Studies on histoplasmosis IV. Elution characteristics of the Histo-inhibitory factor (HIF)

Summary The histo-inhibitory factor (HIF) derived from homogenates of liver or kidney from hamsters infected withHistoplasma capsulatum has been fractionated by column chromatography. It shows maximum absorption at 280 mµ, has a molecular weight of 142,000 and can be eluted from DEAE-cellulose or DEAE-Sephadex A—50 with 0.02 M phosphate — 4 M sodium chloride (1 : 1)HIF can be eluted fromHistoplasma yeast cells at pH 10.0, thus a greater number of positive cultures from chronic histoplasmosis could be expected to result from pretreatment of clinical specimens in glycine buffer pH 10.0 prior to culture.     

The selection and ingestion of epilithic algae byNais elinguis (Oligochaeta: Naididae)

The gut contents ofNais elinguis in an organically polluted river were dominated by epilithic chlorophycean unicells and pennate diatoms with an average cell volume of 1.3 × 10³ µm³. The worm unselectively and opportunistically ingested unicellular algae up to a maximum length of 196 µm and a maximum volume of 24 × 10³ µm³, but colonial and filamentous algae were discriminated against. The morphometry of the pharynx ofN. elinguis probably determined the maximum size of algal cells which could be ingested.     

Effect of light intensity and ammonium-N on carotenogenesis ofTrentepohlia odorata andDunaliella bardawil

AxenicTrentepohlia odorata was cultured at three different NH₄Cl levels (3.5 × 10^–2, 3.5 × 10^–3, 3.5 × 10^–4 M) and three different light intensities (48, 76, 122 µmol m^–2 s^–1). Chloride had no effect on growth over this range of concentration. High light intensity and high NH₄Cl concentration enhanced the specific growth rate. The carotenoid content increased under a combination of high light intensity and low N concentration. WhenD. bardawil was exposed to the same combination of growth conditions, there was an increase in its carotenoid content. The light saturation and the light inhibition constants (K _s andK _i, respectively) for growth, and the saturation constant (K _m) for NH₄Cl were determined. TheK _s andK _i values were higher inT. odorata (66.7 and> 122 mol m^–2 s^–1, respectively) than inD. bardawil (5.1 and 14.7 µmol m^–2 s^–1, respectively). TheK _m value determined at 122 µmol m^–2 s^–1, however, was lower inT. odorata (0.048 µM) than inD. bardawil (0.062 µM).Author for correspondence     

The effect of p-hydroxymercuribenzoate and congeners on microsomal glucose-6-phosphatase

Summary Iodoacetamide, N-ethylmaleimide, p-hydroxy-mercuribenzoate (p-MB) and HgCl₂ were tested as inhibitors of microsomal glucose-6-phosphatase. Iodoacetamide had no effect at 2mm. N-ethylmaleimide inhibited only crude, but not purified microsomal preparations (M₂) or crude microsomes exposed to deoxycholate.¹⁴C-labelled N-ethylmaleimide was not bound by the M₂ protein fraction. p-MB inhibited all types of preparations and the inhibition was not counteracted by detergent. A more detailed study was carried out with the purified M₂ fraction (specific activity: 2–4µmoles P_i/min/mg protein). Glucose-6-phosphate hydrolysis was inhibited 50% by 5 × 10^–5 m p-MB. The inhibition was completely reversible by dithiothreitol except when the enzyme was pre-incubated with p-MB in the absence of substrate. Then p-MB accelerated the temperature-dependent inactivation of glucose-6-phosphatase. Binding studies showed that around 3µmoles¹⁴C-p-MB were incorporated into 100 mg M₂ protein regardless of the concentration of mercurial in the incubation mixture. That is, over a 25 fold range of p-MB concentration, causing up to 80% inhibition of enzyme activity, no difference was seen in the amount of labelled p-MB which was irreversibly bound to M₂ protein. Kinetically p-MB behaved like a reversible inhibitor and this was confirmed by dilution experiments. Several compounds, including some amino acids, antagonized the inhibition by p-MB. The order of effectiveness was EDTA > barbital > tryptophan > histidine > lysine > other amino acids. Glycine, Tris and urea were ineffective competitors of p-MB inhibition. Double reciprocal plots showed that the K_m for glucose-6-phosphate was increased and the V_max reduced in the presence of p-MB. HgCl₂ was a more effective inhibitor than p-MB with a K_i of 6 × 10^–6 m. We conclude that a reaction of p-MB with M₂ sulfhydryls does not play a part in the inhibition of enzyme activity. It is suggested that p-MB may interact with one or more amino acid side chains in such a way that enzyme conformation is altered.Supported by U.S. Public Health Service Grant No. AM11448-08 and General Research Support Grant No. RR05486-12.     

