COMPARISONS OF DIFFERENT MEDIA FOR COUNTING SUGAR TOLERANT YEASTS IN CONCENTRATED ORANGE JUICE

SUMMARY: To select and count the sugar tolerant yeasts which ferment sixfold concentrated orange juice, a high sugar agar medium was developed which contains 50% of glucose, 1% of citric acid and 1% of Tryptone; it is incubated for 4–5 days at 25°.
The medium has disadvantages: it is troublesome to prepare, and colonies grow slowly and are translucent. These properties result directly' from the high sugar concentration, on which the selective action of the medium depends.
Counts on this medium have been compared with those on potato dextrose or nutrient dextrose agars (with 2% and 1% of glucose respectively), with yeasts isolated from fermenting concentrate, in pure culture, and under various practical conditions. As a rule, the counts were virtually the same on the different media; nutrient dextrose agar occasionally failed to record small numbers of these yeasts. If the two low sugar media were acidified to pH 3·5 the counts were reduced.
Potato dextrose agar recorded, besides the above yeasts, sugar intolerant yeasts entering from dirty machines or through bad canning practice: nutrient dextrose agar recorded bacteria in addition. The difference between parallel plating on these media and on the high sugar medium thus yielded useful information about sources of casual contamination.
It is suggested that the above would also be largely applicable to other sugar-rich concentrates of not less than 50° Brix.     

Influence of composition of diluent on populations of yeasts and moulds recovered from raw fruits

AIMS: The aims of this study were (i) to determine the retention of viability of mycoflora removed from raw fruits, and how this affected diluents used to prepare samples for enumeration of propagules, and (ii) to evaluate the performance of recovery media for supporting colony development. METHODS AND RESULTS: Yeasts and moulds removed from seven types of raw fruit were held in seven diluents for 1 h before plating on dichloran rose bengal chloramphenicol (DRBC) agar and plate count agar supplemented with chloramphenicol (100 micro g ml-1) (PCAC). Significant reductions (P=0.05) in populations of yeasts, moulds, and yeasts plus moulds occurred within the 1 h holding period, regardless of diluent composition. Overall, retention of viability was not influenced by diluent composition, and neither DRBC agar nor PCAC were superior in supporting colony development. CONCLUSIONS: The composition of diluents used to prepare food samples for mycological analysis has little affect on the number of yeasts and moulds recovered from seven types of naturally contaminated raw fruit. Both DRBC agar and PCAC are suitable as enumeration media. SIGNIFICANCE AND IMPACT OF THE STUDY: Diluents and media most often recommended for enumerating yeasts and moulds in foods are appropriate for raw fruits.     

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.     

The shelf life and effectiveness of granular formulations of Metschnikowia pulcherrima and Pichia guilliermondii yeast isolates that control postharvest decay of citrus fruit

Our overall objectives were to prepare commercially acceptable formulations of the postharvest biological control yeasts, Metschnikowia pulcherrima and Pichia guilliermondii, which have a long storage life and to determine the effectiveness of these formulations to control postharvest green and blue moulds on citrus fruit. Yeasts, grown on a cane molasses-based medium, were combined with talc or kaolin carriers and various adjuvants and the viability of yeast in 12 formulations was determined over a 6 month period. Formulation no. 11, containing talc, sodium alginate, sucrose, and yeast extract, for both yeasts had a significantly higher viable yeast cell content over a 6 month storage period. Among the formulations, three formulations (formulations no. 5, 6, and 11) were selected for additional in vivo testing because they had higher levels of viability amongst yeast cell populations during storage and were easier to resuspend remained in suspension more easily. These formulations were tested on Satsuma mandarin and grapefruit to control green and blue moulds. Formulations no. 5, 6, and 11 for both yeasts effectively controlled green mould, while only formulation no. 11 with either yeast isolate M. pulcherrima (isolate M1/1) or P. guilliermondii (isolate P1/3) effectively controlled both blue and green moulds.     

