Sterile fish muscle homogenate was prepared from the individually line caught black rockfish (Sebastes melanops) and inoculated with the pure culture of Pseudomonas putrefaciens strain 17. The inoculated homogenate was incubated at 5°C and the growth and production of volatile sulfur compounds determined by a combined gas-liquid chromatography and mass spectrometry. A column containing 90-100 mesh diatomaceous earth coated with 10% Carbowax 20M was developed and tested for the quantitative determination of dimethyl sulfide (DMS), dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS). The DMDS levels in fish homogenate closely paralleled that of P. putrefaciens growth. The maximum DMDS level of 2.00 μg per 100 g fish homogenate was obtained after 10 days at 5°C in samples treated with 1.5% NaCl. NaCl was not required by P. putrefaciens but both the growth and DMDS production were stimulated by 1-2% of NaCl. The DMDS production and the growth of P. putrefaciens were reduced when the homogenate was treated with sodium benzoate (SB) or ethylenediaminetetraacetic acid (EDTA). The maximum levels of DMDS in 0.05% SB treated fish were 0.75 μg per 100 g and 0.85 μg for 0.05% EDTA treatment, respectively. The SB inhibited the growth rate as well as the maximum growth of P. putrefaciens, while EDTA had no effect on the maximum growth but extended the lag period and reduced the rate of growth. Potassium sorbate (PS) had little effect on DMDS production or the growth of P. putrefaciens. The maximum level of DMDS in fish homogenate, treated with 0.1% PS, was 1.80 μg per 100 g and the growth curve was similar to that of the control. / Graduation date: 1974

Graduation date: 1979

The marine vibrio ANT-500 is a typical psychrophile. The growth range of the bacterium at 35��� S is from a maximum growth temperature of 13.5 C to a minimum growth temperature of -2.5 C or less with the optimum near 7 C. The bacterium elaborates its chitinase enzymes between the temperatures of 1.5 and 13.5 C. The enzyme system is composed of three separate enzymes, probably two chitinases and one chitobiase. The rate limiting step of the enzyme-substrate reaction appears to follow Mechaelis-Menton kinetics. Chitin is the only substrate that induces chitinase synthesis in ANT-500. Chitinase activity was found in the digestive tracts of all species of fish sampled in Yaquina Bay, Oregon. Direct correlations were noted between gut content and chitinase activity in the gut, gut content and percent chitinoclasts in the gut, and chitinase activity and percent chitinoclasts. Fish with little or no chitinous material in their gut contents showed low or no detectable levels no chitinase activity and a low percentage of chitinoclasts, while fish with predominantly chitinous material in their gut contents showed high chitinase activity and a large percentage of chitinoclasts. Neither hydrostatic pressure up to 1000 atm nor lack of oxygen had a detrimental effect on chitinase activity from the gut of Raja binoculata. Sterile stomach fluid from Enophrys bison had no effect upon the growth rates of a pure chitinoclastic and a mixed culture of bacteria isolated from the gut of Enophrys bison; however, the gut fluid did allow for greater cell yield. N-acetyl-D-glucosamine, the end product of chitin decomposition, exhibited a ttglucose effect when added to culture medium. Both the pure and the mixed culture showed repression of growth in early log phase when grown in N-acetyl-D-glucosamine. The chitinase isolated from the gut of Enophrys bison is bacterial in origin. Tests in which the bacterial flora of the gut of Enophrys bison was eliminated by use of chloramphenicol revealed no detectable chitinase activity and no chitinoclastic bacterial populations. Fish in the absence of the antibiotic showed both significant chitinase activity and predominating chitinoclast populations. Other data strongly indicate that the chitinase in many species of marine fishes may also be bacterial in origin. / Graduation date: 1976