Conversion of fat into yeast biomass in protein-containing waste-water

Summary Candida tropicalis S001 was grown on the lipid fraction of a protein-containing waste-water in order to (i) remove fat from the water, and (ii) produre yeast biomass for feed. The yeast cells were separated from the waste-water by sedimentation. Defatted waste-water was used for methane production and gave a yield of a 0.3 m³ methane/kg reduced chemical oxygen demand. The maximum specific growth rate (µ_max) of C. tropicalis growing on waste-water fat at pH 4.0 was 0.35 h^–1; the fat content was decreased from 8 g/l to about 0.1 g/l within 24 h. In continous culture a corresponding reduction was maintained at dilution rates up to 0.36 h^–1. The effect on growth of pH, temperature and CO₂ concentration was studied with triolein as the major carbon source. The µ_max was nearly constant (0.16 h^–1) in the pH and temperature range of 3.2–4.0 and 30°–38° C, respectively; 10% CO₂ was optimal for growth. Growth on triolein resulted in a biomass yield of 0.70 g dry weight/g fat. Offprint requests to: S. Rydin     

Optimum growth conditions and light utilization efficiency of Spirulina platensis (= Arthrospira fusiformis) (Cyanophyta) from Lake Chitu, Ethiopia

Spirulina platensis (= Arthrospira fusiformis) was isolated from Lake Chitu, a saline, alkaline lake in Ethiopia, where it forms an almost unialgal population. Optimum growth conditions were studied in a turbidostat. Cultures grown in modified Zarrouk's medium and exposed to a range of light intensities (20–500 µmol photons m^–2s^–1) showed a maximum specific growth rate (µ_max) of 1.78 d^–1. Quantum yield for growth (µ) was 3.8% at the optimum light for growth of 330 µmol photons m^–2s^–1, and ranged from 2.8 to 9.4%. With increase in irradiance, the chlorophyll a concentration decreased, and the carotenoids/chlorophyll a ratio increased by a factor of 2.4. The phosphorus to carbon ratio (P/C) showed some variation, while the nitrogen to carbon ratio (N/C) remained relatively constant, thus causing fluctuations in the N:P ratio (7–11) of cells. An optimum N:P ratio of about 7 was attained in cells growing at the optimum light for growth. Results from the continuous culture experiments agreed well with maximum values of photosynthetic efficiency given in the literature for natural populations of S. platensis in the soda lakes of East Africa, Lake Arenguade (Ethiopia), and Lake Simbi (Kenya).     

Characteristics of plankton communities in Chinese integrated fish ponds: effects of excessive grazing by planktivorous carps on plankton communities

Biomass and production of plankton communities were investigated in two Chinese integrated fish culture ponds in August, Dianshanhu Pond (with high density of planktivorous carp) and Pingwang Pond (with low density of planktivorous carp). The plankton communities were composed of rotifers, protozoans, phytoplankton (<40 µm) and bacteria. The large phytoplankton (>40 µm), cladocerans and copepods were rare because of grazing pressure by the carp. The density or biomass of bacteria (1.93 × 10⁷ and 2.20 × 10⁷ cells ml^–1 on average in Dianshanhu and Pingwang Ponds, respectively), picophytoplankton (24.6 and 18.5 mg m^–3 Chla on average) and rotifers (5372 and 20733 ind. 1^–1 on average) exceeded the maximum values reported for natural waters.The average [³H]thymidine uptake rates were 694 and 904 pmoles 1^–1 h^–1 (13.4 and 20.6 µgC 1^–1) and the bacterial production by the >2 µm fraction amounted 21–28% of total [³H] thymidine uptake rate in both ponds. The mean chlorophylla concentrations were 59.1 and 183 mg m^–3 in Dianshanhu and Pingwang Ponds, respectively. 82.4% and 65.3% of the total Chla was contributed by the <10 µm nano- and picophytoplankton in each pond, respectively. In particular, the picophytoplankton contribution amounted 41.2% of thtal Chla in Dianshanhu Pond. Primary production was 2.5 and 3.4 gC m^–2 d^–1 in each pond, respectively, and >50% of production was contributed by picophytoplankton. The mean biomasses of protozoa were 168 µg 1^–1 and 445 µg 1^–1 and those of rotifers were 763 µg 1^–1 and 1186 µg 1^–1 in Dianshanhu and Pingwang Ponds, respectively. The ecological efficiencies expressed in terms of the ratios of primary production to zooplankton production were 0.22 and 0.31, for the two ponds.     

