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With the increased awareness of the problems associated with the growth dependent analysis of bacterial populations, direct optical detection methods such as flow cytometry have enjoyed increased popularity over the last few years. Among the analyses discussed here are: (1) Bacterial discrimination from other particles on the basis of nucleic acid staining, using sample disaggregation to provide fast reliable enumeration while minimizing data artefacts due to post sampling growth; (2) Determination of basic cell functions such as reproductive ability, metabolic activity and membrane integrity, to characterise the physiological state or degree of viability of bacteria; and (3) The use of single cell sorting onto agar plates, microscope slides or into multi-well plates to correlate viability as determined by cell growth with fluorescent labelling techniques. Simultaneous staining with different fluorochromes provides an extremely powerful way to demonstrate culture heterogeneity, and also to understand the functional differences revealed by each stain in practical applications. Analysis of bacterial fermentations showed a considerable drop (20%) in membrane potential and integrity during the latter stages of small scale (5L), well mixed fed-batch fermentations. These changes, not found in either batch or continuous culture fermentations, are probably due to the severe, steadily increasing stress associated with glucose limitation during the fed-batch process, suggesting 'on-line' flow cytometry could improve process control. Heat injured cells can already show up to 4 log of differences in recovery in different pre-enrichment media, thus contributing to the problem of viable but non-culturable cells (VBNC's). Cytometric cell sorting demonstrated decreasing recovery with increasing loss of membrane function. However, a new medium protecting the cells from intracellular and extracellular causes of oxidative stress improved recovery considerably. Actively respiring cells showed much higher recovery improvement than the other populations, demonstrating for the first time the contribution of oxidative respiration to intracellular causes of damage as a key part of the VBNC problem. Finally, absolute and relative frequencies of one species in a complex population were determined using immunofluorescent labelling in combination with the analysis of cell function. The detail and precision of multiparameter flow cytometric measurements of cell function at the single cell level now raise questions regarding the validity of classical, growth dependent viability assessment methods.

Multi-parameter flow cytometric techniques have been developed for the 'at-line' study of bacterial cultivations. Using a mixture of specific fluorescent stains it is possible to resolve an individual cells physiological state beyond culturability, based on the presence or absence of an intact polarised cytoplasmic membrane, enabling assessment of population heterogeneity. It has been shown that during the latter stages of small-scale (5 l), well mixed fed-batch cultivations there is a considerable drop in cell viability, about 17%, as characterised by cytoplasmic membrane depolarisation and permeability. These phenomena are thought to be due to the severe and steadily increasing stress associated with glucose limitation at high cell densities, during the fed-batch process. Such effects were not found in either batch or continuous culture cultivations. The possibility of using these findings for improved process control using 'on-line' flow cytometry are discussed.

High cell density fed-batch fermentations of Escherichia coli W3110 have been carried out at specific growth rates of less than 0.3 h-1, to investigate the effect of glucose limitation on the physiological state of individual cells. After an initial exponential batch phase, the feed rate was held constant and a final dry cell weight of approximately 50 g per litre was achieved. The fermentations were monitored by mass spectrometry whilst measurements of pH, DOC, CFU/mL, TCN, OD500nm and residual glucose concentrations were made. Satisfactory and reproducible results were obtained. Flow cytometric analysis of cells in broth samples, based on either of two multi-staining protocols, revealed a progressive change in cell physiological state throughout the course of the fermentations. From these measurements it was concluded that the loss in reproductive viability towards the end of the fed-batch process is due to cell death and not due to the formation of a "viable but nonculturable state" as had previously been reported. Since the presence of a high proportion of dead or dying cells at any time during a fermentation has a detrimental effect on the synthesis of any desired product it is proposed that an on-line flow cytometric analysis and control strategy could be used as a means of increasing overall process efficiency., (Copyright 1999 John Wiley & Sons, Inc.)

