Influence of antimicrobial agents on the spoilage of a meat-based entomophage diet

The microbial decomposition of a meat-based entomophage diet presented in Parafilm packets was investigated. Considerable bacteria but not fungi were associated with components used to prepare the diet (i.e., hens' eggs, liver, and ground beef). At the initial sampling time, there were no differences among diet treatments in the size of bacterial or fungal populations. Bacterial populations in diets not containing antibacterial agents rapidly increased and reached an asymptote by 24 h (approximately 10(10) colony-forming units per gram). Bacterial populations also increased in diets containing antibacterial agents, but they were significantly smaller than in diets not containing antibacterial agents. The most prevalent bacteria isolated were Carnobacterium piscicola, Carnobacterium divergens, Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc mesenteroides, and Enterococcus spp., regardless of the antibacterial treatment used. The proliferation of fungi was delayed relative to bacteria, but significant differences were observed among the diet treatments. Fungi were most inhibited by sorbic acid and propionic acid in the absence of antibacterial agents. The most common fungi isolated were the yeasts Candida zeylanoides, Torulaspora globosa, and Yarrowia lipolytica. The pH of diets not containing antibacterial agents decreased rapidly and was highly correlated with increases in bacteria but not fungi. The results of this study demonstrate that antimicrobial agents significantly inhibit spoilage microorganisms in a meat-based diet and that alternative management strategies to delay the decomposition of such diets presented in Parafilm packets should target lactic acid spoilage bacteria, particularly Carnobacterium and Lactobacillus species.     

Direct measurement of acetylesterase in living protist cells

The fluorogenic acetylesterase (acetic ester hydrolase EC 3.1.1.6.) substrate, fluorescein diacetate, was used to measure enzyme activity in living protist cells. The visual enzyme assay was done by monitoring fluorochromasia by fluorescent microscopy. Quantitative fluorogenic assays were done by measuring the evolved fluorescein in a fluorometer. Of 59 strains of bacteria, 35 were fluorochromatically positive. Eight of the fluorochromatically negative strains were fluorogenically positive. Of 22 strains of slime molds and fungi, all were fluorochromatically positive. Three out of 12 different algae were fluorochromatically positive. Several unidentified protozoa were also fluorochromatically positive. Four out of six protozoa were fluorochromatically positive. Structures of special interest showing acetylesterase activity were: the growing hyphal tips of fungi, the vacuolated areas of yeast and protozoa, newly formed bacterial spores or immature fungal spores, "mesosome-like" bodies in Bacillus megaterium, and the cell membrane and nuclear region of green algae. Yeast protoplasts and bacterial protoplasts and spheroplasts were fluorochromatically positive when derived from positive cells and negative when derived from negative cells. There was no correlation between the possession of a capsule and acetylesterase activity. There was no effect on the viability of bacterial cells incubated in the presence of fluorescein diacetate. Paraoxon inhibited bacterial and yeast enzyme at 10(-5)m. Eserine (10(-5)m) and Paraoxon (10(-7)m) inhibited B. megaterium enzyme. Sodium acetate at 10(-2)m did not inhibit bacterial enzyme. The implications of these findings on the location and expression of esterase activity in living cells are discussed.     

Species diversity and relative abundance of vaginal lactic acid bacteria from women in Uganda and Korea

AIMS: Lactobacilli play an important role in maintaining vaginal health of women. The aim of this study was to compare the species richness and relative abundance of Lactobacillus and other lactic acid bacteria in women of two geographically distant countries, Uganda and Korea. METHODS AND RESULTS: Vaginal samples were obtained from two women populations in Uganda and Korea. The Lactobacillus Rogosa SL agar was used for initial isolation of lactic acid bacteria. After phenotypic analyses, the 16S rRNA gene was amplified by polymerase-chain reaction and analysed by the BLAST program and phylogenetic tree construction. A total of 338 (128 Korean and 210 Ugandan) vaginal lactic acid bacterial strains were isolated, including five genera: Lactobacillus, Leuconostoc, Pediococcus, Streptococcus and Weissella. While Lactobacillus crispatus was common in both populations, Lactobacillus fermentum was common only in Korean women, and Lactobacillus gasseri, Lactobacillus reuteri and Lactobacillus vaginalis only in Ugandan women. Among other lactic acid bacteria, Weissella was more common in Ugandan, and Pediococcus in Korean women. All Weissella strains produced hydrogen peroxide, and all Pediococcus strains inhibited Candida species. CONCLUSION: Although many lactic acid bacteria colonize women, their species distributions may be different in women of geographically separated communities. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge of species richness and relative abundance of vaginal lactic acid bacteria, including Lactobacillus, Pediococcus and Weissella, may lead to the design of better probiotic products as bacterial replacement therapy.     

