The microbiology of the eye

Infection of the external structures of the eye is one of the commonest types of eye disease worldwide. In addition, although relatively impermeable to microorganisms, infection within the eye can result from trauma, surgery or systemic disease. This article reviews the general biology of viruses, bacteria, fungi and protozoa and the major ocular infections that they cause. In addition, the effectiveness of the various antimicrobial agents in controlling ocular disease is discussed. Because of changes in the normal ocular flora, continuous monitoring of the microbiology of the eye will continue to be important in predicting future types of eye infection. Basic research is also needed into the interactions of microbes at the ocular surface. There is increasing microbial resistance to the antimicrobial agents used to treat ocular infections and hence, new antimicrobial agents will continue to be needed together with new methods of drug delivery to increase the effectiveness of existing antimicrobial agents.

Role of hexose transport in control of glycolytic flux in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which lias distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.

The use of analysis of variance (ANOVA) in applied microbiology

Experiments combining different groups or factors and which use ANOVA are a powerful method of investigation in applied microbiology. ANOVA enables not only the effect of individual factors to be estimated but also their interactions; information which cannot be obtained readily when factors are investigated separately. In addition, combining different treatments or factors in a single experiment is more efficient and often reduces the number of replications required to estimate treatment effects adequately. Because of the treatment combinations used in a factorial experiment, the DF of the error term in the ANOVA is a more important indicator of the ‘power’ of the experiment than the number of replicates. A good method is to ensure, where possible, that sufficient replication is present to achieve 15 DF for each error term of the ANOVA. Finally, it is important to consider the design of the experiment because this determines the appropriate ANOVA to use. Some of the most common experimental designs used in the biosciences and their relevant ANOVAs are discussed by. If there is doubt about which ANOVA to use, the researcher should seek advice from a statistician with experience of research in applied microbiology.

Fermentation methods for dextransucrase production

Several fermentation methods for the production of the enzyme dextransucrase have been employed. The theoretical aspects of these fermentation techniques have been given in the early chapters of this thesis together with a brief overview of enzyme biotechnology. A literature survey on cell recycle fermentation has been carried out followed by a survey report on dextransucrase production, purification and the reaction mechanism of dextran biosynthesis. The various experimental apparatus as employed in this research are described in detail. In particular, emphasis has been given to the development of continuous cell recycle fermenters. On the laboratory scale, fed-batch fermentations under anaerobic low agitation conditions resulted in dextransucrase activities of about 450 DSU/cm3 which are much higher than the yields reported in the literature and obtained under aerobic conditions. In conventional continuous culture the dilution rate was varied in the range between 0.375 h-1 to 0.55 h-1. The general pattern observed from the data obtained was that the enzyme activity decreased with increase in dilution rate. In these experiments the maximum value of enzyme activity was ∼74 DSU/cm3. Sparging the fermentation broth with CO2 in continuous culture appears to result in a decrease in enzyme activity. In continuous total cell recycle fermentations high steady state biomass levels were achieved but the enzyme activity was low, in the range 4 - 27 DSU/cm3. This fermentation environment affected the physiology of the microorganism. The behaviour of the cell recycle system employed in this work together with its performance and the factors that affected it are discussed in the relevant chapters. By retaining the whole broth leaving a continuous fermenter for between 1.5 - 4 h under controlled conditions, the enzyme activity was enhanced with a certain treatment from 86 DSU/cm3 to 180 DSU/cm3 which represents a 106% increase over the enzyme activity achieved by a steady-state conventional chemostat. A novel process for dextran production has been proposed based on the findings of this latter part of the experimental work.

The production of the enzyme dextransucrase by batch and continuous fermentation techniques