Occurrence and Growth of Yeasts in Yogurts

Yogurts purchased from retail outlets were examined for the presence of yeasts by being plated onto oxytetracycline malt extract agar. Of the 128 samples examined, 45% exhibited yeast counts above 10³ cells per g. A total of 73 yeast strains were isolated and identified as belonging to the genera Torulopsis, Kluyveromyces, Saccharomyces, Candida, Rhodotorula, Pichia, Debaryomyces, and Sporobolomyces. Torulopsis candida and Kluyveromyces fragilis were the most frequently isolated species, followed by Saccharomyces cerevisiae, Rhodotorula rubra, Kluyveromyces lactis, and Torulopsis versatilis. The growth of yeasts in yogurts was related to the ability of the yeasts to grow at refrigeration temperatures, to ferment lactose and sucrose, and to hydrolyze milk casein. Most yeast isolates grew in the presence of 100 μg of sorbate and benzoate preservatives per ml. Higher yeast counts from yogurts were obtained when the yogurts were plated onto oxytetracycline malt extract agar than when they were plated onto acidified malt extract agar.     

Dichloran-glycerol medium for enumeration of xerophilic fungi from low-moisture foods.

A low water activity (alpha omega) medium (0.95 alpha omega) containing 18% (wt/wt) glycerol and 2 micrograms of dichloran per ml was developed for enumerating the fungal flora of dried and semidried foods. The medium, designated DG18, was shown to be significantly better than Christensen malt salt agar when both media were tested with foodstuffs and with pure culture inocula. The need for a medium of reduced alpha omega for enumerating xerophilic fungi from low-moisture foods was demonstrated by comparing fungal counts obtained on both high-alpha omega and low-alpha omega media.     

A selective and indicative medium for groups of Penicillium viridicatum producing different mycotoxins in cereals

A medium, pentachloronitrobenzene-rose bengal-yeast extract-sucrose agar (PRYES), for the isolation of moulds occurring during storage of cereals has been developed and compared with other selective media. The basal medium is yeast extract agar containing 15% sucrose (w/v). In addition to the sucrose content further selective measures include the addition of antibacterial antibiotics chloram-phenicol and chlortetracycline (50 mg/l), the fungicides rose bengal (25 mg/l each), and pentachloronitrobenzene (1 g/l) and a low incubation temperature (20°C). Members of the Mucorales were completely inhibited, and fast-growing species of other moulds were slightly inhibited, allowing important storage moulds to develop. The important ochratoxin A and citrinin-producing Penicillium viridicatum group II was indicated by a typical violet brown reverse on PRYES. Producers of xanthomegnin and viomellein (P. viridicatum group I and P. aurantiogriseum ) were indicated on PRYES by their yellow reverse and obverse colours. The medium was used for screening 40 samples of barley, and moulds with the characteristic colours were all identified as the species mentioned above.     

A selective and indicative medium for groups of Penicillium viridicatum producing different mycotoxins in cereals

A medium, pentachloronitrobenzene-rose bengal-yeast extract-sucrose agar (PRYES), for the isolation of moulds occurring during storage of cereals has been developed and compared with other selective media. The basal medium is yeast extract agar containing 15% sucrose (w/v). In addition to the sucrose content further selective measures include the addition of antibacterial antibiotics chloramphenicol and chlortetracycline (50 mg/l), the fungicides rose bengal (25 mg/l each), and pentachloronitrobenzene (1 g/l) and a low incubation temperature (20 degrees C). Members of the Mucorales were completely inhibited, and fast-growing species of other moulds were slightly inhibited, allowing important storage moulds to develop. The important ochratoxin A and citrinin-producing Penicillium viridicatum group II was indicated by a typical violet brown reverse on PRYES. Producers of xanthomegnin and viomellein (P. viridicatum group I and P. aurantiogriseum) were indicated on PRYES by their yellow reverse and obverse colours. The medium was used for screening 40 samples of barley, and moulds with the characteristic colours were all identified as the species mentioned above.     

Antimicrobial Actions of Degraded and Native Chitosan against Spoilage Organisms in Laboratory Media and Foods

