Growth ofL Research Articles

Modeling of the competitive growth of Listeria monocytogenes and Lactococcus lactis in vegetable broth.

Current mathematical models used by food microbiologists do not address the issue of competitive growth in mixed cultures of bacteria. We developed a mathematical model which consists of a system of nonlinear differential equations describing the growth of competing bacterial cell cultures. In this model, bacterial cell growth is limited by the accumulation of protonated lactic acid and decreasing pH. In our experimental system, pure and mixed cultures of Lactococcus lactis and Listeria monocytogenes were grown in a vegetable broth medium. Predictions of the model indicate that pH is the primary factor that limits the growth of L. monocytogenes in competition with a strain of L. lactis which does not produce the bacteriocin nisin. The model also predicts the values of parameters that affect the growth and death of the competing populations. Further development of this model will incorporate the effects of additional inhibitors, such as bacteriocins, and may aid in the selection of lactic acid bacterium cultures for use in competitive inhibition of pathogens in minimally processed foods.

Specificity of milk peptide utilization by Lactococcus lactis.

To study the substrate specificity of the oligopeptide transport system of Lactococcus lactis for its natural substrates, the growth of L. lactis MG1363 was studied in a chemically defined medium containing milk peptides or a tryptic digest of alpha s2-casein as the source of amino acids. Peptides were separated into acidic, neutral, and basic pools by solid-phase extraction or by cation-exchange liquid chromatogrpaphy. Their ability to sustain growth and the time course of their utilization demonstrated the preferential use of hydrophobic basic peptides with molecular masses ranging between 600 and 1,100 Da by L. lactis MG1363 and the inability to use large, acidic peptides. These peptide utilization preferences reflect the substrate specificity of the oligopeptide transport system of the strain, since no significant cell lysis was inferred. Considering the free amino acid content of milk and these findings on peptide utilization, it was demonstrated that the cessation of growth of L. lactis MG1363 in milk was due to deprivation of leucine and methionine.

Control ofListeria monocytogenesin Salmon; Antimicrobial Effect of Salting, Smoking and Specific Smoke Compounds

Salmon fillets were either salted only, salted and conventionally smoked in a kiln or salted and dried in the kiln without smoke. The processed fillets were experimentally inoculated withListeria monocytogenes(102–3/g), vacuum packed and the growth monitored during storage at 4 °C over a 30-day period. There was no significant growth in the smoked samples, whereas the number of bacteria increased by between 2 and 5 log cycles in the salted-only samples. In samples salted and dried over the time of conventional smoking, the growth was delayed until the second week of storage after which it increased by 2 log cycles. Commercially available phenol components from wood smoke were mixed in concentrations found in smoked products. These mixtures plus formaldehyde were tested for their antimicrobial properties againstL. monocytogenesin tryptone soya broth (TSB) with added salt at a concentration similar to that in smoked salmon (60 g/kg). A range of TSB based phenolic plus formaldehyde growth substrates, each inoculated with 102L. monocytogenes/g, were tested over a 25 day period at 8 °C. A 50 μg/ml formaldehyde concentration inhibited the growth ofL. monocytogenesfor 2 weeks after which growth started. The phenols did not have any synergistic effect with salt or formaldehyde againstL. monocytogenes.

Utilization of oligopeptides by Listeria monocytogenes Scott A.

For effective utilization of peptides, Listeria monocytogenes possesses two different peptide transport systems. The first one is the previously described proton motive force (PMF)-driven di- and tripeptide transport system (A. Verheul, A. Hagting, M.-R. Amezaga, I. R. Booth, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol, 61:226-233, 1995). The present results reveal that L. monocytogenes possesses an oligopeptide transport system, presumably requiring ATP rather than the PMF as the driving force for translocation. Experiments to determine growth in a defined medium containing peptides of various lengths suggested that the oligopeptide permease transports peptides of up to 8 amino acid residues. Peptidase activities towards several oligopeptides were demonstrated in cell extract from L. monocytogenes, which indicates that upon internalization, the oligopeptides are hydrolyzed to serve as sources of amino acids for growth. The peptide transporters of the nonproteolytic L. monocytogenes might play an important role in foods that harbor indigenous proteinases and/or proteolytic microorganisms, since Pseudomonas fragi as well as Bacillus cereus was found to enhance the growth of L. monocytogenes to a large extent in a medium in which the milk protein casein was the sole source of nitrogen. In addition, growth stimulation was elicited in this medium when casein was hydrolyzed by using purified protease from Bacillus licheniformis. The possible contribution of the oligopeptide transport system in the establishment of high numbers of L. monocytogenes cells in fermented milk products is discussed.