丛枝菌根真菌状巨孢囊霉孢子伴生细菌的绿色荧光蛋白标记及定殖分析

摘要：【目的】分析丛枝菌根（Arbuscualr Mycorrhizal, AM）真菌珠状巨孢囊霉(Gigaspora margarita) MAFF 520054孢子伴生细菌的定殖情况,明确其生态位点,以及为进一步分析其种群生态或功能提供信息。【方法】以载体pNF8（gfp-mut1）对6株珠状巨孢囊霉MAFF 520054 孢子伴生细菌进行绿色荧光蛋白（GFP）标记,并通过荧光显微镜和平板计数的方法研究标记菌株对真菌宿主的定殖位点和不同条件下的定殖数量动态。【结果】对粘状芽孢杆菌（Peanibacillus spp.）M060106-1和M061122-6、芽孢杆菌（Bacillus sp.）M061122-10和短小芽孢杆菌（Brevibacillus sp.）M061122-12成功进行了GFP标记,其均具有较好的质粒稳定性,且与出发株的基本性状一致,适合短期内进行环境定殖研究。所有菌株均能定殖珠状巨孢囊霉MAFF 520054孢子壁,而M061122-6和M061122-12还能够定殖其菌丝;不同pH值条件下,各菌株定殖珠状巨孢囊霉MAFF 520054孢子的数量动态均为先上升后下降,pH值对各菌株的定殖数量有不同的影响;各GFP菌株对低活力的珠状巨孢囊霉孢子定殖数量高于高活力的孢子,且对高活力孢子的定殖数量动态不同。【结论】分离的珠状巨孢囊霉孢子伴生细菌能够重新定殖其孢子,菌株的定殖能力受其特性及外界因子的影响,为进一步分析AM真菌伴生细菌的种群生态及功能提供了信息。     

Effect of electrical charges and fields on injury and viability of airborne bacteria

In this study, the effects of the electric charges and fields on the viability of airborne microorganisms were investigated. The electric charges of different magnitude and polarity were imparted on airborne microbial cells by a means of induction charging. The airborne microorganisms carrying different electric charge levels were then extracted by an electric mobility analyzer and collected using a microbial sampler. It was found that the viability of Pseudomonas fluorescens bacteria, used as a model for sensitive bacteria, carrying a net charge from 4100 negative to 30 positive elementary charges ranged between 40% and 60%; the viability of the cells carrying >2700 positive charges was below 1.5%. In contrast, the viability of the stress-resistant spores of Bacillus subtilis var. niger (used as simulant of anthrax-causing Bacillus anthracis spores when testing bioaerosol sensors in various studies), was not affected by the amount of electric charges on the spores. Because bacterial cells depend on their membrane potential for basic metabolic activities, drastic changes occurring in the membrane potential during aerosolization and the local electric fields induced by the imposed charges appeared to affect the sensitive cells' viability. These findings facilitate applications of electric charging for environmental control purposes involving sterilization of bacterial cells by imposing high electric charges on them. The findings from this study can also be used in the development of new bioaerosol sampling methods based on electrostatic principles.     

The effect of prebiotics on production of antimicrobial compounds, resistance to growth at low pH and in the presence of bile, and adhesion of probiotic cells to intestinal mucus

AIMS: Screening of five bile salt-resistant and low pH-tolerant lactic acid bacteria for inhibitory activity against lactic acid bacteria and bacterial strains isolated from the faeces of children with HIV/AIDS. Determining the effect of prebiotics and soy milk-base on cell viability and adhesion of cells to intestinal mucus. METHODS AND RESULTS: Lactobacillus plantarum 423, Lactobacillus casei LHS, Lactobacillus salivarius 241, Lactobacillus curvatus DF 38 and Pediococcus pentosaceus 34 produced the highest level of antimicrobial activity (12,800 AU ml(-1)) when grown in MRS broth supplemented with 2% (m/v) dextrose. Growth in the presence of Raftilose Synergy1, Raftilose L95 and Raftiline GR did not lead to increased levels of antimicrobial activity. Cells grown in the presence of Raftilose Synergy1 took longer to adhere to intestinal mucus, whilst cells grown in the absence of prebiotics showed a linear rate of binding. CONCLUSIONS: A broad range of gram-positive and gram-negative bacteria were inhibited. Dextrose stimulated the production of antimicrobial compounds. Adhesion to intestinal mucus did not increase with the addition of prebiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: The strains may be incorporated in food supplements for HIV/AIDS patients suffering from gastro-intestinal disorders.     