A review of the literature of work carried out on dextransucrase production, purification, immobilization and reactions has been carried out. A brief review has also been made of the literature concerning general enzyme biotechnology and fermentation technology. Fed-batch fermentation of the bacteria Leuconostoc mesenteroides NRRL B512 (F) to produce dextransucrase has formed the major part of this research. Aerobic and anaerobic fermentations have been studied using a 16 litre New Brunswick fermenter which has a 3-12 litre working volume. The initial volume of broth used in the studies was 6 litres. The results of the fed-batch fermentations showed for the first time that yields of dextransucrase are much higher under the anaerobic conditions than during the aerobic fermentations. Dextransucrase containing 300-350 DSU/cm3 of enzyme activity has been obtained during the aerobic fermentations, while in the anaerobic fermentations, enzyme yields containing 450-500 DSU/cm3 have been obtained routinely. The type of yeast extract used in the fermentation medium has been found to have significant effects on enzyme yield. Of the different types studied, the Gistex Standard was found to be the type that favoured the highest enzyme production. Studies have also been carried out on the effect of agitation rate and antifoam on the enzyme production during the anaerobic experiments. Agitation rates of up to 600 rpm were found not to affect the enzyme yield, however, the presence of antifoam in the medium led to a significant reduction in enzyme activity (less than 300 DSU/cm3). Scale-up of the anaerobic fermentations has been performed at up to the 1000 litre level with enzyme yields containing more than 400 DSU/cm3 of activity being produced. Some of the enzyme produced at this scale was used for the first time to produce dextran on an industrial scale via the enzyme route, with up to 99% conversion of sucrose to dextran being obtained. An attempt has been made at continuous dextransucrase production. Cell washout was observed to occur at dilution rates of greater than 0.4 h-1. Dextransucrase containing up to 25 DSU/cm3/h has been produced continuously.

Batch and continuous fermentation methods for the production of dextransucrase

The available literature concerning dextransucrase and dextran production and purification has been reviewed along with the reaction mechanisms of the enzyme. A discussion of basic fermentation theory is included, together with a brief description of bioreactor hydrodynamics and general biotechnology. The various fermenters used in this research work are described in detail, along with the various experimental techniques employed. The micro-organism Leuconostoc mesenteroides NRRL B512 (F) secretes dextransucrase in the presence of an inducer, sucrose, this being the only known inducer of the enzyme. Dextransucrase is a growth related product and a series of fed-batch fermentations have been carried out to extend the exponential growth phase of the organism. These experiments were carried out in a number of different sized vessels, ranging in size from 2.5 to 1,000 litres. Using a 16 litre vessel, dextransucrase activities in excess of 450 DSU/cm3 (21.67 U/cm3) have been obtained under non-aerated conditions. It has also been possible to achieve 442 DSU/cm3 (21.28 U/cm3) using the 1,000 litre vessel, although this has not been done consistently. A 1 litre and a 2.5 litre vessel were used for the continuous fermentations of dextransucrase. The 2.5 litre vessel was a very sophisticated MBR MiniBioreactor and was used for the majority of continuous fermentations carried out. An enzyme activity of approximately 108 DSU/cm3 (5.20 U/cm3) was achieved at a dilution rate of 0.50 h-1, which corresponds to the maximum growth rate of the cells under the process conditions. A number of continuous fermentations were operated for prolonged periods of time, with experimental run-times of up to 389 h being recorded without any incidence of contamination. The phenomenon of enzyme enhancement on hold-up of up to 100% was also noted during these fermentations, with dextransucrase of activity 89.7 DSU/cm3 (4.32 U/cm3) being boosted to 155.7 DSU/cm3 (7.50 U/cm3) following 24 hours of hold-up. These findings support the recommendation of a second reactor being placed in series with the existing vessel.

Statistical analysis in microbiology:statnotes

This book is aimed primarily at microbiologists who are undertaking research and who require a basic knowledge of statistics to analyse their experimental data. Computer software employing a wide range of data analysis methods is widely available to experimental scientists. The availability of this software, however, makes it essential that investigators understand the basic principles of statistics. Statistical analysis of data can be complex with many different methods of approach, each of which applies in a particular experimental circumstance. Hence, it is possible to apply an incorrect statistical method to data and to draw the wrong conclusions from an experiment. The purpose of this book, which has its origin in a series of articles published in the Society for Applied Microbiology journal ‘The Microbiologist’, is an attempt to present the basic logic of statistics as clearly as possible and therefore, to dispel some of the myths that often surround the subject. The 28 ‘Statnotes’ deal with various topics that are likely to be encountered, including the nature of variables, the comparison of means of two or more groups, non-parametric statistics, analysis of variance, correlating variables, and more complex methods such as multiple linear regression and principal components analysis. In each case, the relevant statistical method is illustrated with examples drawn from experiments in microbiological research. The text incorporates a glossary of the most commonly used statistical terms and there are two appendices designed to aid the investigator in the selection of the most appropriate test.