Optical forward-scattering for detection of Listeria monocytogenes and other Listeria species

We demonstrate here the development of a non-invasive optical forward-scattering system, called 'scatterometer' for rapid identification of bacterial colonies. The system is based on the concept that variations in refractive indices and size, relative to the arrangement of cells in bacterial colonies growing on a semi-solid agar surface will generate different forward-scattering patterns. A 1.2-1.5mm colony size for a 1mm laser beam and brain heart infusion agar as substrate were used as fixed variables. The current study is focused on exploring identification of Listeria monocytogenes and other Listeria species exploiting the known differences in their phenotypic characters. Using diffraction theory, we could model the scattering patterns and explain the appearance of radial spokes and the rings seen in the scattering images of L. monocytogenes. Further, we have also demonstrated development of a suitable software for the extraction of the features (scalar values) calculated from images of the scattering patterns using Zernike moment invariants and principal component analysis and were grouped using K-means clustering. We achieved 91-100% accuracy in detecting different species. It was also observed that substrate variations affect the scattering patterns of Listeria. Finally, a database was constructed based on the scattering patterns from 108 different strains belonging to six species of Listeria. The overall system proved to be simple, non-invasive and virtually reagent-less and has the potential for automated user-friendly application for detection and differentiation of L. monocytogenes and other Listeria species colonies grown on agar plates within 5-10 min analysis time.     

Growth kinetics by scanning of granulocytic cell colonies in glass capillaries.

Pure granulocytic colonies were cultivated from mouse bone marrow cells in agar contained in glass capillary tubes using mouse embryo conditioned medium as colony stimulating activity. A random distribution of colonies along the agar gels was achieved under controlled conditions. Only 3 capillaries were needed for a coefficient of variation around 5% provided at least 104 cells were seeded per capillary. The daily growth of single colonies within an agar capillary was followed, using the light scattering properties of the colonies for automatic scanning. The position of the colonies in the capillary greatly affected the scan signal; the consequences of positional changes were studied in detail. Using the mean peak height as growth parameter, the onset of measureable granulocytic colony growth was found between day 2 and 3, the maximum colony size was reached between day 7 and 9, after which the colonies decayed. Other parameters such as colony count and total peak are were determined and their sinificance discussed.     

Enumeration of Escherichia coli in Frozen Samples After Recovery from Injury

More than 90% of the surviving cells of Escherichia coli NCSM were injured after freezing in water at -78 C. Injury was determined by the ability of cells to form colonies on Trypticase soy agar with yeast extract but not on violet red-bile agar and deoxycholate-lactose agar. Exposure of the injured cells to Brilliant Green-bile broth and lauryl sulfate broth prevented subsequent colony formation on Trypticase soy agar with yeast extract. The freeze-injury could be repaired rapidly in a medium such as Trypticase soy broth with yeast extract (TSYB). The repaired cells formed colonies on violet red-bile agar and deoxycholate-lactose agar and were not inhibited by Brilliant Green-bile broth and lauryl sulfate broth. At least 90% of the cells repaired in TSYB within 30 min at 20 to 45 C and began multiplication within 2 h at 25 C. When the cells were frozen in different foods, 60 to 90% of the survivors were injured. Repair of the injured cells occurred in foods during 1 h at 25 C, but generally repair was greater and more reproducible when the foods were incubated in TSYB. The study indicated that the repair of freeze-injured coliform bacteria should be accomplished before such cells are exposed to selective media for their enumeration.     

Biodegradation of triazine herbicides on polyvinylalcohol gel plates by the soil yeast Lipomyces starkeyi

The soil yeast Lipomyces starkeyi was tested for its ability to degrade triazine herbicides. Polyvinylalcohol (PVA) was employed as a solid medium in culture plates instead of agar. The cell sizes of the control (without nitrogen source) on the PVA gel plate were much smaller than those on the agar gel plate. The difference between the diameters of the sample and control colonies on the PVA gel plate were almost twice those of the colonies on the agar gel plate (1.9 and 1.0 mm, respectively). Thus, the PVA gel plate is much better than the agar plate for evaluating the degree of utilization of a sole nitrogen source. The yeast grew well (more than 4 mm in diameter) with 1,3,5-triazine or cyanuric acid as nitrogen source. In addition, melamine and thiocyanuric acid inhibited growth of the yeast, and the sizes of colonies were smaller than those of the control. All triazine herbicides tested (simazine, atrazine, cyanazine, ametryn, and prometryn) could be degraded and assimilated by L. starkeyi.     