Cnida discharge and the mechanism of venom delivery in Anemonia viridis (Cnidaria,Actiniaria)

Cnida discharge in the actinian Anemonia starts with the extrusion of the capsule from the cnidocyte followed by the eversion of the tubule. As the tubule everts, it maintains a tightly closed tip until fully everted. This is considered to be essential for a capsule to discharge as a result of an increase in intracapsular pressure. Venom volumes were measured in 3 types of nematocyst: 408 µm³, 98 µm³, and 9 µm³. Venom flow rates were estimated to range from >43 to 324 µm³ s^–1. It is suggested that the intracapsular pressures required for these flow rates range from 9.7 × 10⁵ to 1.9 x 10⁶ Pa.     

Aerotaxis and chemotaxis ofAzospirillum brasilense: A note

Azospirillum brasilense was attracted to capillaries containing either phosphate buffer, distilled water, or saline. The number of bacteria in these capillaries was 3–4×10⁴, after 1 h of incubation. In the presence of phosphate buffer + attractants, the number of cells accumulated in the capillary increased only to 5×10⁴–1.1×10⁵ cells. It was not possible, therefore, to measure chemotaxis inA. brasilense as distinct from aerotaxis by the capillary method. Chemotaxis was observed in semi-solid agar plates and was determined by a growth band oriented towards the attractant. Positive chemotactic response was obtained with peptone, tryptone, yeast extract, amino acids, organic acids, arabinose and galactose.     

Animal and worker exposure to dust and biological particles in animal care houses

An aerobiological study was performed to evaluate the potential exposure of animals and workers to dust constituents generated during routine animal house work. Different rooms of air conditioned (A, control) and passively ventilated (B, non-air conditioned) animal facilities were sampled, in order to evaluate total airborne culturable fungi and bacteria, fungal spore concentrations and particle levels. Airborne room particles were analyzed gravimetrically and for endotoxin content. All parameters, except for culturable fungi, were higher in facility B and statistically significant, with respect to those from the control facility A. Median values for airborne particle concentration, endotoxin and fungal spores in facility B were: 115 µg m^–3, 25 EU m^–3, and 2173 spores m^–3, respectively. Median values for facility A were: 66 µg m^–3, 9 EU m^–3, and 248 fungal spores m^–3. Broncheoalveolar lavage from rats kept in the rat room of B, presented median concentrations of total cells and lactate dehydrogenase, higher than those found in the control facility (4.4 × 10⁵ vs. 1.1 × 10⁵ and 2.7 UmL^-1 vs. 0.39 UmL^–1, respectively). Values of total and biological particles of both facilities, as well as the time spent in different rooms, showed that worker exposure was higher during cage washing. It was especially high in the passively ventilated facility (airborne particles 686 µg m^–3 3.5 h^–1 vs. 976 µg m^–3 3.5 h^–1, endotoxin 70 EU m^–3 3.5 h^–1 vs. 108 EU m^–3 3.5 h^–1). The type of basidiospores and ascospores found, as well as the significant correlation between particle levels and endotoxin contents suggests that wood chip bedding disturbance during cage washing is an important source for airborne biological particles. The changes in broncheoalveolar lavage components found in rats from these facilities and previously reported changes in pro-inflammatory cellular responses found in workers, indicate that these relatively low levels of exposure are enough to induce a biological response. Studies considering the composition of mixed organic dusts, would be needed to reevaluate current occupational standards.     

