Anomalies in the enumeration of starved bacteria on culture media containing nalidic acid and tetracycline

Culturable counts of antibiotic resistant, genetically engineeredPseudomonas fluorescens were determined on antibiotic-containing plate count agar during starvation in water. Prior to starvation, colony counts obtained on all media separated into two groups. The mean of the colony counts on plate count agar with or without tetracycline (4.9 × 10⁶ ml⁻¹) was significantly higher than the mean colony counts on plate count agar containing either nalidixic acid or nalidixic acid plus tetraclycline (2.5×10⁶ ml⁻¹). After 20 days of starvation the highest mean colony counts continued to be obtained on plate count agar (7.2 × 10⁶ ml⁻¹) with slightly, but significantly, lower counts obtained on plate count agar containing either nalidixic acid (5.6 × 10⁶ ml⁻¹) or tetraclycline (1.5×10⁶ ml⁻¹). A combination of nalidixic acid and tetracycline in plate count agar, however, dramatically reduced colony counts (8.3 × 10² ml⁻¹) after this starvation period. The addition of catalase to plate count agar containing nalidixic acid and tetracycline negated the effect caused by this combination of antibiotics. When colony counts obtained over the entire 20 day incubation were considered, the addition of MgSO₄ to plate count agar containing nalidixic acid and tetracycline resulted in a significant increase in colony counts. Other combinations of antibiotics, nalidixic acid+carbenicillin, nalidixic acid+kanamycin, streptomycin+tetracycline, streptomycin+carbenicillin, rifampicin+tetracycline, rifampicin+carbenicillin, and rifampicin+kanamycin, did not inhibit colony formation of starved cells. Antibiotic resistant strains ofP. putida andEscherichia coli also displayed sensitivity to the combination of nalidixic acid and tetracycline in plate count agar after starvation.     

Genetic variability of aflatoxin B<Subscript>1</Subscript> producing <Emphasis Type="Italic">Aspergillus flavus</Emphasis> strains isolated from discolored rice grains

Twenty-two aflatoxin B₁ (AFB1) producing Aspergillus flavus strains were isolated from 1,200 discolored rice grain samples collected from 20 states across India and tested their potential to produce AFB1 on different agar media. Further these isolates were characterized through randomly amplified polymorphic DNA method. All the strains of A. flavus were produced AFB1 on yeast extract sucrose agar media and none of the strains on A. flavus and A. parasiticus agar. Among the 22 strains, two strains from Tamil Nadu (DRAf 009) and Maharashtra (DRAf 015) produced high amount of AFB1 in all the media tested. To assess the genetic variability in A. flavus, the isolates were analyzed by using random amplified polymorphic DNA markers. Isolates showed 17–80% similarity with standard culture of A. flavus (MTCC 2799).     

Chemical and physiological characterization of taxa in theFusarium sambucinum complex

Forty-one isolates ofFusarium sambucinum sensu lato were screened for production of secondary metabolites in agar cultures. Of 16 strains ofF. sambucinum sensu stricto all but two strains produced diacetoxyscirpenol and two unidentified metabolites, TB1 and TB2 respectively. The two remainingF. sambucinum strains produced T-2 toxin, TB1 and TB2.Fusarium venenotum (6 strains) produced diacetoxyscirpenol and an unidentified metabolite BB.Fusarium torulosum (8 strains) produced wortmannin and antibiotic Y. The three species could be differentiated by their pattern of identified and unidentified metabolites detected by agar plug TLC combined with chemical data from HPLC-diode array detection of fungal extracts, and data on growth rates on potato sucrose agar and tannin sucrose agar.     