The intestinal microflora of persons subjected to a radiation lesion]

The content of large intestine has been studied in persons exposed to radiation injury in consequence of the accident at the Chernobyl Atomic Power Plant. It is stated that bifidobacteria (10(7)-10(10) cells in 1 g of feces) prevailed (as in healthy people), Bifidobacterium indicum being a dominating species. Amount of lactic-acid bacteria in 1 g of defecations of examined patients was within the range of 10(6)-10(9) cells and in certain persons it reached 10(10) cells (primarily fecal Enterococci). A considerable amount of patients with acute radiation sickness of the 3d degree had in their intestine 10(9)/g of lactic-acid bacteria, Lactobacillus casei and L. plantarum prevailing there. The frequency of yeast isolation from defecations of patients constituted 83%, while the number of cells in 1 g of feces--from 10 to 10(4). Yeast of the Candida genus, mainly Candida parapsilosis, prevailed. The species composition of isolated microorganisms has no substantial differences from microcenosis of healthy people. The content of intestine of persons suffered from radiation is characterized only by greater amount of lactic-acid bacteria and enterococci as compared with healthy adults.     

氟康唑对热带念珠菌活性氧和线粒体膜电位的影响

为了探讨氟康唑作用机制,观察它对热带念珠菌作用后存活率、活性氧(Reactive oxygen species,ROS)、线粒体膜电位(Mitochondrial membrane potential,△Ψm)和细胞周期的变化。参照NCCLS M27-A方案的微量稀释法测定氟康唑对热带念珠菌的最低抑菌浓度(MIC);热带念珠菌与不同浓度氟康唑共同培养后用流式细胞术(Flow cytometry,FCM)分析热带念珠菌存活率、ROS、线粒体膜电位△Ψm和细胞周期的变化。结果表明,氟康唑作用后,热带念珠菌氟康唑耐药株的存活率、ROS、线粒体膜电位△Ψm和细胞周期各期比例均没有明显变化;而热带念珠菌氟康唑敏感株的存活率和线粒体膜电位△Ψm明显下降,ROS明显升高,而且大部分热带念珠菌阻滞于G2/M期,并出现明显凋亡峰,呈一定的时间剂量依赖关系。自由基清除剂谷胱甘肽抑制热带念珠菌ROS的产生,阻止细胞周期G2/M期阻滞和降低凋亡。由此可见,氟康唑可能通过刺激热带念珠菌产生过多ROS,并使线粒体膜电位△Ψm下降,从而诱导热带念珠菌凋亡。     

Effects of irrigation treatments on the quality of table olives produced with the Greek-style process

The irrigation of olive orchards is commonly applied to produce table olives with optimal size. No data have been published on the microbiological quality of drupes from irrigated olive groves during fermentation. The trials T100 and T50 (receiving a water amount equivalent to 100 % and 50 % of the required amount, respectively) and control T0 (rainfed trial) were monitored during two consecutive years. The results showed a significant increase of equatorial diameter and flesh:pit ratio of irrigated drupes. The decrease of pH and the numbers of lactic acid bacteria (LAB) registered for the irrigated trials during the fermentation were more consistent than those displayed by control T0. Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus coryniformis, and Pediococcus pentosaceus for LAB and Candida boidinii, Candida diddensiae, and Wickerhamomyces anomalus for yeasts were isolated at the highest concentrations. The global sensory acceptance was better for irrigated trials than control T0. The statistical multivariate analysis showed that the effect of irrigation was independent of the seasonal variability and it clearly distinguished the T100 and T50 trials from rainfed control. Irrigation improves significantly the quality of table olives. Interestingly, all explorative multivariate analyses showed low dissimilarity between irrigated trials; thus, the thesis T50 represents an effective approach to save water in olive orchards without compromising the quality of table olives.     

