A method for comparison of growth media in objective identification of Penicillium based on multi-spectral imaging

We consider the problems of using excessive growth media for identification and performing objective identification of fungi at the species level. We propose a method for choosing the subset of growth media, which provides the best discrimination between several fungal species. Furthermore, we propose the use of multi-spectral imaging as a means of objective identification. Three species of the fungal genus Penicillium are subject to classification. To obtain an objective classification we use multi-spectral images. Previously, RGB images have proven useful for the purpose. We use multi-spectral bands as they provide additional information about the chemistry of the fungal colonies. In this study three media [Czapek yeast extract agar (CYA), oatmeal agar (OAT), and yeast extract sucrose agar (YES)] have been compared on their ability to discriminate between the three species. We propose a statistical method to test which medium or combination of media gives the best discrimination. Statistical tests indicate that YES combined with CYA is the best choice of media in this case. However, for the objective identification one medium is sufficient to discriminate between the species. Statistical tests show that there are significant differences between the species on all individual media, and that these differences are largest on YES. The objective identification has been performed solely by means of digital image analysis. The features obtained from the image analysis merely correspond to macro-morphological features. The species have been classified using only 3-4 of the spectral bands with a 100% correct classification rate using both leave-one-out cross-validation and test set validation.     

Visual clone identification of Penicillium commune isolates

A method for visual clone identification of Penicillium commune isolates was developed. The method is based on images of fungal colonies acquired after growth on a standard medium and involves a high degree of objectivity, which in future studies will make it possible for non-experts to perform a qualified identification of different species as well as clones within a species. A total of 77 P. commune isolates from a cheese dairy were 3-point inoculated on Yeast Extract Sucrose (YES) agar and incubated for 7 days at 25 degrees C. After incubation, the isolates were classified into groups containing the same genotype determined by DNA fingerprinting (AFLP). Each genotype also has a specific phenotype such as different colony colours. By careful image acquisition, colours were measured in a reproducible way. Prior to image analysis, each image was corrected with respect to colour, geometry and self-illumination, thereby gaining a set of directly comparable images. A method for automatic extraction of a given number of concentric regions was used. Using the positions of the regions, a number of relevant features--capturing colour and colour-texture from the surface of the fungal colonies--was extracted for further analysis. We introduced the Jeffreys-Matusitas (JM) distance between the feature distributions to express the similarity between regions in two colonies, and to evaluate the overall (weighted) similarity. The nearest neighbour (NN) classification rule was used. On a dataset from 137 isolates, we obtained a "leave-one-out" cross-validation identification rate of approximately 93-98% compared with the result of DNA fingerprinting.     

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.     

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.     

On the sensitivity of forward scattering patterns from bacterial colonies to media composition

Morphology of colonies is important for taxonomy and diagnostics in microbiology where the response to environmental factors is sensitive enough to support discrimination. In this research, we analyzed the forward scattering patterns of individual Escherichia coli K12 colonies when agar hardness and nutrition levels were varied from the control sample. As the agar concentration increased from 1.2% to 1.8%, the diameter of the forward scattering patterns also increased for the same experimental condition which reflects that the colony thickness at the apex is greater for increased agar concentrations. Regarding nutrition, increasing dextrose resulted in smaller mean colony diameters while the mean diameters of the colonies were proportional to the yeast extract concentration up to 0.5%. The result reveals that ±0.3% agar concentration from the control sample is sufficient to create variations in the scattering patterns. For nutrition –0.25% of yeast extract showed significant variations while +0.25% from control sample showed minimal variations. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

First Isolation of Cryptococcus magnus from a Cat

A 6-month-old male Japanese domestic cat with otitis externa due to Aspergillus fumigatus was treated with antifungal agents for 25 days and appeared to be cured. Many yeast colonies however developed from the ear canal samples on Sabouraud's dextrose agar at 27 degrees C for 5 days, instead of A. fumigatus. This yeast colony was cream-colored and slim in texture with smooth and highly glossy surface after 5-day incubation on Sabouraud's dextrose agar at 27 degrees C. The isolate was identified as Cryptococcus magnus by mycological analysis and 28S ribosomal analysis.     

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.     