An evaluation of straw-extract agar media for the growth and sporulation of Madurella mycetomatis

Four new media, namely Wheat straw extract agar, Bajra straw extract agar, Jowar straw extract agar and Paddy straw extract agar, were evaluated for their potential to stimulate the growth and sporulation of Madurella mycetomatis in comparison with the conventional Sabouraud dextrose agar and Soil extract agar. Vegetative growth of M. mycetomatis on the four types of Straw extract agars was superior to that obtained on Sabouraud dextrose agar. Isolates of M. mycetomatis sporulated better and faster on the Straw extract agars than on the Sabouraud dextrose agar and Soil extract agar. Straw extract agar is recommended as a sporulation medium for M. mycetomatis. It may prove useful especially for studies of the conidium ontogeny of the fungus for elucidating its taxonomic status.     

Evaluation of an aphid‐rearing method using excised leaves and agar medium

The present study evaluated the effectiveness of an aphid‐rearing method devised by Milner in 1981 using Acyrthosiphon pisum and its host plant Vicia faba. In the “agar‐leaf method,” excised leaves of V. faba were attached to the surface of 1% agar gel containing nutrient solution, and test aphids were transferred onto the leaves. Excised leaves grew in size and weight on the agar medium. Fecundity, longevity, body size and developmental time to adulthood were compared between aphids reared using the agar‐leaf method vs. those reared on V. faba seedlings under the same conditions. No significant difference was detected between the two treatments for any of the four parameters, suggesting that the aphids grew and reproduced on excised leaves as successfully as on V. faba seedlings. This method was also useful for inducing males and oviparous females at lower temperature and in short days. Therefore, the present study confirms the effectiveness of using excised leaves on agar and suggests that this method could be applied to the rearing of other aphids, phytophagous mites, leaf miners and leaf‐gall formers.     

Growth and mycotoxin production by <Emphasis Type="Italic">Chaetomium globosum</Emphasis>

Chaetomium globosum, the most common species within this genus, produces chaetoglobosins A and C when cultured on building material. Relatively low levels of these compounds have been shown to be lethal to various tissue culture cell lines. This study had two major objectives: (1) to determine the frequency at which Chaetomium species are isolated in water-damaged buildings and (2) to examine the production of chaetoglobosins A and C in isolates of C. globosum obtained from different buildings. Out of 794 water-damaged buildings, Chaetomium species were isolated in 49% of these structures. C. globosum ATCC 16021 was grown on four different media: oatmeal agar (OA), potato dextrose agar (PDA), corn meal agar (CMA), and malt extract agar (MEA). After 4 weeks, fungal growth was evaluated based on colony diameter and the quantity of spores produced on agar plates. In addition, production of chaetoglobosin A and C was monitored using high performance liquid chromatography. Colony diameter, spore production, and mycotoxin production by C. globosum were the highest on OA. Out of 30 C. globosum isolates cultured on OA for 4 weeks, 16 produced detectable amounts of chaetoglobosin A and every isolate produced chaetoglobosin C.     

Fungal flora and mycotoxins of six kinds of nut seeds for human consumption in Saudi Arabia

A wide range of moulds representing several genera and species, was recorded in this study from 5 seed samples of each almond, cashew nut, chestnut, hazelnut, pistachio nut and walnut collected from different markets in Ar' Ar, Saudi Arabia. The total counts of fungi were widely fluctuated between 1960–7704 and 1948–7434 colonies/g dry seeds on glucose-Czapek's and glycerol agar media at 28°C, respectively, and represented twenty genera, 53 species and 2 varieties of fungi. The prevalent fungi on the 2 agar media wereAspergillus flavus, A. niger andPenicillium chrysogenum. On glucose-Czapek's agar,Rhizopus stolonifer andAspergillus flavus var.columnaris were isolated from all 6 kinds of nut,A. parasiticus from 5 kinds andA. fumigatus from 4 kinds with high frequencies.Eurotium species were completely absent on glucose-Czapek's agar but they were isolated in high frequency from all kinds of nut on glycerol agar medium. The different nut samples were analyzed by thin layer chromatography for the presence of aflatoxins B₁, B₂, G₁ & G₂, citrinin, ochratoxins, patulin, sterigmatocystin, diacetoxyscirpenol, T-2 toxin and zearalenone. Aflatoxins B₁ & G₁ were detected in 3 out of the 5 samples tested of chestnut at concentrations ranging between 20 to 60 µg/kg. All other samples of almond, cashew nut, hazelnut, pistachio nut, and walnut that were analyzed were mycotoxin free.     