乳杆菌优良菌株的筛选及对小鼠阴道白假丝酵母菌过度增殖干预

目的探讨乳杆菌优良菌株的筛选及其对小鼠阴道白假丝酵母菌过度增殖干预作用。方法对德氏乳杆菌DM8909菌株进行诱变筛选,得到耐抗生素突变株208,以ICR小鼠为实验对象,用甲硝唑进行小鼠阴道菌群脱污染,将白假丝酵母菌接种到小鼠阴道内,构建小鼠阴道白假丝酵母菌过度增殖模型。采用阴道接种德氏乳杆菌DM8909突变株208菌液（2×108CFU/mL）处理,取其阴道冲洗液进行阴道菌群分析,并进行阴道上皮细胞的电镜检查。结果乳杆菌干预后显著清除阴道内的假丝酵母菌,肠杆菌恢复正常水平,与模型组比较,差异有统计学意义（P〈0.05）。结论德氏乳杆菌DM8909抗性菌株208能调整白假丝酵母菌在小鼠阴道内定植,对小鼠阴道上皮细胞有恢复作用。     

Use of sulfite and hydrogen peroxide to control bacterial contamination in ethanol fermentation.

Lactic acid bacteria isolated from an industrial-scale ethanol fermentation process were used to evaluate sulfite as a bacterial-contamination control agent in a cell-recycled continuous ethanol fermentation process. The viabilities of bacteria were decreased by sulfite at concentrations of 100 to 400 mg liter-1, while sulfite at the same concentrations did not change the viability of the Saccharomyces cerevisiae strain used in this process. Sulfite was effective only in the presence of oxygen. Bacteria showed differences in their susceptibilities to sulfite. Facultatively heterofermentative Lactobacillus casei 4-3 was more susceptible than was obligatory heterofermentative Lactobacillus fermentum 7-1. The former showed higher enzyme activities involved in the production and consumption of hydrogen peroxide than did the latter. The viability of L. fermentum 7-1 could be selectively controlled by hydrogen peroxide at concentrations of 1 to 10 mM. Based on these findings, it is hypothesized that the sulfur trioxide radical anions formed by peroxidase in the presence of hydrogen peroxide are responsible for the control of contaminating bacteria. Sulfite did not kill the yeast strain, which has catalase to degrade hydrogen peroxide. A cell-recycled continuous ethanol fermentation process was run successfully with sulfite treatments.     

地衣芽胞杆菌对白色念珠菌等的拮抗作用

目的了解地衣芽胞杆菌在试管内与阴道正常菌群共生关系的情况。方法将地衣芽胞杆菌菌液分别与葡萄球菌、大肠埃希菌、白色念珠菌、德氏乳杆菌混合培养,定量计数各菌在不同时间内单独培养和混合培养时各菌的活菌数。结果地衣芽胞杆菌生长不受金黄色葡萄球菌、白色念珠菌和大肠埃希菌的影响,金黄色葡萄球菌和白色念珠菌在有地衣芽胞杆菌存在的情况下,其生长受到明显的抑制（P〈0.05）;乳杆菌在12-48 h内,有显著的抑制地衣芽胞杆菌生长的作用,而乳杆菌的生长不受地衣芽胞杆菌的存在与否而正常生长。结论地衣芽胞杆菌对金黄色葡萄球菌及白色念珠菌在体外具有明显的拮抗作用,地衣芽胞杆菌对大肠埃希菌、乳杆菌无明显的体外拮抗作用。     

Cellular injuries upon exposure of Escherichia coli and Lactobacillus rhamnosus to high-intensity ultrasound

AIMS: To examine cellular injuries occurring in cells of Escherichia coli (Gram-negative bacteria) and Lactobacillus rhamnosus (Gram-positive bacteria) in response to a high-intensity ultrasound treatment using classical plate count technique and flow cytometry. METHOD AND RESULTS: According to plate count results, E. coli (D-value 8.3 min) was far more sensitive than L. rhamnosus (D-value 18.1 min) in their response to the ultrasound intensity applied (20 kHz, 17.6 W). The dye precursor carboxyfluorescein diacetate (cFDA) could freely diffuse across the cytoplasmic membrane of intact cells of Gram-positive bacteria L. rhamnosus, resulting in its intracellular enzymatic conversion and emission of green fluorescence. In contrast, the presence of an outer membrane on E. coli, which represents the class of Gram-negative bacteria, apparently disabled the penetration of viability marker cFDA. Ultrasound application on E. coli yielded in an increasing population with disintegrated outer membrane, which allowed penetration of cFDA and its intracellular enzymatic conversion as well as accumulation. In both organisms evaluated only a small population was labelled by propidium iodide upon exposure to ultrasound for up to 20 min. Within the experimental conditions investigated ultrasound did not considerably affect the cytoplasmic membrane, although according to plate count results viability loss occurred. CONCLUSIONS: The results compiled suggest, that ultrasound induced cell death, which may not be related to membrane damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Limitation on the use of bacteriocins, which are aimed on destabilization of cytoplasmic membrane but inhibited by the outer membrane, could be overcome by ultrasound-assisted physical disruption of the outer membrane.     