The importance of fish culture has been increasing since 1990’s. The steady growth of fish culture helps to ensure a stable supply of fish for human consumption. However, when compared with capture fisheries, production from fish culture is greatly influenced by fish diseases. Outbreaks of fish diseases have caused great economic loss to fish culture. Research has been conducted to understand and reduce the occurrence of fish diseases in fish culture. In Hong Kong, bacterial infection is the most common cause of fish diseases. This project is therefore directed to isolate and identify the causative bacterial pathogen of some fish disease cases with the aim of setting up a local fish disease database for assisting the identification of diseases and improving the understanding of fish diseases in fish farms in Hong Kong. In this project, seven fish disease cases caused by bacteria were investigated with the AFCD officials in Hong Kong. Nine fish disease bacterial pathogens were isolated and identified using different methods (including commercial biochemical test kits, automated system and DNA sequencing). The bacteria identified included Aeromonas hydrophila, Lactococcus garvieae, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus iniae, Vibrio vulnificus and Aeromonas salmonicida. Sensitivity tests to 10 common antibiotics conducted for the identified bacteria showed that spectinomycin is the most broad spectrum antibiotics. In addition, a long-term physical storage of bacterial stock with glycerol and glass beads was established for further research of the identified bacteria. For efficient data analysis, an electronic database using Microsoft Access to hold the identification results and case history of each isolated bacteria was developed. Different data entry forms and reports were also constructed to facilitate easy data entry and data access for users. The three bacteria identification methods were compared for their efficiency and accuracy. Some limitations encountered in this project including time constraints and low accuracy of some biochemical identification tests were discussed and recommendations to overcome these limitations and improvements to the constructed database were made. / published_or_final_version / Environmental Management / Master / Master of Science in Environmental Management

Comparatively little work has been done to determine the ecology of Clostridium botulinum type E since its initial isolation in the nineteen-thirties. This spore forming, anaerobic microorganism is relatively heat labile and has been missed in ecological surveys in which heat was used to selectively screen for spore formers. Use of gentler methods has, however, facilitated its demonstration in marine sediments throughout the Northern Hemisphere. The type E organism elaborates a highly potent neurotoxin and has been isolated as the causative agent in recent fatalities involving the consumption of fish products. Until recently the organism was not believed to be present in the United States south of the Canadian border. The purpose of this investigation was to determine if the organism could be demonstrated in shellfish, inland lake fish, and sediment samples throughout the Northwest. Samples of coastal shellfish-- including various species of clams, crabs and oysters, varieties of smoked fish products, species of inland lake fish and inland lake, river and coastal sediment samples were examined for the presence of the type E organism. The organism was found, to some extent, in almost every type of sample tested. Type E toxin was demonstrated in incubated samples of shellfish and smoked fish products collected from eleven sites along the Oregon and Washington coast. The organism was found in shore sediments from the tidewater and freshwater areas of the Columbia, Alsea, and Umpqua rivers but could not be demonstrated in sediments taken from saltwater beaches at the mouths of these rivers. The type E organism was also demonstrated in fish from inland lakes in the Oregon Cascade Mountains, in sediments from the shores of these lakes, and along the shores of a river and three reservoirs in this area. These samples were collected between 95 and 120 linear miles from the coast. The isolation of the type E botulinum organism in fish and shellfish products demonstrates that the organism does present a potential hazard which should be recognized by Northwest processors and distributors of these products. High concentrations of the spores of this organism, such as those found in bottom sediments of some of the inland lakes and reservoirs may serve to contaminate fish and other wildlife. Much of the data collected supports a terrestial distribution of the spores. / Graduation date: 1966

Corrigenda pasted onto back end-paper. / Bibliography: leaves 167-189. / 191, [229] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / In this study the wbh region responsible for O-antigen biosynthesis was isolated and partially characterised. The operon appears to be made up of genes that were acquired from other bacteria. The presence of IS1358 indicates that it may have played a role in the acquisition or rearrangement of the polysaccharide biosynthesis genes in V. anguillarum 01. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1999

Corrigenda pasted onto back end-paper. / Bibliography: leaves 167-189. / 191, [229] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / In this study the wbh region responsible for O-antigen biosynthesis was isolated and partially characterised. The operon appears to be made up of genes that were acquired from other bacteria. The presence of IS1358 indicates that it may have played a role in the acquisition or rearrangement of the polysaccharide biosynthesis genes in V. anguillarum 01. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1999

