Effectiveness of certain sterilizing agents for seedling incubation in radiothymidine

Failure of seed sterilization methods and breakdown in sterile technique can result in catabolism of thymidine by microorganisms during seedling incubation in radioisotope. The effectiveness of some sterilizing agents was monitored by microbiological methods and by thin layer chromatography for the presence of thymidine degradation products. Sodium hypochlorite and ethylene oxide each sterilized pea and sunflower seeds. Sodium hypochlorite failed to sterilize onion seeds while ethylene oxide was only occasionally effective and often retarded germination. Captan, commonly used before germination to prevent fungal blight of seedlings, did not reduce the bacterial flora of onion seeds.     

Microbiological Aspects of Ethylene Oxide Sterilization: I. Experimental Apparatus and Methods

A specially built thermochemical death-rate apparatus is described which can be used to determine the resistance of microorganisms to ethylene oxide under controlled conditions. The apparatus was designed to provide instantaneous exposure of microorganisms to ethylene oxide and to eliminate variables that could result in errors when death kinetic reaction rates are calculated. The apparatus is used to obtain ethylene oxide resistance data which are useful in evaluating and developing sterilizing cycles for materials with known bacterial concentrations, as well as for calculating probability factors on which a given test condition can be expected to provide sterilization.     

Comparison of Two Methods for Enumeration of Anaerobe Numbers on Forages and Evaluation of Ethylene Oxide Treatment for Forage Sterilization

Experiments were conducted to (i) compare most-probable-number (MPN) procedures with roll tube procedures for enumeration of forage anaerobic bacteria and (ii) evaluate the efficacy of using ethylene oxide to sterilize wet herbage. Alfalfa, corn, and alfalfa-orchardgrass silages and alfalfa and orchardgrass herbages were analyzed for total anaerobic bacteria (medium pH, 6.8) and acid-tolerant anaerobic bacteria (medium pH, 4.5) by both roll tube and MPN procedures. No difference was found between the roll tube and MPN procedures for total bacteria; however, higher counts were obtained for acid-tolerant bacteria when the MPN procedure was used. Although MPN procedures require less time to obtain an estimate of bacterial numbers, isolation and identification of the microbial population is not possible. Alfalfa herbage was treated with ethylene oxide for 12, 24, or 36 h, incubated for 7 days at 37°C with or without addition of a bacterial inoculant, and analyzed for total bacteria by MPN procedures. Microbial growth after inoculation of ethylene oxide-treated herbage indicated that there was insufficient residual ethylene oxide to inhibit subsequent microbial growth. The results also indicated that 24 h was required to adequately sterilize fresh herbage. Thus, ethylene oxide can be used to sterilize wet herbage for use as a substrate for pure cultures of silage bacteria.     

Chitinolytic enzymes and the bacterial microflora in the digestive tract of cod, Gadus morhua

The levels of chitinolytic enzymes and chitinolytic bacteria in the digestive tract of feeding and fasting cod were compared. Enzyme activities within a given tissue were of a similar order irrespective of the presence of chitinolytic bacteria and/or chitin. Cod chitinase and chitobiase are therefore endogenous and constitutive enzymes. Fasting cod had similar numbers of bacteria within the gastro-intestinal compartments as feeding fish. Representative bacterial isolates from fasting fish were characterized.     

Bacterial Colonization of Cod (Gadus morhua L.) and Halibut (Hippoglossus hippoglossus) Eggs in Marine Aquaculture