Direct identification of pure Penicillium species using image analysis

This paper presents a method for direct identification of fungal species solely by means of digital image analysis of colonies as seen after growth on a standard medium. The method described is completely automated and hence objective once digital images of the reference fungi have been established. Using a digital image it is possible to extract precise information from the surface of the fungal colony. This includes color distribution, colony dimensions and texture measurements. For fungal identification, this is normally done by visual observation that often results in a very subjective data recording. Isolates of nine different species of the genus Penicillium have been selected for the purpose. After incubation for 7 days, the fungal colonies are digitized using a very accurate digital camera. Prior to the image analysis each image is corrected for self-illumination, thereby gaining a set of directly corresponding images with respect to illumination. A Windows application has been developed to locate the position and size of up to three colonies in the digitized image. Using the estimated positions and sizes of the colonies, a number of relevant features can be extracted for further analysis. The method used to determine the position of the colonies will be covered as well as the feature selection. The texture measurements of colonies of the nine species were analyzed and a clustering of the data into the correct species was confirmed. This indicates that it is indeed possible to identify a given colony merely by macromorphological features. A classifier (in the normal distribution) based on measurements of 151 colonies incubated on yeast extract sucrose agar (YES) was used to discriminate between the species. This resulted in a correct classification rate of 100% when used on the training set and 96% using cross-validation. The same methods applied to 194 colonies incubated on Czapek yeast extract agar (CYA) resulted in a correct classification rate of 98% on the training set and 71% using cross-validation.     

pH gradients through colonies of Bacillus cereus and the surrounding agar.

pH-sensitive microelectrodes, constructed with a tip diameter of about 4 microns, were deployed through 24 h and 48 h colonies of Bacillus cereus incubated on CYS medium (Casamino acids, yeast extract, salts), with and without glucose. Measurements of pH were used to construct pH profiles through the colony and the surrounding agar. pH gradients could be detected for at least 800 microns into the agar beneath a 24 h colony, and to approximately 10 mm horizontally away from the edge of the colony. In older colonies, the lateral gradient extended for over 20 mm. The pH of the underlying agar was increased by up to 1.45 pH units after 48 h growth without glucose. When colonies were grown with glucose, a significant area of acidification was observed within the colony in addition to a zone of alkalinization present at its periphery. Acidification was thought to be due to the anaerobic fermentation of glucose producing organic acids whilst alkalinization was due to the aerobic oxidation of amino acids releasing ammonia.     

Induction of L-Phase Variant from Protoplast of Staphylococcus aureus

Protoplasts of Staphylococcus aureus 209P and Cowan 1 were induced by treatment with lysostaphin. These protoplasts were sensitive to detergent, a low concentration of sodium chloride and low temperature. Almost all protoplast cells spread on CLYS agar medium (casein hydrolysate, yeast extract, Na-lactate, and NaCl) formed typical L-form colonies. Horse serum (0.25%) and Mg²⁺ (109 mm) are essential factors for formation of the L-form colonies of 209P. In the case of Cowan 1, Mg²⁺ was not required. The active factor(s) in horse serum was heat-resistant and protein in nature.     

Effect of yeast extract on fluoranthene degradation and aromatic ring dioxygenase expressing bacterial community structure of a fluoranthene degrading bacterial consortium

Fluoranthene (Fla) is a high molecular weight polycyclic aromatic hydrocarbon that exerts hazardous effects on living organisms. An efficient Fla degrading bacterial consortium LP was enriched from an oil contaminated soil sample, with and without yeast extract as a supplement. Objective of the present study was to see if there was any differential effect of yeast extract addition on Fla degradation potential and aromatic ring dioxygenase expressing bacteria (ARDB) of the enrichments. Primary enrichment of the soil sample was carried out in minimal salt medium (MSM) added with 500 mg l⁻¹ Fla and 0.05% yeast extract (YMSM). Secondary, tertiary and subsequent enrichments were prepared in YMSM and MSM after every sixteen days of incubation. Fla was efficiently degraded by YMSM enriched culture than MSM enriched culture. However, when MSM enrichment was incubated longer instead of further subculturings, it also degraded Fla efficiently. All three enrichments exhibited growth of bacterial colonies on Fla sprayed minimal agar plates however only YMSM enrichment showed clear zone forming bacterial colonies. A positive effect was observed of yeast extract on ARDB population of LP consortium. To our limited knowledge this is first time that effect of yeast extract on ARDB population was studied.     