Performance of Selective and Differential Media in the Primary Isolation of Yeasts from Different Biological Samples

In view of the increase in yeast infections, especially polymicrobial ones, differential culture media have acquired increasing importance. The present study evaluated the Sabouraud chloramphenicol, Biggy agar, Pagano Levin agar and CHROMagar Candida media in terms of isolation, number of yeast colony forming units per plate, and inhibition of bacteria and filamentous fungi. To this end, we used 223 biological samples, including feces, and oral, vaginal and anal mucosae from 86 patients presenting or not symptoms of fungal infections. The four media did not differ significantly in terms of detection of yeast-positive cultures. The number of colony forming units per plate ranged from zero to 2.380, with a predominance of counts of 1 to 9 colonies per plate. No significant differences were observed among the four culture media in terms of number of colonies counted, for each kind of biological material. Fifteen species belonging to the genera Candida, Saccharomyces, Cryptococcus, Trichosporon and Rhodotorula were isolated, with C. albicans being the predominant species, followed by C. parapsilosis and R. rubra. CHROMagar Candida and Biggy agar were complementary in the isolation of the different species and favored a greater recovery of polymicrobial cultures. Pagano Levin agar isolated the smallest variety of species. Sabouraud chloramphenicol agar was the least effective in terms of bacterial inhibition and favored a greater development of filamentous fungi. The results suggest that more than one culture medium should be used for an adequate primary isolation.     

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.     

Uptake of Glucose and Phosphorus by Growing Colonies of Fusarium oxysporum as Quantified by Image Analysis

Olsson, S. 1994. Uptake of glucose and phosphorus by growing colonies of Fusarium oxysporum as quantified by image analysis. Experimental Mycology 18, 33-47. The simplest of all heterogeneous environments for fungal colony growth is the petri dish with an agar medium. As the colony grows there will be a depression of nutrient concentrations under the colony caused by the uptake of nutrients by the growing colony. Image analysis methods have been developed for measuring medium concentrations of glucose and phosphorus with simultaneous biomass density determinations in agar systems. Maps of the concentrations in the agar medium under the colony and of colony biomass density were produced. A new method for weighing fungal colonies grown on agar is also presented. For Fusarium oxysporum phosphorus and glucose uptake from the medium was the same irrespective of the C/mineral ratios in the medium within the measured range of ratios. Even the concentration profiles of the nutrients under the colony were the same irrespective of nutrient ratios. Distribution of biomass density was affected by differences in glucose concentrations, being highest at the colony margin at the lower concentrations. The results indicate that the fungal colony is able to take up nutrients at the margin in excess of the local needs.     

Fungal Air Spora at Ibadan, Nigeria

The fungal air spora at Ibadan, Nigeria, was investigated by using Casella Slit Samplers. Three sites, incorporating three locations at each site, were selected for the exposure of replicate plates during sampling. To provide data on a wide range of saprophytic and pathogenic fungal spores, isolations were made on Sabouraud dextrose agar and malt agar plates incubated at 26 and 37 C. Altogether over 60,000 fungal colonies were isolated and counted during the 12-month sampling period. The prevalent fungal genera recorded were: Cladosporum, Curvularia, Fusarium, Aspergillus, Penicillium, Pithomyces, Aureobasidium, Geotrichum, Phoma, Nigrospora, Epicoccum, and Neurospora. The wet and dry seasons (indicated by the temperature, relative humidity, and rainfall data) caused seasonal periodicity in colony numbers. The influence of culture media on the isolated colonies was not significant when the total number of isolated colonies were considered on a monthly basis, but in reviewing a few of the fungal genera there were marked differences between the two media, especially with Pithomyces. Attempts were made to identify some of the isolated colonies by species, e.g., Aspergillus carneus, Aspergillus flavus, Aspergillus fumigatus, Curvularia geniculata, Fusarium oxysporum, Penicillium herquei, Pithomyces chartaum, Rhizopus arrhizus, and Syncephalastrum racemosum. Such identifications provide a basis for further studies on the role of these fungal species in the frontier problem of contamination and biodegradation of drugs and pharmaceuticals, allergies and other problems in the local environment.     