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Manipulation or control of the microbiological aquaculture environment has been identified as an important focus area for future hatchery development. Subsequently, alternatives to obtain control of the microbiological environment are being sought of which the field of probiotics appears highly promising. Probiotics are usually selected based on various in vitro characteristics, however, the methods used differ and are sometimes unsuccessful due to poor experimentation. The aim of this work is to contribute towards the development of a protocol for the in vitro screening of bacterial candidate probiotics for marine fish larviculture. To reduce the number of candidate probiotics to be tested in vivo, various in vitro experiments need to be conducted, each screening for a particular mode of action – antagonism towards pathogen through production of antimicrobial compounds, growth and attachment to fish intestinal mucus, and the production of other beneficial compounds such as vitamins, fatty-acids and digestive enzymes. A total of 108 bacteria species were isolated from the digestive tract of the adult common clownfish, Amphiprion percula to screen for potential probiotics to be used in clownfish larval rearing. The antagonistic compounds assay identified twelve isolates which showed antagonism towards two or more aquatic pathogens. This was followed by an in vitro test that involved growing the organisms in fish intestinal mucus and modeling their growth parameters. A ranking index (RI) was developed using the lag period (λ) and doubling time (td) of the organism, where (1over λ x td) x 100. Five candidate probionts (AP1-AP5) with varied growth parameters were used for further in vitro experiments. The attachment to mucus assay introduced a novel tool for quantifying competition for attachment sites between candidate probionts and pathogens on mucus. Candidate probiont Pseudoalteromonas AP5 reduced the attachment ability of Vibrio alginolyticus when added before the pathogen and partially out-competed the pathogen for attachment sites when added second. In vitro screening for the production of beneficial compounds tested the candidate probiotics’ ability to produce digestive enzymes - trypsin, lipase and alkaline phosphatase as well as carotenoids and vitamin C. Candidate probiont Pseudoalteromonas AP5 produced high levels of the enzymes (98.2, 34.1 and 91.3 mU product liberated.ml⁻¹, respectively) and contained carotenoids while Kocuria AP4 contained carotenoids but produced low quantities of enzymes (7.8, 0 and 59 mU product liberated.ml⁻¹, respectively). None of the candidate probiotics produced vitamin C. To eliminate potential pathogenic or toxic candidate probionts, Artemia nauplii were exposed to each candidate probiont and the percentage Artemia mortality after 24-hours was determined. Candidate probiont AP2 caused high mortality of Artemia nauplii (98.4%) and was excluded from further studies. Identification of candidate probionts AP3-AP5 was performed using 16S-rDNA molecular techniques and the bacteria were assigned the names Bacillus AP3, Kocuria AP4 and Pseudoalteromonas AP5, respectively. Two methods of larval probiont delivery were tested – attachment to Artemia, and in-water delivery. Attachment to Artemia was high for both Kocuria AP4 and Pseudoalteromonas AP5 (7.2 x10³ and 2.7x10⁴ bacteria.nauplius⁻¹, respectively) while the in-water viability experiment showed that Kocuria AP4 comprised 23.9% of the total culturable water microflora after 24 hours while Pseudoalteromonas AP5 contributed 100%. To validate the findings from the in vitro experiments, in vivo trials using clownfish larvae were performed. Of the four candidate probiotics tested, only Kocuria AP4 showed potential to increase larval survival. In vitro tests produced a better understanding of the possible mode of action and strategies of competition between bacteria, however, the number of criteria in which a candidate probiont is successful in vitro may not be the best predictor for its effectiveness in vivo. Commercial studies that reduce between-treatment variation are required to test predictions about the most suitable probiont or combinations thereof.