Aquaculture has brought about increased interest in mass production of marine fish larvae. Problems such as poor egg quality and mass mortality of fish larvae have been prevalent. The intensive incubation techniques that often result in bacterial overgrowth on fish eggs could affect the commensal relationship between the indigenous microflora and opportunistic pathogens and subsequently hamper egg development, hatching, larval health, and ongrowth. Little information about the adherent microflora on fish eggs is available, and the present study was undertaken to describe the microbial ecology during egg development and hatching of two fish species of potential commercial importance in marine aquaculture. Attachment and development of the bacterial flora on cod (Gadus morhua L.) eggs from fertilization until hatching was studied by scanning electron microscopy. The adherent microflora on cod (G. morhua L.) and halibut (Hippoglossus hippoglossus) eggs during incubation was characterized and grouped by cluster analysis. Marked bacterial growth could be demonstrated 2 h after fertilization, and at hatching eggs were heavily overgrown. Members of the genera Pseudomonas, Alteromonas, Aeromonas, and Flavobacterium were found to dominate on the surface of both cod and halibut eggs. The filamentous bacterium Leucothrix mucor was found on eggs from both species. While growth of L. mucor on halibut eggs was sparse, cod eggs with a hairy appearance due to overgrowth by this bacterium close to hatching were frequently observed. Vibrio fischeri could be detected on cod eggs only, and pathogenic vibrios were not detected. Members of the genera Moraxella and Alcaligenes were found only on halibut eggs. Caulobacter and Seliberia spp. were observed attached to eggs dissected from cod ovaries under sterile conditions, indicating the presence of these bacteria in ovaries before spawning. Adherent strains did not demonstrate antibiotic resistance above a normal level. Attempts to regulate the egg microflora by incubation of gnotobiotic eggs with defined antibiotic-producing strains did not result in persistent protection against subsequent colonization by the microflora of the incubator.     

Isolation of mycobacteria from frozen fish destined for human consumption

Mycobacteria were isolated from defrost water and tissue of sole (Solea solea), hake (Merluccius merluccius), cod (Gadus morhua), ling (Genypterus blacodes), and monkfish (Lophius piscatorius) on L?wenstein-Jensen medium after incubation at different temperatures. Samples of frozen fish were obtained under sterile conditions inside a refrigeration chamber (-18 to -22 degrees C) in a wholesale market from which these products are distributed to shops for retail sale and human consumption.     

Screening and characterisation of potentially pathogenic bacteria associated with Atlantic cod Gadus morhua larvae: bath challenge trials using a multidish system

In intensive aquaculture systems, high concentrations of nutrients and high densities of fish larvae provide favorable conditions for opportunistic pathogenic bacteria to flourish. We screened potentially pathogenic bacterial strains isolated from moribund Atlantic cod Gadus morhua larvae, pollack Pollachius pollachius, coalfish Pollachius virens, Atlantic halibut Hippoglossus hippoglossus, rotifers, algae and water samples from different hatcheries. Three identical challenge experiments tested a total of 53 strains. A multidish system was used: cod eggs were placed in single wells, together with 2 ml of sterile seawater, and exposed to the bacterial cultures. Final bacterial concentrations in the wells were 10(6) and 10(4) CFU ml(-1). Eggs and larvae not exposed to bacteria were used as unchallenged controls. Challenged controls were exposed to Vibrio anguillarum strain 610. Eggs were challenged approximately 48 h prior to hatching and mortality was recorded daily throughout the yolk-sac period. In spite of the high challenge dose of 106 CFU ml(-1), only 5 bacterial strains tested caused higher mortality than the unchallenged controls. Four of these strains were identified by 16S rDNA and gyrase B gene (GyrB) sequencing as resembling V. anguillarum and 1 strain resembled Carnobacterium sp. Most of the larvae exposed to these strains died within 10 d of challenge. Serotyping of the strains resembling V. anguillarum gave inconclusive results. This indicates differences in serology compared to the serotypes O1, O2 and O3, associated with disease. Three bacterial strains seemed to have a slower infection rate, indicating a longer incubation period. The remaining 45 strains did not seem to have a negative effect on larval survival, suggesting that these are not primary pathogens.     

On the disinfection of catheters,cystoscopes, bronchoscopes and similar instruments by means of a mixture of ethylene oxide and carbon dioxide

Summary An apparatus and a method are described for a simple and reliable disinfection of catheters, cystoscopes, bronchoscopes, etc. by means of a gas mixture consisting of 10% ethylene oxide and 90% carbon dioxide. The air is evacuated by suction from a disinfection vessel containing the instruments and replaced by the ethylene oxide-carbon dioxide mixture, which must be allowed to act for 8 hours at 37°C. After disinfection, the instruments which have previously been packed in cellophane bags can be stored and transported sterile for a considerable period. Bacterial spores with a resistance of 5 minutes at 100°C. are killed by this method, which has no damaging effect on the instruments.     