We have developed a technique for laser tuning at rates of 100 kHz or more using a pair of acousto-optic modulators. In addition to all-electronic wavelength control, the same modulators also can provide electronically variable Q-switching, cavity length and power stabilization, chirp and linewidth control, and variable output coupling, all at rates far beyond what is possible with conventional mechanically tuned components. Tuning rates of 70 kHz have been demonstrated on a radio-frequency-pumped CO2 laser, with random access to over 50 laser lines spanning a 17% range in wavelength and with wavelength discrimination better than 1 part in 1000. A compact tuner and Q-switch has been deployed in a 5-10-kHz pulsed lidar system. The modulators each operate at a fixed Bragg angle, with the acoustic frequency determining the selected wavelength. This arrangement doubles the wavelength resolution without introducing an undesirable frequency shift.

Continuous culture fermentations of Escherichia coli W3110 have been carried out at controlled dissolved oxygen levels of 40% and 10% of saturation. Satisfactory and reproducible results were obtained. Agitation speeds of 400 and 1200 rpm at an aeration rate of 1 vvm have been used as well as an aeration rate of 3 vvm at 400 rpm. The upper levels of these variables represent much higher agitation and aeration intensities than those normally used in practical fermentations. The fermentations were monitored by mass spectrometry and optical density, and cell samples were studied by flow cytometry, SEM, and TEM. Protocols were developed so the state of both cell membranes and cell size could be measured by flow cytometry. Under all the conditions of agitation and aeration, flow cytometric analysis indicated that both cell membranes were intact and that a cytoplasmic membrane potential existed; also the cell size did not change, results confirmed by SEM and TEM. There were no detectable changes in off-gas analysis or optical density during the continuous fermentation nor in the cell structure as revealed by SEM or TEM, except at the highest agitation intensity. Under the latter conditions, after 7 h, the outer polysaccharide layer on the cell was stripped away. It is concluded that any changes in biological performance of this E. coli cell line due to variations in agitation or aeration intensity or scale of operation cannot be attributed to fluid dynamic stresses associated with the turbulence generated by impellers or with bursting bubbles., (Copyright 1998 John Wiley & Sons, Inc.)

Background: Sheepskin bedding might increase house dust mite exposure and so explain some of the increasing prevalence of severity of childhood asthma., Methods: Relationships between use of different types of bedding, and diagnoses of asthma, symptoms of wheezing, skin prick test evidence of house dust mite sensitivity, and airway responsiveness to methacholine, were examined retrospectively in a birth cohort of children followed longitudinally to age 15 years., Results: In the whole cohort, no associations were identified to suggest a causal relationship between use of any type of bedding and development of features of asthma. Although not an a priori hypothesis, we noted that among children with a family history of atopic disease, those who were house dust mite sensitive were more likely to have used an innerspring mattress (29.6% vs 10.2% who had not used an innerspring mattress, p = 0.005)., Conclusion: In this subgroup, increased airway responsiveness and mite sensitivity were significantly associated with use of innerspring mattresses, although whether this is a causal or secondary association is not certain. Use of a sheepskin in the bed in early childhood was not an additional risk factor for the development of asthma.

The Otago photoscreener is an optical instrument which gives a very sensitive indication of the accuracy with which a subject's eyes are fixing and focusing. Early experience suggested that this instrument could be used effectively to screen for the presence of amblyogenic factors in pre-verbal infants. This communication describes the development of ocular fixation and focusing in 137 normal infants who were followed at regular intervals during the first year of life. Accurate fixation and focusing was found in 13% of three-month-old infants, in 68% of six-month-old infants and in 76% of one year olds. The levels of 'focusing' visual acuity obtained from the photoscreen data are considerably better than the levels of cortical acuity measured by the standard behavioral and electrophysiological methods. This suggests that human infants fix and focus accurately for a considerable period before they are able to perceive all the details obtained in their retinal images.