Pathogenesis of paracoccidioidomycosis: A histopathological study of the experimental murine infection

The pathogenesis of primary pulmonary P. brasiliensis infection, the systemic dissemination which followed, and the histopathology of the main organs involved was studied in a murine model of chronic paracoccidioidomycosis. Adult male BALB/C mice, were challenged intranasally with 26×10^–6 viable P. brasiliensis yeast cells. We inoculated 86 animals which were sacrificed from 0 h to 20 weeks. As controls, 11 mice were instilled with saline solution, and 48 with 26×10^–6 heat-killed. P. brasiliensis yeast cells. None of the animals receiving saline, exhibited pathologic alterations; 11.6% of those inoculated with the heatkilled cells, revealed mild, transitory acinopulmonary neutrophilic infiltrates. The animals infected with viable cells, developed a systemic process affecting mainly the lungs (46.5%), liver (18.6%), lymphnodes (18.6%), and spleen (3.5%). In this group of animals, lung lesions were detected regularly at all time periods from 3 h to 20 weeks. A multiple bronchoneumonic process was initially observed at 6 h, reached its maximum intensity around the third day, subsided thereafter but did not disappear and reactivated after the fifth week to become stationary until the end of experiments. Dissemination to other organs occurred early, and apparently by the hematogenous route. Initially the inflammatory cell infiltrate was mainly neutrophilic. With time, these cells were gradually replaced by lymphocytes, histiocytes and plasmocytes. Granuloma configuration of the cell infiltrate was distinctly seen around the fifth week, with multinucleated giant cells appearing at the ninth week. Hiliary lymphnode involvement was rare (7%) and primary lung lesions, as seen in tuberculosis and histoplasmosis, were not observed.     

Intracellular chloride activity of leech neurones and glial cells in physiological,low chloride saline

Leech blood apparently contains considerably less chloride than generally used in physiological experi ments. Instead of 85–130 mM Cl^– used in experimental salines, leech blood contains around 40 mM Cl^– and up to 45 mM organic anions, in particular malate. We have reinvestigated the distribution of Cl^– across the cell membrane of identified glial cells and neurones in the central nervous system of the leech Hirudo medicinalis L., using double-barrelled Cl^–- and pH-selective micro electrodes, in a conventional leech saline, and in a saline with a low Cl^– concentration (40 mM), containing 40 mM malate. The interference of anions other than Cl^–to the response of the ion-selective microelectrodes was estimated in Cl^–-free salines (Cl^– replaced by malate and/or gluconate). The results show that the absolute intracellu lar Cl^– activities (aCl_i) in glial cells and neurones, but not the electrochemical gradients of Cl^– across the glial and the neuronal cell membranes, are altered in the low Cl^–, malate-based saline. In Retzius neurones, aCl_i is lower than expected from electrochemical equilibrium, while in pressure neurones and in neuropil glial cells, aCl_i is distributed close to its equilibrium in both salines, re spectively. The steady-state intracellular pH values in the glial cells and Retzius neurones are little affected (0.1 pH units) in the low Cl^–, malate-based saline.     

Large forms of histoplasma

Summary Various types of large forms ofHistoplasma were studied in experimentally infected hamster and mouse tissues.Histoplasma can be found in the yeast phase in 5 distinct forms: 1. the classicalHistoplasma capsulatum form (ovoid, 2–5 µ in size); 2. occasional large forms in necrotic tissue and old cultures (up to 9 µ); 3. induced large forms in tissue explants (up to 20 µ); 4. small or large yeast cells surrounded by halos (up to 30 µ) and 5. the duboisii form (up to 22 or more) found exclusively inHistoplasma duboisii. Correlations between the above large forms with the classical small form were discussed.Histoplasma duboisii and the classicalHistoplasma capsulatum can be separated (1) by its size in the parasitic phase in vivo and in vitro; (2) by its different pathogenicity for experimental animals and (3) by the most particular tissue response in hamsters and in mice.This project was in part supported by Research Grant E-576 of the National Institutes of Health and a grant of the Squibb Institute for Medical Research.     

Non-symmetrical cytosine methylation in tobacco pollen DNA

We have detected sequence-specific non-symmetrical cytosine methylation within a 140 bp region of the promoter for the tobacco auxin-binding protein gene T85 in pollen DNA. Direct sequencing of the population of bisulphite reaction products showed that, in this region, 10 out of a possible 49 cytosine residues were methylated at a high frequency in pollen whereas the corresponding region from somatic cells (leaf DNA) did not show a detectable level of methylation. The context of these sites was 1×m⁵CpTpC, 1×m⁵CpGpT, 1×m⁵CpCpT, 2×m⁵CpTpT, 2×m⁵CpGpG, and 3×m⁵CpApT of which only m⁵CpGpG and m⁵CpGpT fitted the consensus sequence for symmetrical methylation in plants.     