Viral and bacterial diseases of Atlantic cod Gadus morhua, their prophylaxis and treatment: a review

This review summarises the state of knowledge of both viral and bacterial diseases of Atlantic cod Gadus morhua, and their diagnosis, prophylaxis and treatment. The most important losses have been at the larval and juvenile stages, and vibriosis has long been the most important bacterial disease in cod, with Listonella (Vibrio) anguillarum dominant among pathogenic isolates. Vaccination of cod against pathogens such as L. anguillarum and Aeromonas salmonicida clearly demonstrates that the cod immune system possesses an effective memory and appropriate mechanisms sufficient for protection, at least against some diseases. Well-known viruses such as the nodavirus that causes viral encephalopathy and retinopathy (VER), infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) have been isolated from Atlantic cod and can be a potential problem under intensive rearing conditions. No commercial vaccines against nodavirus are currently available, whereas vaccines against IPNV infections based upon inactivated virus as well as IPNV recombinant antigens are available. A number of investigations of the pharmacokinetic properties of antibacterial agents in cod and their efficacy in treating bacterial infections have been reviewed.     

Antimicrobial activity in serum of Atlantic cod Gadus morhua L.: comparison between cultured and wild fish

The present investigation determined the antibacterial activities in the sera of both cultured and wild Atlantic cod Gadus morhua . Serum samples from both groups of fish were quantified for total protein, and their effects against fish pathogens Listonella anguillarum , Aeromonas salmonicida and Yersinia ruckeri were determined using co-incubation assay. Total serum protein concentrations were not significantly different between farmed and wild Atlantic cod. Sera of cultured Atlantic cod significantly decreased growth of L. anguillarum by at least two-log₁₀ reductions in bacterial count, while those of the wild Atlantic cod were able to inhibit the growth of all three fish bacterial pathogens. The present study showed that sera from wild fish possess broader antibacterial activities than cultured Atlantic cod and that these could provide an explanation for the differences regarding their immunity to bacterial infections as well as their health status.     

Resistance of Micro-organisms to Inactivation by Gaseous Ethylene Oxide

A simple method for the exposure of micro-organisms to ethylene oxide on membrane filters in a modified desiccator has been devised and used to study microbial resistance to the gaseous sterilant and the term ' R -value' is suggested to express this. The resistance of many known species and isolates has been assessed and compared. Several species of Bacillus were isolated from natural habitats and their spores were found to be more resistant than the strain of Bacillus subtilis var. niger (NCTC 10073) frequently used to monitor ethylene oxide sterilization. However, endospores of some bacterial species exhibited little resistance. Fungal spores and vegetative bacteria exhibited low resistance to the sterilant except after drying in organic material when they appeared more resistant than spores of B. subtilis var. niger. It was concluded that resistance to ethylene oxide did not correlate with resistance to heat, irradiation or other chemical disinfectants, or to the existence in the endospore form per se.     

Immunohistochemistry of Atlantic cod larvae Gadus morhua experimentally challenged with Vibrio anguillarum

Farming of Atlantic cod Gadus morhua is one of the most rapidly growing sectors of Norwegian aquaculture. Classical vibriosis caused by Vibrio anguillarum is a problem in cod aquaculture. To prevent disease outbreaks, a thorough understanding of the infection route and the impact of the bacteria on the host is important. The intestinal tract, skin and gills have all been proposed as routes of entry for bacterial infections such as vibriosis. We aimed to further develop understanding of V anguillarum serotype O2alpha infections in cod larvae by elucidation of a possible route of entry, the pattern of infection and its histopathology. Cod eggs were transferred to a 24-well polystyrene multi-dish with 2 ml of sterile aerated 80% (28 per thousand salinity) seawater. Challenge doses were 10(4) and 10(6) CFU ml(-1). Unchallenged larvae were used as controls. Larvae for immunohistochemical examination were sampled daily from each group. In most of the larvae, either no or very few bacteria were observed. Typical findings were clusters of bacteria in the spaces between the primary gill lamellae. None of these bacteria seemed to have adhered to the gills. Intestines of 3 out of 161 larvae examined contained positively immunostained bacteria. Some bacteria appeared attached to the microvilli, but none was observed inside epithelial cells. Only 2 larvae from the low-challenge dose group showed clear signs of histopathology, which occurred in the intestine. It is not possible to draw any conclusions regarding the portal of entry.     