The objective of this study was to determine whether chitosan (poly-β-1,4-glucosamine) and hydrolysates of chitosan can be used as novel preservatives in foods. Chitosan was hydrolyzed by using oxidative-reductive degradation, crude papaya latex, and lysozyme. Mild hydrolysis of chitosan resulted in improved microbial inactivation in saline and greater inhibition of growth of several spoilage yeasts in laboratory media, but highly degraded products of chitosan exhibited no antimicrobial activity. In pasteurized apple-elderflower juice stored at 7°C, addition of 0.3 g of chitosan per liter eliminated yeasts entirely for the duration of the experiment (13 days), while the total counts and the lactic acid bacterial counts increased at a slower rate than they increased in the control. Addition of 0.3 or 1.0 g of chitosan per kg had no effect on the microbial flora of houmous, a chickpea dip; in the presence of 5.0 g of chitosan per kg, bacterial growth but not yeast growth was substantially reduced compared with growth in control dip stored at 7°C for 6 days. Improved antimicrobial potency of chitosan hydrolysates like that observed in the saline and laboratory medium experiments was not observed in juice and dip experiments. We concluded that native chitosan has potential for use as a preservative in certain types of food but that the increase in antimicrobial activity that occurs following partial hydrolysis is too small to justify the extra processing involved.     

Dyes as fungal inhibitors: effect on colony enumeration

The effects of three dyes on the colony enumeration of nine fungal strains (including members of the Deuteromycetes and Zygomycetes) in pure and mixed cultures were investigated. Using malt extract agar as basal and control medium, the following dyes and concentrations were assayed: auramine (25 ppm), gentian violet (5 ppm) and malachite green (1 ppm). The chemicals commonly used in commercial media dichloran (2 ppm) and rose bengal (50 ppm) were included in the study as reference mould-spreading inhibitors. Higher counts were usually obtained in the media containing dichloran, rose bengal or auramine, including the control medium in the absence of chemical when the mixed-conidium inocula did not include a spreading mould. Nevertheless in most cases no significant differences were observed between them. Malachite green (1 ppm) performed mainly as a strong inhibitor of spreading moulds, only allowing adequate colony development and recoveries of both Fusarium and Aspergillus strains tested.     

Effect of heat treatment on the antimicrobial properties of tef dough, injera, kocho and aradisame and the fate of selected pathogens

A known population from each of a 24h culture of Bacillus cereus, Pseudomonas aeruginosa, Salmonella spp., Shigella spp., Klebsiella spp. and Staphylococcus aureus was inoculated into tef flour–water/kocho–water mixtures in screw-capped flasks and allowed to ferment for 30h at room temperature (18–21°C). The flasks were then heat-treated. Cultures of the test bacteria were inoculated into tubes containing graded volumes of 30-h-fermented tef dough/kocho extracts which had been heat-treated at 45, 61 and 80°C in assay broth containing aqueous extracts from injera and aradisame. They were incubated for 24h at 32°C and optical densities determined. Populations of the major indigenous bacteria, yeasts and moulds in fermented tef dough (30h), kocho samples, injera and aradisame were determined from other control portions of the same samples. Higher temperature (80°C) heat-treatment promoted the inhibitory potential of extracts from doughs of both foods as compared with lower temperature heat-treatments (45 and 61°C). Asporogenous test bacteria were affected more than the spore-formers. Better efficacy of extracts from injera and aradisame suggested improved antimicrobial properties of the baked products than in doughs. Heat of baking inactivated all vegetative cells although spores of B.cereus, the yeasts and moulds survived the heat (100°C) applied for 5min. The c.f.u./g of food for B. cereus was below the disease-causing level (0.5×101 and 1.5×103, in injera and aradisame, respectively). Actual baking temperatures in homes are higher than the ones used here; if post-baking contamination is minimized or prevented, the products would be microbiologically safe with respect to the asporogenous pathogens when served fresh. Further studies on aflatoxins and improved storage conditions for kocho are recommended.     

Microbial removal from the separated liquid fraction of anaerobically digested pig manure in meso-scale integrated constructed wetlands

The aim was to investigate microbial removal from the liquid fraction of anaerobically digested pig manure in meso-scale integrated constructed wetlands (ICW’s) over a 13 month period. Four treatments were investigated: T1 (standard), T2 (effluent recycling), T3 (high nutrient loading), and T4 (high flow rate). Mean counts of yeasts and moulds and spore-forming bacteria were higher in T3 and T4 than in T1 and T2 (P < 0.05). Flow through the cells reduced mean counts of coliform, yeasts and moulds and spore-forming bacteria across all treatments (P < 0.01). Counts varied with season; coliform were highest in the Summer (P < 0.001), with yeasts and moulds highest in the Summer and Autumn (P < 0.01) and spore-formers lowest in the Autumn (P < 0.001). As Salmonella was undetectable in the influent and Escherichia coli and Enterococcus were rarely detected it is difficult to make conclusions regarding pathogen removal. Further investigations using marked strains would allow pathogen tracking within the ICW’s.     