Agarose/agar assay system for the selection of bacteriocin-producing lactic fermentation bacteria

The direct selection of bacteriocin-producing lactic fermentation bacteria was possible by plating diluted cultures of Pediococcus acidilactici on mixed agarose agar layers with the amount of each component incrementally adjusted to 1.2% (w/v). Between 0.5 and 1% agarose, the increased flexibility of the solidified support layer allowed its removal from Petri dishes without tearing and its smooth layering on the surface of 1.5% (w/v) standard agar medium seeded with Listeria innocua as the test organism. Selection of bacteriocin-producing clones was based on the size of inhibition zones visible in the bottom agar layer under colonies growing on the agarose/agar top layer. The lack of contact with the test organism permitted the transfer of superior clones from the surface of the agarose/agar layer directly into an appropriate nutrient medium.     

The influence of medium aeration on in vitro rooting of Australian plant microcuttings

Media with different air filled porosity were compared with standard agar medium for root induction and root elongation for two Australian plants Grevillea thelemanniana and Verticordia plumosa×Chamelaucium uncinatum. Microcuttings from shoot cultures were pulsed for 7 days on a high auxin (40 M IBA), agar-solidified medium in the dark. The rooting of the microcuttings was then compared on standard agar medium (M1, 1/2 MS, no hormones) and on three experimental treatments: – porous-agar medium (1/2 MS, no hormones, 30 g agar l^–1, solidified then blended to provide aeration); – white sand, or white sand wet with M1 medium; and – a sterile propagation mix. The protocol using the propagation mix is referred to as IVS (In Vitro Soil). A separate experiment involved flushing the IVS soil profile with low or normal oxygen. The controls on M1 medium showed low and variable rooting percentages. The rate of root induction and the average total root length per microcutting at final harvest was significantly higher using the IVS protocol, porous-agar or white sand, while addition of agar medium to sand suppressed the percentage rooting and elongation as did flushing the air space in the IVS rooting medium with low oxygen. Other species tested on M1 medium and IVS including Pimelea physodes, Conospermum eatoniae, Verticordia grandis, and a Chamelaucium megalopetalum×C. uncinatum hybrid all showed a significant improvement on the IVS system. The IVS culture technique reduces plant-handling costs.     

Comparison of Nostocean hormogonium induction and its motility on solid plates between agar and gellan gum at varying gel matrix concentrations

To establish a sensitive bioassay for Nostocean hormogonium induction, we compared the effectiveness of the morpho-differentiation induction on two gelled plates, agar and gellan gum, for anacardic acid C15:1-Δ⁸ decyl ester (1) (100 nmol/disc). On BG-11₀ (nitrogen-free) medium-based 0.6 and 0.8% agar plates, Nostoc sp. strain Yaku-1 isolated from a coralloid root of Cycas revoluta in Yakushima Island showed clear morpho-differentiation from filamentous aggregates into hormogonia, and the induced hormogonia dispersed within 24 h; however, similar hormogonium formation was not observed at agar concentrations of 1.0% or higher. Conversely, hormogonium induction was considerably more pronounced on gellan gum plates than those on agar plates through concentrations ranging from 0.6 to 1.6% even after 12 h of incubation, particularly active on the 0.8–1.0% gellan gum plates. Thus, gellan gum plates can achieve clear results within 12 h and are thus highly useful for primary screening for hormogonium-inducing factors (HIFs).     