Effect of Moisture on Ethylene Oxide Sterilization

Bacterial cells dehydrated beyond a critical point no longer react uniformly to ethylene oxide sterilization. The percentage of cells resistant to the lethal effect of ethylene oxide after desiccation is often as small as 0.1 to 0.001%. However, 5% resistant cells were observed with one type of microorganism dried in broth. The presence of organic matter increases the percentage of cells that become resistant to ethylene oxide after dehydration. The phenomenon is produced by exposing cells to a vacuum or a chemically desiccated atmosphere. It is not a permanent change, because the resistant cells rapidly become susceptible if wetted with water. On the other hand, mere exposure to a high relative humidity (RH), i.e., 75 to 98%, after desiccation requires 6 and 4 days, respectively, to overcome this resistance. Moisture studies showed that there is less water in bacterial cells that have been desiccated and then equilibrated to successively high RH values up to 100% RH, than in cells that have not been desiccated, but allowed to dry naturally until equilibrated to the same RH values.     

Comparative bacterial clearances of muscle and skin/subcutaneous tissues with and without dead bone: a laboratory study

Seventeen New Zealand White rabbits underwent implantation of three different concentrations of bacteria and a sterile saline control solution with and without dead autologous bone in eight separate muscular and eight separate subcutaneous sites. Following a period of 1 week, each site was surgically explored and samples of tissue were taken for histology and quantitative culture. Results reveal that final bacterial concentrations in the subcutaneous sites were significantly lower than in the muscle sites (p less than or equal to 0.0001) for each concentration of bacteria, with and without dead bone. Dead bone resulted in very significantly greater bacterial concentrations in both subcutaneous and muscle sites. Clinically, these results indicate that a thorough bony wound debridement is more important than the type of tissue used to close the wound. Flap tissue should be selected with regard to the perfusion, contour, and appearance of the recipient site.     

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Thailand

Tissue banking started in Thailand in 1979. Five years after this, the Bangkok Biomaterial Centre (BBC) was established in the Faculty of Medicine, Siriraj Hospital, with the support of the IAEA program. The objective of the Centre was to provide sterile bones and tissues for clinical use. Through the passage of time, the Bangkok Biomaterial Centre has gained confidence from the end user and by 2007 has processed 33,872 allografts from 491 deceased donors and 4,035 live donors were used in medical treatment in 3,596 patients in more than 79 different hospitals. More than 305 surgeons from Thailand used the tissue produced in the BBC. At the beginning of its work the BBC concentrate its activities on the production of the following tissues: freeze dried bone, freeze dried dura mater and freeze dried fascia lata. All of these tissues were sterilised using ethylene oxide gas until the end of year 1984. Since 1985 the BBC sterilise tissue using ionising radiation. The BBC is now producing deep-frozen; bone tendon, cartilage, trachea and soft tissue; freeze-dried; bone, fascia lata, dura mater, amniotic membrane, bone hydroxyapatite, bone tablet and fresh preserved amniotic membrane Yongyudh Vajaradul is a Founder of Bangkok Biomaterial Centre and also a President of TATB, Bangkok, Thailand. Jorge Morales Pedraza is a former IAEA Interregional Project Manager, Vienna, Austria.     

Factors influencing the resistance of biological monitors to ethylene oxide

The resistance of bacterial spore monitors is markedly influenced by the environmental conditions existing during development of the spores and, subsequently, in the preparation and evaluation of the monitor. Sporulation medium, suspending medium, pasteurization and storage conditions influence resistance of spores of Bacillus subtilis var. niger to ethylene oxide, but incubation temperature and age of sporulating culture appear to be unimportant. The conditions under which the spore suspension is dried on the supporting medium of the monitor exerts a major influence on resistance. Spores exposed to ethylene oxide are abnormally susceptible to damage by shaking with Ballotini, a method frequently used to recover spores from monitors. Nutritional conditions, pH and temperature of incubation influence the ability of survivors to form colonies on solidified media.     