Further Studies on the Suitability of Various Media Containing Antibacterial Antibiotics for the Enumeration of Moulds in Food and Food Environments

Oxytetracycline–glucose–yeast extract agar (OGYA), gentamicin–glucose–yeast extract agar (GGYA) with the antibiotic added separately or sterilized with the medium, chlortetracycline–Rose Bengal agar (CRA), chloramphenicol-streptomycin agar (PYA) and to a lesser extent, oxytetracycline–gentamicin–glucose–yeast extract agar (OGGYA) with or without Rose Bengal added, have been compared for the selective enumeration of moulds in foods. The results obtained from dried cereal products show that the media are almost equally productive and selective when applied to such foods, but Rose Bengal limits the size of mould colonies. When examining fresh proteinaceous foods, such as minced meat and chicken, CR agars and to a certain extent gentamicin-containing agars show the distinct advantage of being much more inhibitory towards the psychrotrophic Gram negative rods that predominate in the associated flora of such foods. Oxytetracycline–glucose–yeast extract agar lost its bacteriostatic properties when heavily challenged with proteinaceous substrates and/or incubated for longer periods at 37° or even at 25°. For such applications chloramphenicol was found to be the antibiotic of choice.     

Optimal growth temperature for the isolation of Plesiomonas shigelloides, using various selective and differential agars.

The growth characteristics of known strains of Plesiomonas shigelloides were compared with those of Aeromonas species (the major competing species in environmental waters) on plesiomonas differential agar, inositol brilliant green bile salt, and modified salmonella-shigella agar at incubation temperatures of 37, 42, and 44 degrees C. Using local isolates from clinical and environmental sources, optimal growth conditions, as determined by colony counts and the colony characteristics, plesiomonas differential agar proved to be ideal when incubated at 44 degrees C. Contrary to earlier recommendations for 48 h incubation, the colonies could be recognized readily after an incubation of 24 h.     

The Identification of Wild Yeast Colonies on Lysine Agar

The most common wild yeasts infecting pressed baker's yeast in Great Britain are Candida tropicalis, C. krusei, C. mycoderma, Trichosporon cutaneum, Torulopsis candida and Rhodotorula mucilaginosa. Wild yeasts are readily detected and quantitatively estimated by plating infected baker's yeast on lysine agar, which permits of only limited growth of baker's yeast.
Morphology of wild yeast colonies on lysine agar is affected by duration of incubation, location in the agar plate, and sometimes by temperature of incubation, density of infection and numbers of baker's yeast cells present. It is therefore possible to identify each species by at least one characteristic type of colony produced under specified conditions. Ability to grow at 30° and 37° serves to distinguish further between certain species.     

Identification of NephrotoxicPenicillium Species from Cereal Grains

A system is described for identifying grain-inhabiting nephrotoxicPenicillium spp. based on their colony characters on Czapek yeast extract agar, yeast extract sucrose agar, and malt extract agar media, and their secondary metabolite profiles on thin layer chromatography plates. Using this system, the identity of 11Penicillium species, or their chemotypes, producing nephrotoxic metabolites could be confirmed. The species areP. verrucosum chemotype I, P.verrucosum chemotype II,P. expansum, P. citrinum, P. aurantiogriseum, P. freii, P. tricolor, P. polonicum, P. viridicatum, P. cyclopium, and P. melanoconldlum. Other non-nephrotoxicPenicillium species present on stored grains were separated from nephrotoxic species by their colony characters and metabolite profiles.     

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.     

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.     

A new preferential medium for enumeration and isolation of desert actinomycetes

In order to facilitate the discovery of novel actinomycetes from the Egyptian deserts, which can be useful as new sources for bioactive metabolites, different media for enumeration and isolation of desert actinomycetes have been tested. For this purpose, 30 soil samples from different six sites representing the Western and Eastern deserts of Egypt were collected. The two deserts are considered hyper-arid and the soil characteristics were determined. The media used were glucose–yeast extract agar, soil extract agar and a new minimal medium (MM) containing glucose, yeast extract and mineral salts. The effects of the soil characteristics on the total viable actinomycete counts on the three media were evaluated. The results showed that the highest actinomycete count in samples from five out of six sites was obtained on MM. Also MM was more selective for actinomycetes and significantly decreased the number of fungal colonies and to a lower extent the number of bacterial colonies. Moreover, it supported the development of different and diverse groups of actinomycetes. From the results obtained in this study, MM is a new useful medium for enumeration and selective isolation of actinomycetes from the desert soils.     

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。