Elimination of the non-specific binding of avidin to tissue sections

Summary A simple procedure is described for eliminating non-specific staining with avidin—peroxidase conjugates. Murine ovaries were embedded in either paraffin wax or epoxy resin and, after blocking endogenous peroxidase activity, were treated with 10 µg/ml biotinylatedPisum sativum agglutinin. Avidin—peroxidase conjugates (5 µg/ml), diluted in standard 0.05m tris-buffered saline, pH 7.6, containing 0.139m NaCl, produced considerable background coloration and intense mast cell staining in controls without the lectin. This background diminished as the ionic strength of the buffer was raised. At 0.125m Tris-buffered saline (containing 0.347m NaCl) the background was completely unstained, with elimination of all binding to mast cells and only minimal loss of specific lectin binding.     

Detection of an antigenic cell wall layer inHistoplasma capsulatum

Histoplasma capsulatum yeast cells have been studied by immunoelectron microscopy using rabbit polyclonal antisera and a biotin-avidin-peroxidase detection system. An antigenic surface layer has been visualized in the cell wall of immunostained organisms. This layer was not seen in samples prepared by standard electron microscopic methods or in negative controls used with the immunocytochemical technique. Without immunostaining the cell wall ofHistoplasma appeared almost transparent. In contrast, after immunoperoxidase staining the cell wall was conspicuous, bounded by the darkly stained outer layer. This electron dense layer, appeared to be a reservoir of surface antigens that were recognized by anti-Histoplasma antibodies.Abbreviations CHHA Cystine-heart-hemoglobin agar - PBS phosphate buffered saline - Ig immunoglobulin - TBS Tris buffered saline - DAB 3,3-diaminobenzidine tetrachloride - FITC fluorescein isothiocyanate - M199 tissue culture medium 199, according to Morgan et al. (1950)     

Spironolactone antagonism of aldosterone action on Na+ transport and RNA metabolism in toad bladder epithelium

Summary In earlier studies, aldosterone increased the incorporation of precursors into a class of cytoplasmic RNA with the characteristics of messenger RNA (mRNA), in toad bladder epithelium. In the present studies, this effect was analyzed further with a competitive antagonist, spironolactone (SC-9420). Paired hemibladders were labeled with³H-uridine (30 min pulse–140 min chase), with or without aldosterone (3.5×10^–8 m, 7×10^–8 m) in the presence or absence of SC-9420 (7×10^–6 m, 2.5×10^–5 m) at molar ratios of 2001 to 2801. Cytoplasmic RNA, either the total phenol-SDS extract or polyadenylated-RNA (poly(A)(+)-RNA) obtained by oligo-deoxythymidylate-cellulose (oligo(dT)-cellulose) chromatography was analyzed in linear 5–20% sucrose gradients. Eight sets of experiments were completed in which the short-circuit current (scc) was monitored for 180 min and the incorporation of³H-uridine (30 min pulse–150 min chase) was simultaneously determined on pools of epithelia from 5 to 10 hemibladders. The fractional change inscc correlated linearly with the fractional change in³H-uridine of 12S cytoplasmic RNA (r=0.95,p<0.001). The poly(A)(+)-RNA fraction had no detectable rRNA or tRNA and gave a heterogeneous pattern, typical of mRNA, in the sucrose gradients. In the presence of exogenous aldosterone, SC-9420 inhibited the incorporation of³H-uridine into poly(A)(+)-RNA (particularly 12S). These results support the inference that induction of mRNA mediates the action of aldosterone on Na⁺ transport.     

Transport-limited fermentation and growth of Saccharomyces cerevisiae and its competitive inhibition

Summary The anaerobic glucose uptake (at 20°, pH 3.5) by resting cells of Saccharomyces cerevisiae followed unidirectional Michaelis-Menten kinetics and was competitively inhibited by l-sorbose; K _m and K _i were respectively 5.6×10^-4 m and 1.8×10^-1 m; V _max was 6.5×10^-8 moles mg^-1 min^-1. The aerobic uptake of glucose by resting yeast was also inhibited by l-sorbose but did not follow unidirectional Michaelis-Menten kinetics. Glucose-limited growth in the chemostat of a respiration-deficient mutant of S. cerevisiae was competitively inhibited by l-sorbose. As predicted by theory for transport-limited growth in the chemostat (van Uden, 1967) the steady state glucose concentrations were linear functions of the l-sorbose concentrations with different slopes at different dilution rates; K _m and K _i were respectively 7.2×10^-4 m and 1.8×10^-1 m. It is concluded that glucose transport was the rate-limiting step of anaerobic fermentation of S. cerevisiae and of growth of the mutant and that l-sorbose is a competitive inhibitor of active glucose transport in this yeast. The latter conclusion is accommodated in the transport model of van Steveninck and Rothstein (1965).     