Development of a bacteriophage model system to investigate virus inactivation methods used in the treatment of bone allografts

Bone allografts are commonly used in a variety of surgical procedures, to reconstruct lost bone stock and to provide mechanical support during the healing process. Due to concerns regarding the possibility of disease transmission from donor to recipient, and of contamination of grafts during retrieval and processing procedures, it is common practice to sterilise bone allografts prior to issue for clinical use. It is vital that the sterilisation processes applied to allografts are validated to demonstrate that they achieve the required level of bioburden reduction, and by extension that validated models are used for these studies. Two common sterilisation protocols applied to bone allografts are gamma irradiation and ethylene oxide gas sterilisation, and there are currently no validated models available for measuring the anti-viral efficacy of ethylene oxide treatment with regard to bone allografts or readily useable models for assessing the anti-viral efficiency of gamma irradiation treatment. We have developed and validated models for both these sterilisation processes, using the bacteriophage ϕ×174, and utilised the models to measure the antiviral activity of the standard ethylene oxide and gamma irradiation sterilisation processes applied to bone allografts by the National Blood Service. For the irradiation model, we also utilised bacterial spores (Bacillus pumilus). Our results show that ethylene oxide sterilisation (which can only be applied to lyophilised grafts) inactivated >6.1log₁₀ of the model virus, and gamma irradiation (at 25–40 kGy and applied to frozen allografts) inactivated 3.6–4.0log₁₀ of the model virus and >4log₁₀ of the bacterial spores. Gamma irradiation at this dosage is therefore not in itself a sterilisation process with respect to viruses.     

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Asia and the Pacific and the Latin American regions

The technical assistance program of the International Atomic Energy Agency (IAEA) for its member states in the framework of the implementation of its program on radiation and tissue banking focuses on ensuring the availability of quality radiation-sterilised tissue grafts. The IAEA also helps its member states to develop quality control capabilities in order to ensure the safe use of the processed tissues in certain medical treatments. The majority of developing countries does not have such capacity, and must import expensive sterilised tissues from developed countries. The IAEA’s core contribution to its program on radiation and tissue banking in Asia and the Pacific and the Latin American regions is a technology for sterilisation by gamma radiation and a training program for tissue bank operators and medical personnel. The Agency develops capabilities for radiation sterilisation of tissue grafts, both for reducing the pre-processing bacterial load, and as a terminal sterilisation process. Sterilising tissue grafts offers a clear advantage in terms of safety. Moreover, compared to alternative sterilisation methods, radiation sterilisation is considered particularly safe in relation to environmental concerns, and the deposition of harmful residuals in the tissue, which occurs for example in the use of chemical such as ethylene oxide gas. Radiation sterilisation, thus, has become the method of choice for an increasing number of tissue banks. Radiation sterilisation of tissue grafts is a critical component in the chain connecting donors to recipients of high quality tissue grafts. Due to this fact, the IAEA has evolved as the only organisation in the UN System with expertise related to tissue banking.     

Isolation of Mycobacteria from Frozen Fish Destined for Human Consumption

Mycobacteria were isolated from defrost water and tissue of sole (Solea solea), hake (Merluccius merluccius), cod (Gadus morhua), ling (Genypterus blacodes), and monkfish (Lophius piscatorius) on Löwenstein-Jensen medium after incubation at different temperatures. Samples of frozen fish were obtained under sterile conditions inside a refrigeration chamber (−18 to −22°C) in a wholesale market from which these products are distributed to shops for retail sale and human consumption.     

Ethylene: potential key for biochar amendment impacts

Significant increases in root density, crop growth and productivity have been observed following soil additions of biochar, which is a solid product from the pyrolysis of biomass. In addition, alterations in the soil microbial dynamics have been observed following biochar amendments, with decreased carbon dioxide (CO₂) respiration, suppression of methane (CH₄) oxidation and reduction of nitrous oxide (N₂O) production. However, there has not been a full elucidation of the mechanisms behind these effects. Here we show data on ethylene production that was observed from biochar and biochar-amended soil. Ethylene is an important plant hormone as well as an inhibitor for soil microbial processes. Our current hypothesis is that the ethylene is biochar derived, with a majority of biochars exhibiting ethylene production even without soil or microbial inoculums. There was increased ethylene production from non-sterile compared to sterile soil (215%), indicating a role of soil microbes in the observed ethylene production. Production varied with different biomass sources and production conditions. These observations provide a tantalizing insight into a potential mechanism behind the biochar effects observed, particularly in light of the important role ethylene plays in plant and microbial processes.