Colony pattering ofAspergillus oryzae on agar media

To clarity the effects of nutrient concentration and diffusion on the pattern formation of fungal colonies, the colony patterning ofAspergillus oryzae at various nutrient and agar levels was studied experimentally and was summarized in a colony morphology diagram. Roles of the nutrient content and the relaxation of nutrient distribution on the colony patterning were discussed based on a computer model of the mycelial growth. The colony morphology changed from compact to ramified as the nutrient and agar levels were lowered. No clear boundary was found between these two morphologies. The deterioration of substrate around the growing colony was detected when the morphic switching from homogeneous into splitting patterns emerged in the growth of ramified colonies. In the mycelial growth model, dense compact colonies developed at low growth rates and high nutrient influx into the colonized area. Under low nutrient levels, splitting colonies appeared at high growth rates as compared with the nutrient influx.     

Dichloran as an Inhibitor of Mold Spreading in Fungal Plating Media: Effects on Colony Diameter and Enumeration

Problems associated with overgrowth by spreading molds are not addressed by currently recommended fungal enumeration media. Twenty-two fungi, including 12 mold and 3 yeast genera, were evaluated for the effects of dichloran (2,6-dichloro-4-nitroaniline), previously identified as a mold-spreading inhibitor, on colony diameter and enumeration. On malt agar or antibiotic-potato-dextrose agar (APDA), colony diameters were effectively reduced when dichloran was added. Colony diameters decreased as the dichloran concentration increased. Counts obtained with mixed mold spore suspensions were lower on APDA supplemented with 25 μg of dichloran per ml than on APDA and were higher than APDA with the addition of 5 μg of dichloran per ml (APDA-D-5). Overall counts of mixed and individual mold spore and yeast suspensions were higher in APDA-D-5 than in APDA. The additional advantages of APDA-D-5 may be useful in routine enumeration of fungi.     

Improvement of human melanoma colony formation in soft agar using boiled instead of autoclaved agar

The effect of agar sterilized by either boiling or autoclaving on human melanoma colony formation in soft agar was compared using cells from 17 biopsies of metastatic malignant melanoma. The frequency of colony formation was significantly increased for cells grown in boiled agar in 8 samples (47%), unchanged in 8 samples (47%), and decreased in only one sample (6%). There were increases in both cluster and colony formation for the melanomas which had augmented colony formation when grown in boiled agar. There was also qualitative morphological improvement, including rounder, smoother cells and less extracellular debris surrounding the colonies. These data suggest that melanoma colony formation is enhanced when cells are grown in agar which has been sterilized by boiling rather than autoclaving.     

Influence of Structural Properties and Kinetic Constraints on Bacillus cereus Growth

The influence of structural properties and kinetic constraints on the behavior of Bacillus cereus was investigated on agar media. Dimensional criteria were used to study the growth in bacterial colonies. The architecture of the agar gel as modified by the agar content was found to influence the colony size, and smaller colonies were observed on media containing 50 to 70 g of agar liter⁻¹. Except at low nutrient levels, colonies responded to nutrient gradients by decreasing in size the farther away they were from the nutrient source, and the decrease in colony size was influenced by the agar content. The diffusivities of glucose and a protein (insulin-like growth factor) were not affected by the gel architecture, suggesting that other factors, such as mechanical factors, could influence microbial growth in the agar systems used. Increasing the viscosity of the liquid phase of the agar media by adding polyvinylpyrrolidone resulted in a reduction in colony size. When the agar concentration was increased, the colony areas were not influenced by the viscosity of the system.     

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.     

Wall mannoproteins in cells from colonial phenotypic variants of Candida albicans

Candida albicans ATCC 26555 switched at high frequency (10(-1) to 10(-3)) between several phenotypes identified by colony morphology on a defined mineral amino-acid-containing agar medium supplemented with arginine and zinc (LAZ medium). When cells taken from colonies exhibiting distinct morphologies were plated directly onto LAZ agar, spontaneous conversion to all the variant phenotypes occurred at combined frequencies of 2.1 x 10(-1) to 9.5 x 10(-3). However, when cells taken from the different colonial phenotypes were plated directly onto an undefined medium (yeast extract/peptone/dextrose; YPD medium), or first incubated in liquid YPD medium and then cloned on YPD agar, all colonies observed exhibited the same phenotype (smooth-white). When cells from the smooth-white colonies were plated as clones on LAZ agar, the original switch phenotype reappeared. These results suggest that environmental conditions such as the growth medium (and possibly the temperature) influence switching by suppressing phenotype expression, but have no effect on genotype. The variant colony morphologies also appeared to be associated with differences in the relative proportions of yeast and mycelial cells. Zymolyase digests of wall preparations obtained from cells belonging to different colonial phenotypes were analysed by SDS-PAGE. After blotting to nitrocellulose paper, the mannoproteins were stained with Concanavalin A, with a polyclonal antiserum enriched in antibodies against mycelium-specific wall components, and with a monoclonal antibody raised against a high-molecular-mass mannoprotein band (260 kDa) specific to the walls of mycelial cells. The results suggest that phenotypic switching might be associated with changes in the degree of glycosylation in high-molecular-mass mannoproteins, or in the way these mannoproteins are bound to other cell wall components.     