Inhibitory activity of vaginal Lactobacillus bacteria on yeasts causing vulvovaginal candidiasis

Growing frequency of therapeutical failures of vulvovaginal candidiasis, resulting from resistance of certain species of Candida to imidazole agents, raises interest in the use of probiotics from Lactobacillus genera as prophylaxis. Unfortunately, little is known about inhibitory mechanisms of Lactobacillus on Candida. The aim of this study was to compare the activity of selected Lactobacillus species, representing the physiological vaginal flora, against Candida as well as investigation whether their inhibitory activity against Candida is related strictly to hydrogen peroxide and lactic acid production. 125 strains from vaginal smears of healthy women were classified by making use of phenotypic and genotypic methods. The majority of strains belonged to L. acidophilus: L. acidophilus sensu stricto, L. crispatus, L. gasseri and L. johnsonii as well as L. fermentum and L. plantarum species. Culture supernatants of selected 25 strains representing the isolated species were examined for their inhibitory activity against the growth of Candida albicans and C. glabrata. The results showed that the strongest and the fastest activity against C. albicans was demonstrated by L. delbrueckii strains, producing the largest quantities of hydrogen peroxide. On the other hand, extended activity, demonstrable after 24 hours, was shown by non-H2O2 producing L. plantarum supernatants. Growth of C. glabrata was not inhibited by any of the examined strains of Lactobacillus. Comparison of activity of live active cultures of Lactobacillus strains and their mixtures with this of pure H2O2 and lactic acid has shown that pure chemical compounds were less active than the cultures. This suggests that mixtures of Lactobacillus strains are in cooperation with each other using many different metabolites.     

Isolation and characterization of bacteriocin-like inhibitory substances from lactic acid bacteria isolated from Azerbaijan cheeses

The search for lactic acid bacteria (LAB) producing bacteriocin-like inhibitory substances (BLISs) was performed in traditional Azerbaijan cheeses. Eight strains have been isolated and identified, four of which (S1a, S2, Q2b, and M1b) were identified as Lactobacillus buchneri. The remaining four strains (M3a, A4b, Q4a, and A3) were identified as L. pentosus, L. brevis, L. fermentum, and L. collinoides, respectively. Antimicrobial agents of all isolated LAB strains suppressed the growth of Lactobacillus bulgaricus 340, Escherichia coli, Enterococcus durans, and Listeria innocua. Saccharomyces cerevisiae and Candida pseudotropicalis were resistant to these BLISs. Proteinase K and trypsin completely inactivated BLIS whereas lipase and amylase partially inactivated BLIS.     

2株动物微生态制剂菌的分离鉴定及其生物学特性初步研究

目的从饲料中进行微生物的分离与培养,筛选动物微生态制剂候选菌株。方法利用变性梯度凝胶电泳(DGGE)筛选得到的7株微生物区分为2种菌,经测序确定其为热带假丝酵母和植物乳杆菌;检测了不同pH、温度、胆盐、金属铜和需/厌氧对其生长的影响。结果当pH小于2.5或铜离子高于150 ppm时,植物乳杆菌无法生长,而热带假丝酵母数量略有下降;胆盐和金属离子对2种菌影响较小,42℃培养条件下相对于30℃培养时热带假丝酵母数量下降了6个数量级。结论筛选得到的2株菌具有应用于动物微生态制剂的潜力,为动物微生态制剂候选菌筛选和评价提供了基础数据。     

Selection of Potential Probiotic <Emphasis Type="Italic">Lactobacillus</Emphasis> with Inhibitory Activity Against <Emphasis Type="Italic">Salmonella</Emphasis> and Fecal Coliform Bacteria