We examined growth of spirometric lung function in 696 children of European ancestry who were followed from ages 9 to 15 years and stratified according to their degree of responsiveness to methacholine inhalation challenge, atopic status, and respiratory symptoms. Subjects were participants in the longitudinal Multidisciplinary Health and Development Study in Dunedin, New Zealand. Forced expired volume in 1 second (FEV1), and vital capacity (VC) were measured at 9, 11, 13, and 15 years of age, concurrently with assessment of airway responsiveness determined by the concentration of methacholine causing a 20% fall in FEV1 (PC20 FEV1). Atopic status was assessed at age 13 by skin-prick testing to 11 allergens. In children demonstrating airway hyperresponsiveness, FEV1 increased with age at a slower rate, and the FEV1/VC ratio had a faster rate of decline through childhood, compared to non-responsive children. Subjects with positive skin tests to house dust mite and cat dander also had lower mean FEV1/VC ratios than the control group. Any reported wheezing was associated with slower growth of FEV1 and VC in males. We conclude that in New Zealand children with airway responsiveness and/or atopy to house dust mite or cat growth of spirometric lung function is impaired.

Background: Although asthma diagnosed by a physician is known to be related to serum IgE levels, it is not known whether there is a relation between the level of IgE and airway hyperresponsiveness to a methacholine challenge. The characteristics of asymptomatic persons that predispose them to airway hyperresponsiveness are also unknown., Methods: We studied the relation between the serum total IgE level and airway hyperresponsiveness in the presence or absence of asthma and other atopic diseases in a birth cohort of children. Data from a questionnaire regarding respiratory symptoms, plus measurements of the serum total IgE level and airway responsiveness to inhaled methacholine, were obtained for 562 11-year-olds in New Zealand., Results: The boys had a higher prevalence than the girls of current diagnosed asthma (13 percent vs. 6 percent), current symptoms of wheezing (22 percent vs. 15 percent), and airflow obstruction at base line (6 percent vs. 1 percent) and had a wider distribution of IgE levels, although mean IgE levels (120.8 IU per milliliter in the boys and 98.1 IU per milliliter in the girls) did not differ significantly between the sexes. The prevalence of diagnosed asthma was strongly related to the serum IgE level (P for trend less than 0.0001). No asthma was reported in children with IgE levels less than 32 IU per milliliter, whereas 36 percent of those with IgE levels greater than or equal to 1000 IU per milliliter were reported to have asthma. This relation with the serum IgE level was not explained by a concomitant diagnosis of allergic rhinitis or eczema. Airway hyperresponsiveness to a methacholine challenge also correlated very highly (P less than 0.0001) with the serum IgE level. This relation remained significant even after the exclusion of children with diagnosed asthma (P less than 0.0001) and of all children with a history of wheezing, allergic rhinitis, or eczema (P less than 0.0001)., Conclusions: Even in children who have been asymptomatic throughout their lives and have no history of atopic disease, airway hyperresponsiveness appears to be closely linked to an allergic diathesis, as reflected by the serum total IgE level.

To determine the prevalence and severity of childhood asthma in New Zealand we studied 815 children from a birth cohort by questionnaire, clinical examination and pulmonary physiological measurements at age 9 years. More than 19% of the sample had experienced wheezing in the previous year, and 11% had wheezed in the month before assessment. In all, 220 of 815 children (27.1%) had had wheezing episodes by age 9; in 34 (4.2%) episodes had been of sufficient frequency to warrant regular anti-asthma treatment. Only 32% of all wheezing children were reported by their parents to suffer from asthma, and, in groups matched for frequency of symptoms, treatment given for wheezing was influenced strongly by whether or not the label of 'asthma' had been given. The detailed history provided most information useful in diagnosing asthma; clinical examination, peak flow records, spirometry and bronchial provocation testing provided only limited additional information. A wide spectrum of frequency and severity of recurrent wheezing disorders was evident in New Zealand children. Throughout all degrees of severity, prevalence rates appeared higher than those reported in studies from the United Kingdom.

Children with moderate and severe, mild, and trivial asthma were compared with a group of children who did not have asthma on a range of measures including family social background (socioeconomic status, family adversity, family environment), intelligence, reading, spelling, and behaviour. None of the background measures significantly differentiated among the groups. The group of children with moderate and severe asthma was found to have a significantly lower mean score for verbal intelligence, full scale intelligence, and spelling than those with mild asthma, but did not differ significantly from the remainder of the sample. Children with mild asthma were found to have slightly higher reading scores.