Colony variation in Sinorhizobium meliloti inoculant strain U 45

A culture of Sinorhizobium meliloti strain U 45, maintained on yeast extract-mannitol (YM) agar, produced a mixture of Congo red-absorbing (R1) and non-absorbing (W1) colonies when grown on YM medium containing Congo red. The original freeze-dried (FD) culture formed gummy (G), white (W2) and small red (R2) colony types on the above medium. All colonies were stable except G, which segregated into G and W2-like types. Immune diffusion patterns of all colony types were identical. The W1 colony type dominated R1 when a 1:1 combination was sub-cultured on YM agar. The parent cultures and their variants exhibited a range of N₂-fixing effectiveness and competitiveness when inoculated onto two cultivars of Medicago sativa. Variant R2 from the FD culture was ineffective on both cultivars. Genomic DNA fingerprinting with insertion elements ISRm3 and ISRm2011-2 suggested that transposition of these elements was not a cause of variation, but a DNA band was absent in the profiles of two out of three W2-like colonies. Protein profile comparisons showed high similarity (r = 0.98) between the colony types when grown in YM broth. When grown on Tryptone-Yeast extract medium, variants from the FD and agar-maintained cultures formed separate clusters with r = 0.79. Polymerase chain reaction fingerprinting using repetitive, site-directed and arbitrary primers failed to differentiate the variants. The results emphasize the need to monitor culture variability to maintain the quality of legume inoculants.     

Yeasts associated with the infrabuccal pocket and colonies of the carpenter ant Camponotus vicinus

After scanning electron microscopy indicated that the infrabuccal pockets of carpenter ants (Camponotus vicinus) contained numerous yeast-like cells, yeast associations were examined in six colonies of carpenter ants from two locations in Benton County in western Oregon. Samples from the infrabuccal-pocket contents and worker ant exoskeletons, interior galleries of each colony, and detritus and soil around the colonies were plated on yeast-extract/ malt-extract agar augmented with 1 M hydrochloric acid and incubated at 25 C. Yeasts were identified on the basis of morphological characteristics and physiological attributes with the BIOLOG(?) microbial identification system. Yeast populations from carpenter ant nest material and material surrounding the nest differed from those obtained from the infrabuccal pocket. Debaryomyces polymorphus was isolated more often from the infrabuccal pocket than from other material. This species has also been isolated from other ant species, but its role in colony nutrition is unknown.     

WL营养琼脂对葡萄酒相关酵母的鉴定效果验证

利用WL营养琼脂对采自葡萄园和葡萄汁发酵过程中的35株酵母菌进行了分类鉴定,同时进行了5.8S-ITS和26S rDNA D1/D2区的扩增与测序。结果表明利用WL营养琼脂的鉴定结果与测序结果基本符合。WL营养琼脂是一种较为有效的葡萄酒相关酵母菌的分类鉴定培养基。     

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.     

Agar Concentration in Counting Clostridium Colonies

Decreasing the agar concentration of a counting medium from the usual 1.5% resulted in larger colonies with less interference from gas in Clostridium botulinum 115B and C. sporogenes PA 3679. Optimal agar concentration was 0.65% for C. botulinum with 24-hr incubation and 0.50% for C. sporogenes with 48-hr incubation. Lower concentrations yielded growth too diffuse for counting. Motility was considered the explanation for increased colony size in softer agar. The greater the degree of motility, the greater would be the diffusibility expected, and thus the higher the agar concentration required to insure discrete colonies. For quantitating motility, evaluations were made by use of microscopic examination of liquid cultures and rate of diffusion in a semisolid medium. With both criteria, the degree of motility of C. botulinum 115B clearly exceeded that of C. sporogenes PA 3679. Small-colony variants of C. botulinum in 0.65% agar yielded only small colonies on subculture, with a corresponding decrease in degree of motility of the cells by both criteria. Colony size of the nonmotile C. perfringens ATCC 3624 was unaffected by lowered agar concentrations.