Monocyte chemoattractant protein-1 (MCP-1) gene transduction: an effective tumor vaccine strategy for non-intracranial tumors

Recently, there has been renewed interest in the concept of tumor vaccines using genetically engineered tumor cells expressing a variety of cytokines to increase their immunogenicity. Human MCP-1 (JE) is a potent chemoattractant and activator of monocytes and T lymphocytes and thus a good candidate gene for a tumor vaccine. We therefore evaluated the efficacy of vaccines consisting of irradiated tumor cells transduced with the murine MCP-1 gene in the syngeneic 9L gliosarcoma brain tumor model. 9L cell lines stably expressing murine MCP-1 (9L-JE) and control cell lines expressing neomycin 3 phosphotransferase (9L-Neo) were generated by infection with a Moloney murine leukemia retroviral vector. Fisher 344 rats were immunized with intradermal injections of 5×10⁵ or 2×10⁶ irradiated (5000 cGy) 9L-JE, 9L-Neo, and wild-type 9L (9L-WT) cells. Two weeks later immunized an non-immunized animals were challenged with varyious doses of intradermal (5×10⁶–5×10⁷) or intracerebral (2×10⁴–5×10⁵) 9L-WT cells. Intradermal tumors grew in all non-immunized animals. No tumors grew in animals immunized with irradiated 9L-JE or 9L-Neo cells and challenged with inocula of fewer than 5×10⁵ 9L-WT cells. With higher inocula up to 10⁷ cells, tumors appeared in all the animals. Tumors in animals immunized with 9L-JE were always smaller than tumors in the other groups. In addition, only the 9L-JE vaccine protected against tumor inocula of 5×10⁷ cells. Thus vaccination with MCP-1-expressing cells was able to protect animals against at least a 100-fold larger number of challenge tumor cells than vaccination with control cells. In contrast to studies with intradermal tumors, immunization with 9L-JE and 9L-Neo produced only minimal protection against intracerebral tumors. There was no significant difference between the 9L-JE and 9L-Neo vaccines in intracerebral challenge. This study suggests that tumor vaccines expressing cytokine genes such as MCP-1 can increase the antitumor response. However, the protective effect of these vaccines appears to be largely limited to intradermal tumors rather than intracerebral tumors.     

Effect of yeast inoculation rate on the metabolism of contaminating lactobacilli during fermentation of corn mash

Two separate 4 (bacterial concentrations)×6 (yeast concentrations) full factorial experiments were conducted in an attempt to identify a novel approach to minimize the effects caused by bacterial contamination during industrial production of ethanol from corn. Lactobacillus plantarum and Lactobacillus paracasei, commonly occurring bacterial contaminants in ethanol plants, were used in separate fermentation experiments conducted in duplicate using an industrial strain of Saccharomyces cerevisiae, Allyeast Superstart. Bacterial concentrations were 0, 1×10⁶, 1×10⁷ and 1×10⁸ cells/ml mash. Yeast concentrations were 0, 1×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, and 4×10⁷ cells/ml mash. An increased yeast inoculation rate of 3×10⁷ cells/ml resulted in a greater than 80% decrease (P<0.001) and a greater than 55% decrease (P<0.001) in lactic acid production by L. plantarum and L. paracasei, respectively, when mash was infected with 1×10⁸ lactobacilli/ml. No differences (P>0.25) were observed in the final ethanol concentration produced by yeast at any of the inoculation rates studied, in the absence of lactobacilli. However, when the mash was infected with 1×10⁷ or 1×10⁸ lactobacilli/ml, a reduction of 0.7–0.9% v/v (P<0.005) and a reduction of 0.4–0.6% v/v (P<0.005) in the final ethanol produced was observed in mashes inoculated with 1×10⁶ and 1×10⁷ yeast cells/ml, respectively. At higher yeast inoculation rates of 3×10⁷ or 4×10⁷ cells/ml, no differences (P>0.35) were observed in the final ethanol produced even when the mash was infected with 1×10⁸ lactobacilli/ml. The increase in ethanol corresponded to the reduction in lactic acid production by lactobacilli. This suggests that using an inoculation rate of 3×10⁷ yeast cells/ml reduces the growth and metabolism of contaminating lactic bacteria significantly, which results in reduced lactic acid production and a concomitant increase in ethanol production by yeast.