The prevalence of bronchial hyperreactivity to inhaled methacholine and of a clinical history of symptoms of asthma was determined in a birth cohort of 9 year old New Zealand children. A history of current or previous recurrent wheezing was obtained in 220 of 815 children. Of 800 who had spirometric tests, 27 (3.4%) had resting airflow obstruction (FEV1/FVC less than 75%). Methacholine challenge was undertaken without problem in 766 children, the abbreviated protocol being based on five breaths and four concentrations. A fall in FEV1 of more than 20% was observed in 176 children (23% of challenges, 22% of the full cohort) after inhalation of methacholine in concentrations of up to 25 mg/ml. The prevalence of bronchial reactivity in children with symptoms was related to the frequency of wheezing episodes in the last year, and the degree of reactivity to the interval since the last episode. Sixty four children (8.0%) with no history of wheeze or recurrent dry cough were, however, also responsive to methacholine 25 mg/ml or less, while 35% of children with current or previous wheezing did not respond to any dose of methacholine. Bronchial challenge by methacholine inhalation was not sufficiently sensitive or specific to be useful as a major criterion for the diagnosis of asthma in epidemiological studies. The occurrence of airway reactivity in children without symptoms of asthma, however, raises the possibility that adult onset asthma and the development of airways obstruction in some subjects with chronic bronchitis could have origins in childhood.

Fibrosing alveolitis is a rare, diffuse lung disease characterized by varying combinations of two histological features: thickening of alveolar walls and the presence of large mononuclear cells in the alveolar spaces. Clinical details of 10 children with fibrosing alveolitis are reported. The main symptoms in children are tachypnoea or dyspnoea, cough, poor weight gain, and cyanosis. The condition is similar to that in adults, but it is usually a more acute illness, and if untreated, more predictably fatal. Respiratory failure, pulmonary hypertension, and cardiac failure are the major complications. Less commonly, superimposed bacterial infection and pneumothorax occur. Chest x-rays often show a sequence of changes with a ground-glass appearance and fine mottling in the early stage of the disease, progressing to a picture of mainly hilar linear markings in those children who recover. The histological features at lung biopsy or necropsy are described; these correlated poorly with the radiological features, steroid responsiveness, and clinical course. Lung function tests in 3 older children showed evidence of markedly reduced lung volumes in 2. Static lung compliance in 4 children in the acute stage of the illness was normal in 3 and diminished in one. The response to steroid therapy was analysed in cases from the literature and the 10 reported cases. No spontaneous remissions occurred, all the survivors having been treated with corticosteroids. In children fibrosing alveolitis is almost always a corticosteroid-responsive disease. An appropriate course of prednisolone would be of at least 4 week's, but preferably of 8 weeks' duration, at a minimum daily dose of 2 mg/kg. After improvement the steroid withdrawal should be cautious and protracted, comprising at least a year's continuous treatment.

This retrospective study of coeliac disease in Otago showed that the cumulative prevalence in childhood was low at 35 per 100,000. The overall prevalence of the condition in Otago was also low at nine per 100,000. This low prevalence is probably the result of underdiagnosis. Only 35% of patients diagnosed during the 10 year period of the study were members of the Coeliac Society.

The associations between skin sensitivity to various common allergens and the development of childhood asthma were ascertained in a longitudinal study of a birth cohort of New Zealand children up to the age of 13 years. Of 714 children skin-tested, 45.8% were sensitive to at least one of 11 allergens, the most common responses being to rye grass pollen (32.5%), house dust mite (30.1%) and cat dander (13.3%). Allergen-specific relative risk analysis, controlled for the effect of sensitivity to other allergens, demonstrated that sensitivity to house dust mite and to cat dander were highly significant independent risk factors associated with the development of asthma (whether defined as recurrent typical respiratory symptoms, increased airway responsiveness, or the concurrent presence of both), whereas grass sensitivity was not a significant independent risk factor for asthma.