In strain SM101, hypermotile variants are often found arising from the edges of colonies on agar plates. Hypermotile cells are longer than wild-type cells, and video microscopy of their gliding motility suggests that they form long, thin filaments that move rapidly away from a colony, analogously to swarmer cells in bacteria with flagella. To identify the cause(s) of the hypermotility phenotype, the genome sequences of normal strains and their direct hypermotile derivatives were determined and compared. Strains SM124 and SM127, hypermotile derivatives of strains SM101 and SM102, respectively, contained 10 and 6 single nucleotide polymorphisms (SNPs) relative to their parent strains. While SNPs were located in different genes in the two sets of strains, one feature in common was mutations in cell division genes, an ftsI homolog in strain SM124 (CPR_1831) and a minE homolog in strain SM127 (CPR_2104). Complementation of these mutations with wild-type copies of each gene restored the normal motility phenotype. A model explaining the principles underlying the hypermotility phenotype is presented.     

Random amplified polymorphic DNA (RAPD) analysis reveals extensive natural chimerism in a marine protochordate

Random amplified polymorphic DNA (RAPD) analysis was applied to individual modules (zooids) of a colonial ascidian to investigate the presence and extent of chimerism, the parabiotic association of different genetic entities. The technique proved to be rapid and efficient for distinguishing different genotypes present in a colony, and revealed genetic mosaicism in wild material, as well as in laboratory cultures following planned fusion. Approximately one-third of colonies in the natural population studied possessed multiple genotypes, presumably as the result of fusion of different colonies. Furthermore, individual zooids of different genetic origin often intermingled after colony fusion, spreading each genotype throughout a larger total area.     

Development of a Method for Detection of Lactic Acid Bacteria Producing Exclusively the l-(+)- Isomer of Lactic Acid

A method was developed for the detection and isolation, within a population of lactic acid bacteria, of strains producing exclusively the l-(+)- isomer of lactic acid; the visual detection of colonies of these particular strains can be carried out directly on agar plates (50 to 70 colonies per plate). The method is based on an enzymatic stereospecific reaction involving d-(-)-lactate dehydrogenase and linked to a staining reaction; the diffusion area of the d-(-)- isomer stains red around the d-(-)- and the dl-lactic acid-producing colonies, while the colonies producing exclusively l-(+)-lactic acid are detected by the absence of the colored halo. The intensity of staining was increased when cellulose powder and Tween 20 were added to the agar medium.     

Identification of a major glucose transporter in Flavobacterium johnsoniae: Inhibition of F. johnsoniae colony spreading by glucose uptake

Many members of the phylum Bacteroidetes, such as Flavobacterium johnsoniae, can glide over a solid surface: an ability called gliding motility. It can be usually observed on agar plates as thin, flat, spreading colonies with irregular, feathery edges; this phenomenon is called colony spreading. Colony spreading of F. johnsoniae on 1.5% agar plates containing poor nutrients is dose‐dependently inhibited by addition of D‐glucose, as previously reported. Accordingly, here, we created mutants (by transposon mutagenesis) that partially suppressed glucose‐mediated inhibition of colony spreading. Among the isolates, we found that one had a transposon insertion in Fjoh_4565, tentatively named mfsA, which encodes a major facilitator superfamily (MFS) transporter previously shown to be required for growth on glucose, N‐acetyl‐glucosamine, and chitin. We constructed an mfsA deletion mutant and found that the mutant showed no glucose‐mediated acceleration of growth or glucose uptake. The mfsA gene complemented the phenotype of a glucose‐negative Escherichia coli. These results suggest that the mfsA gene encodes the sole MFS transporter of glucose in F. johnsoniae and that glucose uptake is partially required for the glucose‐mediated inhibition of F. johnsoniae colony spreading.

     

A visual method for rapid screening of xylose-fermenting ethanolic strains of Klebsiella pneumoniae.

A simple and rapid screening method for selecting hyper-ethanolic strains of Klebsiella pneumoniae is described. The method involves a novel biological screening marker, namely, the yeast Candida ethanothermophilum. The screening marker was seeded on an agar plate to the surface of which agar blocks, each containing a colony of K. pneumoniae, were subsequently fixed. This seeded plate lacked sources of carbon and energy. Ethanol formed in the agar blocks by the K. pneumoniae colonies diffused into the seeded medium and served as a carbon and energy source for the ethanotrophic yeasts. Colonies of yeasts appeared around the agar blocks in regions of ethanolic diffusion. Hyper-ethanolic strains of K. pneumoniae were thus selected on the basis of the number of colonies of the screening marker that appeared around the blocks containing the ethanolic colonies of K. pneumoniae.     