Medium for the Enumeration and Isolation of Bacteria from a Swine Waste Digester

A habitat-simulating medium was developed for the enumeration and isolation of bacteria from a swine waste digester. A roll tube medium with growth factors for strict anaerobes from previously studied anaerobic ecosystems was used to evaluate the effects of deletion, addition, or level of digester fluid, digester fluid treated with acid or base, rumen fluid, fecal extract, anaerobic pit extract, tissue extract, carbohydrates, peptones, short-chain fatty acids, minerals, vitamins, N and P sources, reducing and solidifying agents, buffers, and gases on colony counts. Decreasing the agar concentration from 2.5 to 1.0% increased the counts twofold. Blending increased the counts 1.7-fold. With a medium (174) containing digester fluid, peptones, minerals, cysteine, sodium carbonate, and agar, colony counts were 60% of the microscopic count and improved yields 2.5 to 20 times those obtained with media previously used for digesters or developed for other anaerobic ecosystems. Colony counts continued to increase for up to 4 weeks of incubation. Medium 174 permits the enumeration of total, methanogenic, and, with deletion of reducing agent, aerotolerant bacteria. The results suggest that the predominant bacteria grow slowly and have requirements different from those of bacteria from other ecosystems.     

Isolation of Ureolytic Peptostreptococcus productus from Feces Using Defined Medium; Failure of Common Urease Tests

Colony counts of fecal samples from three persons, obtained by using a chemically defined anaerobic roll-tube medium (containing glucose, maltose, glycerol, minerals, hemin, B-vitamins, methionine, volatile fatty acids, sulfide, bicarbonate, agar, carbon dioxide (gas phase), and 1 mM NH(4) (+) as main nitrogen source), averaged 60% of the 8.8 x 10(10) bacteria per g obtained when 0.2% Trypticase and 0.05% yeast extract were added to the otherwise identical medium. When 0.2% vitamin-free Casitone replaced Trypticase and yeast extract, counts were 94% those of the more complex medium. When urea-nitrogen was added to the defined medium as the main nitrogen source in place of NH(4) (+), counts of relatively large colonies averaged 1.0 x 10(9) per g of feces from five persons-1.1% of counts on the medium containing Trypticase and yeast extract. All of the organisms from the large colonies in the urea roll tubes were morphologically similar, and all six representative strains isolated were identified as urease-forming Peptostreptococcus productus, a species not previously known to produce urease. Ureolytic strains of Selenomonas ruminantium and P. productus were negative for urease activity in three assay media when inocula were from media containing complex nitrogen sources. The study documents that P. productus is the most numerous ureolytic species so far found in human feces and suggests that NH(4) (+) and more complex organic nitrogen sources strongly repress its production of urease. The study also indicates the efficacy of chemically defined media for direct selective isolation of nutritional groups of bacteria from feces.     

Inhibition of Histoplasma capsulatum by Candida albicans and Other Yeasts on Sabouraud''s Agar Media

The inhibition of growth of Histoplasma capsulatum by Candida albicans and other yeasts on Sabouraud's agar was investigated. Histoplasma (yeast-phase inoculum) was grown alone and in mixtures with yeasts at 25 C for 4-week periods. As few as 10 colonies of C. albicans completely inhibited the growth of approximately 50,000 potential colonies of Histoplasma. The pH was determined in cultures of 36 colonies of Candida on media containing 1, 2, and 4% glucose by spotting the agar with pH indicators. A drop in the pH became noticeable in all three media about the 3rd day of incubation, and a pH of 3.5 was reached in about 7 days. Subsequently, the pH remained almost stationary in the 4% glucose-agar, rose slowly in the 2% glucose-agar, and rose sharply in the 1% glucose-agar. The growth of Histoplasma was inhibited completely at pH 4 and below. When the pH was controlled in mixed cultures, some growth of Histoplasma was obtained. Substitution of maltose for glucose delayed the development of acidity and allowed the appearance of numerous mycelial colonies in the presence of Candida. This growth was arrested as soon as the medium became acid. Four other species which also acidified the Sabouraud's medium effected similar inhibition. It was thus shown that severe and prolonged acidity produced by some yeasts in the sugar-rich Sabouraud's media is alone sufficient to completely inhibit Histoplasma during the standard 4-week incubation of specimens such as sputum.     