Aflatoxin production by Aspergillus flavus isolates pathogenic to coconut insect pests

Aspergillus flavus isolated from naturally infected leaf-eating caterpillar (Opisina arenosella W.), lace bug (Stephanitis typica D.) and plant hopper (Proutista moesta Westwood), insect pests of the coconut palm, were tested for aflatoxin (AT) production by employing various media formulations. These A. flavus isolates were earlier found to be entomopathogenic in laboratory bioassays. A laboratory contaminant and four standard aflatoxigenic A. flavus isolates were also included in this study as reference strains. All A. flavus isolates were tested on seven AT detection media: coconut extract agar, coconut extract-sodium desoxycholate agar, coconut extract-ascorbic acid agar, coconut extract-Czapek Dox agar, coconut extract-milk powder agar, 10% commercial coconut milk powder agar (CCMPA) and 20% CCMPA. Only two isolates of A. flavus, originally isolated from O. arenosella and P. moesta, produced ATs. AT production was detected within 48 h of incubation and was detected continually up to 1 month. These AT-producing A. flavus isolates also produced bright yellow pigmentation in the medium. Of all the seven media used for AT detection, CCMPA (10%) was found to be the best one, followed by 20% CCMPA, for direct and rapid AT detection. AT production was not necessary for pathogenicity in the insects. This revised version was published online in July 2006 with corrections to the Cover Date.     

Efficient rooting for establishment of papaya plantlets by micropropagation

A low cost micropropagation protocol to produce high quality root systems which are easy and economical to acclimatize is essential for large-scale micropropagation of papaya (Carica papaya L.). In this study, individual shoots (>0.5 cm) with 2_∼3 leaves from in vitro papaya multiple shoots were cultured on MS agar medium containing 2.5 μM IBA under dark conditions for 1 week for root induction. They were then transferred to agar or vermiculite media, containing half strength MS medium, under aerated or non-aerated conditions, for root development. Rooting percentage of shoots cultured for 2 weeks in aerated vermiculite was 94.5%, compared with 90.0% in non-aerated vermiculite, 71.1% in aerated agar, and 62.2% in non-aerated agar. Shoots with roots were acclimated in vermiculite under 100% RH for 1 week and then under ambient conditions for 2 weeks in a temperature-controlled growth chamber (28 °C). The survival rates of the plantlets were 94.5% from aerated vermiculite, 87.8% from non-aerated vermiculite, 42.2% from aerated agar, and 35.6% from non-aerated agar. Thus, root induction in low-concentration IBA agar medium followed by root development in vermiculite containing half strength MS medium under aerated conditions results in efficient rooting of in vitro papaya shoots. This revised version was published online in June 2006 with corrections to the Cover Date.     

A Selective Medium for Isolation and Presumptive Identification of the Bacteroides fragilis Group

A new selective medium, Bacteroides fragilis ammonium-sulfate gentamicin (BFAG) agar, for isolation and presumptive identification of the Bacteroides fragilis group is presented in this paper. This semisynthetic medium includes 0.2 g of ammonium sulfate, 0.7 g of lactose, 10 mg of gentamicin, 0.1 mg of aminobenzylpenicillin, 60 units of bacitracin, 20 mg of sodium cholate and 1 mg of sodium azide per 100 ml of medium. Stock cultures of the B. fragilis group grew well on this medium. None of the other 126 gram-positive or negative strains belonging to 40 aerobic or 45 anaerobic species tested grew on this medium. Three of the seven specimens in the clinical trials yielded colonies of only the B. fragilis group on BFAG agar plates. Also BFAG agar plates inoculated with human feces and contents of the alimentary tract (stomach, small intestine, cecum and colon) of mice gave rise to colonies of only the B. fragilis group. The high selectivity and good plating efficiency of BFAG agar enabled us to isolate the B. fragilis group rapidly from various clinical specimens.     