Three hundred and sixty presumptive lactic acid bacteria (LAB) isolated from pregnant sows, newborn, suckling, and weaned piglets were preliminarily screened for anti-Salmonella activity. Fifty-eight isolates consisting of Lactobacillus reuteri (n = 32), Lactobacillus salivarius (n = 10), Lactobacillus mucosae (n = 8), Lactobacillus johnsonii (n = 5), and Lactobacillus crispatus (n = 3) were selected and further characterized for probiotic properties including production of antimicrobial substances, acid and bile tolerance, and cell adherence to Caco-2 cells. Eight isolates including Lact. johnsonii LJ202 and Lact. reuteri LR108 were identified as potential probiotics. LJ202 was selected for further use in co-culture studies of two-bacterial and multiple-bacterial species to examine its inhibitory activity against Salmonella enterica serovar Enteritidis DMST7106 (SE7106). Co-culture of LJ202 and SE7106 showed that LJ202 could completely inhibit the growth of SE7106 in 10 h of co-culture. In co-culture of multiple-bacterial species, culturable fecal bacteria from pig feces were used as representative of multiple-bacterial species. The study was performed to examine whether interactions among multiple-bacterial species would influence antagonistic activity of LJ202 against SE7106 and fecal coliform bacteria. Co-culture of SE7106 with different combinations of fecal bacteria and probiotic (LJ202 and LR108) or non-probiotic (Lact. mucosae LM303) strains revealed that the growth of SE7106 was completely inhibited either in the presence or in the absence of probiotic strains. Intriguingly, LJ202 exhibited notable inhibitory activity against fecal coliform bacteria while LR108 did not. Taken together, the results of co-culture studies suggested that LJ202 is a good probiotic candidate for further study its inhibitory effects against pathogen infections in pigs.     

Functional and safety characterization of autochthonous Lactobacillus paracasei FS103 isolated from sheep cheese and its survival in sheep and cow fermented milks during cold storage

The main objective of this study was to evaluate some probiotic characteristics of Lactobacillus spp. isolated from traditional sheep cheese, and to investigate the fermentative ability and viability in sheep and cow milks of a selected potential probiotic Lactobacillus (L.) strain, i.e., L. paracasei FS103. A total of 54 autochthonous Lactobacillus isolates were characterized for (i) acidity and bile salt resistance, (ii) tolerance to gastric and intestinal juice models, and (iii) antagonistic activity against pathogens and antibiotic resistance. Potential probiotic Lactobacillus has been used in sheep and cow milks for the manufacturing of experimental fermented milks. In these latter, pH value, microbial count, and sensory analysis were carried out. Lactobacillus FS103 classified as L. paracasei subsp. paracasei had a good survival in gastric and intestinal juice models, inhibited the growth of undesirable bacteria, and was susceptible to chloramphenicol, clindamycin, penicillin, amoxicillin, erythromycin, tetracycline, and ampicillin. Moreover, when used to produce experimental sheep and cow fermented milks, L. paracasei FS103 was able to acidify both milk types leading to a continuous pH decrease during all fermentation time (24 h). FS103 population remains viable at a level > 108 CFU mL−1 after 21 days of cold (4 °C) storage. The results of sensory analysis showed that scores related to consistency, taste, and astringent were significantly higher in sheep fermented milk while animal-like was less acceptable compared to cow fermented milk. Lactobacillus paracasei FS103 isolated from sheep cheese exhibited potential probiotic properties and suitable features for sheep and cow fermented milks maintaining high vitality during cold storage.     

Reassessment of tetrazolium bromide as a viability stain for spores of vesicular-arbuscular mycorrhizal fungi

Techniques for the determination of viability of field-collected vesicular-arbuscular mycorrhizal spores must be valid for spores of different ages and of varying levels of peridium (outer wall of unispored sporocarp) development. The use of the dehydrogenase-activated stain 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) for determining viability of endogonaceous spores was reassessed. Freshly isolated and stored spores of three isolates of Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe were stained using two different protocols: Miller, Torres, and McClean (Mycological Research, in press) and An and Hendrix (Mycologia 80: 259–261). Time course results from selected experiments were analyzed graphically, and pooled results from all experiments were analyzed by factorial analysis of variance to determine statistically significant differences among the protocols, storage regime of spores, control killing method, and the effect of added cobalt ions. Significant nondehydrogenase-specific staining occurred in both types of controls of freshly isolated spores using Miller, Torres, and McClean's method, rendering it inadequate for viability determination. With An and Hendrix's protocol, fresh nonkilled spores stained significantly more than ethanol-treated spores, although these results underestimated viability as determined by two other assessment procedures, and autoclaving of fresh spores was not a satisfactory control treatment. Addition of cobalt to the incubation solution effectively drove all formazan production to the blue color but did not change the total percentage of colored spores produced, regardless of protocol used. Results corroborate the use of An and Hendrix's protocol with stored spores if color interpretation was corrected to include both blue and red colors as indicators of viability, results were read before nonspecific staining starts, and timing was extended to 75 hours. Both staining methods work with uniformly stored spores using specific incubation times, but not with spores of mixed ages, which could include young spores, making MTT not useful as a viability stain for field-isolated spores.