Ethanol tolerance, sugar tolerance and invertase activities of some yeast strains isolated from steep water of fermenting cassava tubers

E kunsanmi , T.J. & O dunfa , S.A. 1990. Ethanol tolerance, sugar tolerance and invertase activities of some yeast strains isolated from steep water of fermenting cassava tubers. Journal of Applied Bacteriology 69 , 672–675.
Thirteen yeasts isolated from the steep water of fermenting cassava tubers were screened for ethanol tolerance. Three strains which showed measurable growth in medium containing 10% (v/v) ethanol were also sugar-tolerant and grew well in medium containing 25% (w/v) glucose. One of the strains, YC3, was found to possess much higher invertase activity than the other two and could be of value in ethanol production from molasses. Further search for industrially useful yeasts in African fermented foods is suggested.     

Biochemical Studies on the Mineral Components in Sake Yeast

Analysing the mineral components in yeasts (sake yeast and some of other brewer’s yeasts), the author found that no remarkable difference was seen on the composition of the major mineral components. The potassium content of yeast cultured in koji extract is lower than that cultured in malt extract or in the synthetic medium. Potassium concentration of koji extract is lower than that of each of other media, and it was possible to increase the potassium content of the cells to the normal level by the enrichment of the koji extract with potassium chloride. From these facts, it is assumed that the potassium concentration of koji extract is insufficient for the saturation in the cells with potassium.

Phosphorus and magnesium contents in the cells are not so much affected by pH of the medium as potassium.     

Effect of heat and acid decontamination treatments on the recovery of Legionella pneumophila from drinking water using two selective media.

Two different decontamination systems, heat and acid, and two isolation media, GVPC and MWY agar were tested for the recovery of Legionella pneumophila from drinking water. The samples were concentrated by filtration through 0.2 micron polyamide filter and the membranes were resuspended in the original water samples. The suspension was divided into three parts: the first was placed in a 50 degrees C water bath, the second was acidified in HCl-KCl solution and the third did not undergo any treatment. The isolation was made by means of media containing charcoal, yeast extract and glycine with cycloeximide (GVPC) or vancomycin, polimixin B, anysomicin and dyes (MWY). Heating at 50 degrees C for 30 minutes was seen to be the best decontamination system above all when used with GVPC agar. Moreover, with this pretreatment higher counts were obtained both on MWY and GVPC agar. The MWY agar produced the highest isolatin percentages and the highest counts.     

Production of Concentrated Lactococcus lactis subsp. cremoris Suspensions in Calcium Alginate Beads

The effect of simultaneous modification of medium composition and growth conditions on the production of Lactococcus lactis subsp. cremoris biomass in calcium alginate beads was studied by the response surface method. Statistical methods of data analysis for unbalanced experiments are illustrated. The media tested were whey, whey supplemented with yeast extract and/or meat extract, milk, and the commercial medium Gold Complete (Nordica). Fermentations were performed at 23°C under pH control (5.6, 6.0, 6.4, or 6.8). In one complete series, 1% CaCO₃ was added to the growth media. There were strong interactions between CaCO₃ and media, CaCO₃ and pH level, and CaCO₃, media, and pH level. In media with CaCO₃, all first-order interactions between media, pH, and sampling time were significant. The addition of CaCO₃ increased cell counts in whey-meat extract medium, but no significant difference was found with the other media. Uncoupling between growth and acidification occurred between 16 and 22 h. Highest counts were obtained on milk and Gold Complete (6 × 10¹⁰/g). In CaCO₃-containing media, pH influenced cell counts only in whey and in Gold Complete (pH 5.6 and 6.0 giving the best results); pH also influenced the bead mass obtained at the end of the fermentation. Biomass production in alginate gels is proposed as a method of obtaining concentrated cell suspensions without centrifugation or filtration.     

Control of mites during isolation of yeasts from natural habitats

Summary Addition of 6.6 ml /liter of cygon (dimethoate, commercial) to acidified malt extract agar (Difco YM medium) suppressed mite activity completely and did not appear to affect the number or species of yeasts isolated. Rose Bengal suppressed filamentous fungi to some extent.