Penicillium siamensis sp. nov., from Thailand soil

Fungal flora of 20 samples of lentil seeds collected from Assiut governorate, Egypt, were studied. Seventeen genera and 13 species were isolated on glucose- (15 genera and 27 species), cellulose-(15 genera and 25 species)Czapek's agar media at 28 °C. The most common species were as follows: on glucose-Czapek's agar, Aspergillus fumigatus, A. niger, A. flavus, A. terreus, Penicillium notatum and Rhizopus oryzae and on cellulose agar, A. fumigatus, A. niger, A. flavus and P. notatum. Thin layer chromatographic (TLC) analysis and a biological test (Artemia salina) indicated the presence and the toxicity of aflatoxin in the extract of one sample (aflatoxins B₁, B₂, G₁ and G₂, at 20 mg/kg, total). This is the first report of the natural occurrence of aflatoxins in lentils.     

Oil Cakes as Media for Growing Catenaria anguillulae Sorokin, a Facultative Endoparasite of Nematodes

Summary Growth, morphology, visibility of sporangia and colony colour of 10 isolates of Catenaria anguillulae were compared on six media: linseed oil-cake agar, mustard oil-cake agar, neem oil-cake agar, beef extract agar, Emerson agar and YPSS agar with a view to selecting the best growth medium. In general, maximum radial growth of most of the isolates was recorded on linseed oil-cake agar medium, whereas neem oil-cake agar medium supported least growth of all the isolates of C. anguillulae. Linseed oil-cake agar medium also maintained the typical characters of the fungus and clear visibility of morphological details.     

Slime Production by Pseudomonas aeruginosa

Adequate experimental conditions for slime production by Pseudomonas aeruginosa were investigated using a cellophane plate method. Definite slime production was observed on heart infusion agar, brain heart infusion agar, yeast extract agar and synthetic agar, but not on nutrient agar. The addition of phosphate to the nutrient agar above 0.05% caused visible slime formation. Incubation at 37 C resulted in a higher yield of slime than at 25 C. Longer incubation seemed more favorable for slime production, while the pH reaction of the test media did not effect the slime yield. All the test cultures of P. aeruginosa produced large amounts of slime by this procedure. Cultures of Pseudomonas fluorescens, other Pseudomonas spp. and certain vibrios also produced slime under these experimental conditions.     

Hyphal and mycelial interactions betweenAgaricus bisporus andScytalidium thermophilum on agar media

The interaction betweenAgaricus bisporus andScytalidium thermophilum on agar media was studied by differential interference contrast and phase contrast microscopy.A. bisporus combatively replacesS. thermophilum in culture on agar media. The antagonistic effect ofA. bisporus is transmissible through a cellophane membrane and causes irreversible disintegration ofS. thermophilum protoplasm, resulting in a total loss of viability after prolonged interaction between the two fungi. On compost extract agar, but not on other media, the growth rate ofA. bisporus increased from 2.7 to 5.3 mm·d^–1 following contact withS. thermophilum mycelium.     

SIVB 2003 Congress Symposium Proceeding: Plant Growth and Sugar Utilization in an Agitated,Thin-Film Liquid System for Micropropagation