Thesis (MSc Food Sc )--Stellenbosch University, 2002. / ENGLISH ABSTRACT: The focus of this study was the development of food products produced from the neck flesh of Cape hake (Merluccius capensis) as a means of adding value to hake heads. The product prototypes that have been developed include curried fish chowder (packaged in stand-up pouches), fish spread (packaged in plastic casings) and Chakalaka hake (packaged in both cans and retortabie pouches). A formula for fish stock, which was used as a base ingredient in the three product prototypes, has also been standardised. Shelf life testing was regarded an essential part of the development processes for the refrigerated product prototypes i.e. the curried fish chowder and the fish spread. Shelf life determinations involved microbiological testing based on set microbiological standards as well as sensory monitoring and pH testing. Proximate chemical- and mineral analyses were performed on freeze-dried samples of the developed product prototypes. The efficiency of the antimicrobial peptides enterocin 1071 A and 1071 B, as biological preservatives, versus that of conventional artificial preservatives was evaluated in the fish spread prototype. Three batches of fish spread were prepared: one containing the enterocin crude extract; the second a combination of sodium benzoate and potassium sorbate, while the third batch containing no preservatives served as the control. Microbiological- and histamine tests coupled with organoleptic monitoring and pH testing were carried out over a 21-day period. It was concluded that although enterocins 1071A and 1071B had some preserving effect in the fish spread, the artificial preservative combination was the superior preserving agent. However, neither the biological preservatives nor the artificial preservative combination succeeded in providing a satisfactory shelf life. The preserving agents used in this study were however not necessarily included at optimum levels and higher levels could possibly lead to an improved shelf life. The influence of two processing temperatures [121°C (249.8°F) and 116°C (240.8°F)] and two food container types (retortable pouch and can) on the sensory quality characteristics of the Chakalaka hake-prototype was investigated. The results indicated that the shorter processing time obtained with a higher processing temperature resulted in a product of better sensory quality. The sensory quality characteristics of Chakalaka hake processed in cans at 121°C were closest to that considered desirable for the product. / AFRIKAANSE OPSOMMING: Die fokus van hierdie studie was die ontwikkeling van voedselprodukte geproduseer uit die nekvleis van stokvis (Merluccius capensis) met die doeI om waarde by stokviskoppe te voeg. Die volgende produk-prototipes is ontwikkel: 'n dik vissop met 'n kerriegeur (verpak in regopstaande sakkies), 'n vissmeer (verpak in plastiekomhulsels) en Chakalaka hake (verpak beide in blikkies en retortbestande sakkies). 'n Formule vir visaftreksel, wat as 'n basis-bestanddeel in die drie produk-prototipes gebruik is, is ook gestandaardiseer. Rakleeftydstudies het 'n belangrike deel van die ontwikkelingsprosesse van die verkoelde produk-prototipes, d.i. die dik vissop en die vissmeer, uitgemaak, Rakleeftydbepalings het mikrobiologiese toetsing, gebaseer op vasgestelde mikrobiologiese standaarde, sowel as die monitor van sensoriese eienskappe en pH metings behels. Proksimale chemiese- en mineraal analises is uitgevoer op gevriesdroogde monsters van die ontwikkelde produk-prototipes. Die effektiwiteit van die antimikrobiese peptiede enterosien 1071 A en 1071 B, as biologiese preserveermiddels, is ondersoek in vergelyking met dit van konvensionele kunsmatige preserveermiddels in die vissmeer-prototipe. Drie mengsels vissmeer is berei waarvan die eerste enterosien kru-ekstrak bevat het; die tweede 'n kombinasie van natrium bensoaat en kalium sorbaat, terwyl 'n derde mengsel geen preserveermiddels bevat het nie en gedien het as kontrole. Mikrobiologiese- en histamien toetse is gelyklopend met organoleptiese monitering en pH metings oor 'n tydperk van 21 dae op monsters van die drie vissmeermengsels uitgevoer. Die ondersoek het getoon dat enterosien 1071 A en 1071 B wel 'n mate van bederfwering in die vissmeer meegebring het, maar dat die kunsmatige preserveermiddelkombinasie 'n beter preserveringseffek gehad het in die produk. Nie die biologiese preserveermiddels óf die kunsmatige preserveermiddelkombinasie kon 'n bevredigende rakleeftyd teweegbring nie. Die preserveermiddels in hierdie studie is egter nie noodwendig in optimale hoeveelhede gebruik nie en hoër vlakke kan moontlik tot 'n verbeterde rakleeftyd lei. Die effek van twee prosesseringstemperature [121 °C (249.8°F) en 116°C (240.8°F)], sowel as twee verpakkingstipes (retortbestande sakkie en blik), op die sensoriese kwaliteitseienskappe van die 'Chakalaka hake' -prototipe is ondersoek. Die resultate het aangedui dat die korter prosesseringstyd verkry met 'n hoër prosesseringstemperatuur, gelei het tot 'n produk van beter sensoriese gehalte. Die sensoriese kwaliteitseienskappe van 'Chakalaka hake' geprosesseer in blikke by 121°C was die naaste aan dit wat beskou word as gewens vir die produk.