Analysis of recombinant protein expression by MALDI-TOF mass spectrometry of bacterial colonies

E. coli expressing soluble recombinant HIV antigens were analyzed directly by MALDI-TOF mass spectrometry (MS) from bacterial colonies picked from agar plates. An HIV envelope (ENV) antigen construct, penvA, was expressed in E. coli by transformation of the plasmid pPL/penvA-M. The plasmid was co-transformed into E. coli DH5 alpha cells with an equal quantity of the plasmid pKRR826, the parent vector without the penvA insert, and plated at medium density on L-agar plus ampicillin plates. A total of 24 colonies from four agar plates (six colonies per plate) were picked and transferred into 50% acetonitrile--0.1% trifluoroacetic acid aliquots for analysis by MALDI-TOF MS. The MS analysis detected 10 of 24 colonies expressing the recombinant protein; one colony expressed a mutant penvA protein; eleven of 24 colonies showed ions only from E. coli; and two of 24 colonies showed no detectable proteins. When E. coli transformed only with plasmid pPL/penvA-M were examined, all (10 of 10) colonies showed the penv insert by the MALDI-TOF MS method. The method is fast (less than 1.5 h for 24 colonies) and allows identification of colonies expressing intact or mutant proteins directly from culture plates without sample purification.     

Tandem coagulase/thermonuclease agar method for the detection of Staphylococcus aureus.

In optimizing previously reported coagulase agar media to obtain a rapid, reliable, and inexpensive coagulase test agar, variations in plasmas, pH, buffer system, fibrinogen, and fibrinolytic inhibitor were investigated. The agar with the following composition was determined best for the demonstration of coagulase production by Staphylococcus aureus: 25 ml of 15% bovine fibrinogen (fraction I, type I, citrated, Sigma Chemical Co.), 25 ml of rehydrated rabbit plasma (coagulase plasma ethylenediaminetetraacetic acid, Difco), 10.0 mg of soybean trypsin inhibitor (Schwarz/Mann), and 450 ml of brain heart infusion agar (Difco). In additional studies involving 7 different temperatures and 11 heating times, the thermal destruction of microbial nucleases on plate count agar and coagulase test agar was investigated. Heating the plates for 2.5 h at 65 degrees C destroyed all heatlabile nucleases, but not thermonucleases of S. aureus. A tandem agar plate method for the identification of S. aureus was developed. Coagulase and thermonuclease activity of 50 colonies can be detected on a single agar plate. Suspect S. aureus colonies isolated on various selective media are transferred to coagulase test agar, the plates are incubated at 37 degrees C for 18 h, and the coagulase reaction is recorded. The plates are then heated at 65 degrees C for 2.5 h, overlaid with toluidine blue-metachromatic diffusion agar, and reincubated at 37 degrees C for 3 h, and the thermonuclease reaction is recorded. Studies based on 88 enterotoxigenic S. aureus strains and 133 and 48 suspect S. aureus strains isolated from fresh salami mixtures on mannitol salt and tellurite-polymyxin-egg yolk agars, respectively, demonstrated 100% agreement between the tandem agar plate method and standard coagulase and thermonuclease tests. Overall, the tandem agar plate method is a rapid and convenient approach contributing to the identification of S. aureus from foods.     

Application of robotics and image processing to automated colony picking and arraying.

We describe a system that applies image processing and robotic techniques to automatically pick individual colonies from square petri dishes and array them in 96-well microtiter plates. Digital images of the colony distribution in the dishes are acquired using a video camera and frame buffer. Commercial image processing software is used to identify individual colonies and determine their locations. A Hewlett-Packard Microassay System robot reads the resulting coordinate file for each dish, picks cells from each identified colony and transfers the cells into a microtiter plate well. A disposable pipet tip is used as the sterile implement for colony picking. Custom holders position the dishes accurately and provide common coordinate systems for imaging and picking. The system is calibrated to account for the depth of agar in the dishes. The robot can process up to 10 dishes and 20 plates (1920 colonies) in a single run. It has successfully arrayed a cosmid library of the S. pombe genome consisting of approximately 6000 colonies in 30 petri dishes in about 40 hours of robot time. Future enhancements to the system are discussed.     

CAS平板覆盖法检测氢氧化细菌铁载体

【目的】用CAS平板覆盖法检测氢氧化细菌铁载体,解决通用CAS琼脂平板法中十六烷基三甲基溴化铵对真菌和某些细菌的生长抑制问题。【方法】将改良的CAS检测培养基覆盖在长满菌落的无铁培养基上,生长抑制问题因微生物未与十六烷基三甲基溴化铵直接接触而解决。【结果】3株氢氧化细菌SDW-5、SDW-9和AaP-13均能产生单菌落,加入CAS检测培养基1 h后,菌落周围产生明显的铁载体晕圈。【结论】本方法成功解决了生长抑制问题,可以作为检测微生物铁载体的通用方法。     

Requirement of the agr locus for colony spreading of Staphylococcus aureus

The important human pathogen Staphylococcus aureus is known to spread on soft agar plates. Here, we show that colony spreading of S. aureus involves the agr quorum-sensing system. This finding can be related to the agr-dependent expression of biosurfactants, such as phenol-soluble modulins, suggesting a connection between spreading motility and virulence.     

Relationship between colonial surface and density on agar plate

The size of a colony on an agar plate is influenced by the number of colonies ( N ) on this plate. When N is small, colonies reach a larger size. The relationship between colonial surface and density of bacteria on agar plate was studied for nine bacterial and two yeast strains. A mathematical model describing the relationship between the logarithm base 10 of the number of colonies on the agar plate and the average colonial diameter was built. This model is shown to be adapted to most of the strains studied and could be a tool for media quality control.