Summary Agitated layers of liquid medium were created on platform shakers in jars with 25–30 ml of medium (similar to conventional agar culture) rotating at 90 rpm. Thin films were scaled up in larger rectangular vessels on tilted shelves that periodically rock. In jars of liquid medium with a density of 180 explants per liter, multiplication rates of Hota tokudama var. ‘Newberry Gold’ were optimal with a media sucrose concentration of 5% [both with and without 1 μM benzyladenine (BA)]. Endogenous levels of soluble sugars were directly related to the concentration of sucrose in the medium. Three Hosta cultivars (‘Striptease’, ‘Minuteman’, and ‘Stiletto’) with plant densities of 40–200 explants per liter of medium were tested in larger, agitated, thin-film vessels in media with 5% sucrose and directly compared to agar medium. Higher rates of multiplication were observed in liquid than agar with the magnitude of the difference dependent on explant density. Pooled results for the three varieties with 200 explants per liter showed multiplication rates of 1.7x and 2.3x for agar and thin-film liquid, respectively. At 40 explants per liter, the multiplication rate was increased to 2.1x for agar and 3.4x for thin-film liquid. Sugar uptake was greater in liquid than agar and was greater in the higher densities, with the magnitude of the effect dependent on plant variety. Increased vessel size in the liquid, thin-film system and greater sugar uptake allowed more, larger plants to be harvested. Alocasia macrorrhizos was cultured in growth medium containing 1μM BA and 5% sucrose with plant densities in the range of 33–330 explants per liter. Dry weight and multiplication rate were greater in the liquid system than agar with the magnitude of the difference dependent on plant density. With approximately 165 explants per liter, and greater at the initiation of culture, plant density limited growth in both agar and liquid thin-film systems. In a multiplication medium (3 μM BA and 3 μM ancymidol) plant size was reduced by 50% and 60% (fresh weight) in liquid and agar, respectively. Initial density in the range of 165–330 explants per liter did not limit growth with the smaller plants in liquid or semisolid multiplication medium. Sugar uptake was greater in liquid than agar. While ample sugar was present in media for growth at any density on agar, sugar depletion was limiting growth at highest densities with the larger plants in liquid growth medium. In semisolid agar medium, sugar uptake by plants was more rapid than diffusion across the agar medium, resulting in non-equilibrium conditions following the culture cycle. In agitated, liquid medium, a greater transfer of sugars to plant tissue was related to accelerated growth.     

<Emphasis Type="Italic">Candida neustonensis</Emphasis> sp. nov., a novel ascomycetous yeast isolated from the sea surface microlayer in Taiwan

A new ascomycetous yeast species, Candida neustonensis is proposed in this study based on four strains (SN92^T, SN47, SJ22, SJ25) isolated from sea surface microlayer in Taiwan. These four yeast strains were morphologically, physiologically and phylogenetically identical to each other. No sexual reproduction was observed on 5% malt extract agar, corn meal agar, V8 agar, McClary’s acetate agar and potato-dextrose agar. Phylogenetic analysis of the sequences of the D1/D2 domain of the large subunit (LSU) rRNA gene places C. neustonensis as a member of the Pichia guilliermondii clade, it also reveals that the phylogenetically closest relatives of C. neustonensis are C. fukuyamaensis (4.4% divergence), C. xestobii (4.4% divergence) and P. guilliermondii (4.5% divergence). C. neustonensis also is clearly distinguished from other known species in the P. guilliermondii clade based on the results of physiology tests. From these comparison analyses, the following novel yeast species is proposed: Candida neustonensis sp. nov., with strain SN92^T (= BCRC 23108^T = JCM 14892^T = CBS 11061^T) as the type strain.     

Gracilaria species (Gracilariaceae, Rhodophyta) from southeastern Australia, including a new species, Gracilaria perplexa sp. nov.: Morphology, molecular relationships and agar content

Select species of the agarophyte Gracilaria were studied from southeastern Australia. The morphology and anatomy of species is described and molecular relations are inferred based on plastid and mitochon‐drial DNA sequence data. Agar yields and qualities are determined for each species. Gracilaria chilensis, found in Tasmania and Victoria, is morphologically and molecularly similar to G. chilensis from New Zealand and Chile and has low agar yields of 11–16%. Gracilaria cliftonii from Victoria, has high crude agar yield (52%) and is molecularly uniform. Gracilaria perplexa sp. nov., known only from Botany Bay, New South Wales, has an agar yield of 39%. The agar of G. perplexa is unusual in requiring the addition of 0.1 mol L^?1 NaCl for alcohol precipitation and is cold‐water (25°C) soluble because of the very high sulfate ester content. Molecular phylogeny shows that G. perplexa is closely related to Gracilaria preissiana from western Australia, but differs from the latter in its reduced branching and narrower more terete axes.