In Vitro and In Vivo Evaluation of the Antimicrobial Activity of Azithromycin against Legionella Species

The antimicrobial activity of azithromycin (AZM) was evaluated againstLegionella species using an experimental model of legionellosis. The minimum inhibitory concentration90s (MIC90s) of AZM against 35 standard strains (0.25 mg/L) and 22 Japanese clinical isolates ofLegionella pneumophila (0.063 mg/L) were lower than those of erythromycin (EM) and almost equal to those of clarithromycin and roxythromycin. Using14C-labeled antibiotic, AZM and EM were observed concentrated inside human polymorphonuclear leukocytes (PMN) with intracellular/extracellular concentration ratios of AZM and EM at 120 minutes after dosing of 27.3 and 22.2, respectively. AZM inhibited the growth ofL. pneumophila (80-045 strain) from cultured guinea pig alveolar macrophages, even when the extracellular AZM was washed out on day 2 of culture. However, the addition of identical concentrations of EM failed to produce a similar inhibition. Pharmacokinetic studies of AZM tissue distribution in guinea pigs infected withL. pneumophila 80-045 revealed high drug concentrations in the lung and liver and a longer half-life in these organs compared with those in plasma. Treatment of guinea pigs with experimentally-induced legionellosis with oral AZM (10 mg/kg/day for 2 days) was more effective than EM treatment (10 mg/kg/day for 4 days) or a placebo, and resulted in a significant improvement in the survival rate. Our results suggest that AZM is a promising new drug for the treatment of legionellosis using a short-term dosing regimen.

Photoinduced Toxicity of PAHs to the Foliar Regions ofBrassica napus(Canola) andCucumbis sativus(Cucumber) in Simulated Solar Radiation

Simulated solar radiation and natural sunlight can enhance polycyclic aromatic hydrocarbons (PAH) toxicity, previously reported in terms of inhibited production ofLemna gibbaleaves and diminished growth ofBrassica napusseedling roots. This work has been extended to examine the photoinduced impact of PAHs on the foliar regions of terrestrial plants. To carry out these experiments two crop species,B. napus(canola) andCucumbis sativus(cucumber), were chosen to test the photoinduced toxicity of six PAHs (anthracene, phenanthrene, benzo(a)anthracene, benzo(a)pyrene, flouranthene, and pyrene). Aqueous solutions containing PAHs were sprayed on the foliage of the plants. It was found that all the PAHs tested had negative impacts on the foliage of the plants and the concentrations that induced toxicity were consistent with those observed for inhibition of growth ofL. gibba.The impacts were observed as diminished biomass accumulation, induction of chlorosis, and inhibition of photosynthesis. It may be concluded from this work that PAHs in rain and surface waters could be harmful to photosynthetic tissues of terrestrial plants.

Effects of solution pH and bicarbonate on the growth and nodulation of a range of grain legume species

Defining plant adaptation to soil conditions is critical for the successful introduction of grain legume species into farming systems. This nutrient solution study examined the effects of pH (4, 5, 6, 7 and 8) and bicarbonate (5 mM KHCO3) on the growth and nodulation of 14 grain legume species supplied with N or reliant on N2-fixation. Species includedPisum sativum L.,Cicer arietinum L.,Lens culinaris Med., and a range ofLupinus, Vicia andLathyrus species. Species differed greatly in response to solution pH. For both N-fertilized and N2-fixing plants, shoot growth ofL. culinaris was very sensitive to low pH (pH < 7), whereas shoot growth ofLupinus angustifolius L. andLupinus albus L. was sensitive to higher pH (pH ≥ 6). Other species had a broader optimal pH range for growth when supplied with N, but were generally sensitive to low pH (pH < 7 forC. arietinum andVicia sativa L., pH < 6 forP. sativum, Vicia faba L.,Lathyrus sativus L. andLathyrus cicera L., and pH < 5 forVicia benghalensis L. andVicia narbonensis L.) when reliant on N2-fixation. For these other species, symbiotic N2-fixation appeared to be more sensitive than host plant growth to low pH. This finding was supported by lower nodule numbers and mass, and lower N concentrations in shoots of sensitive species at low pH relative to higher pH. ForL. culinaris, nodule numbers and mass were relatively unaffected by pH 5–8, N concentrations in shoots were high at low pH and plants developed symptoms relating to H+ toxicity at pH as high as 7. These results indicate that host plant growth ofL. culinaris is more sensitive to low pH than theRhizobium symbiosis. ForL. albus andL. angustifolius, both host plant growth and symbiotic N2-fixation appeared to be equally sensitive to pH ≥ 6.Lupinus pilosus Murr. was more tolerant of high pH than the otherLupinus species. At pH 4, two genotypes ofC. arietinum had better early nodulation than other species.Vicia ervilia L. nodulated poorly at all levels of solution pH, indicating that the commercial Group E inoculum (Rhizobium leguminosurum bv.viceae SU303) may not be effective for this species in solution culture. Addition of bicarbonate decreased shoot growth, nodulation and N concentrations in shoots of most species. Early nodulation (nodule number) ofLathyrus ochrus (L.) DC was not affected by the bicarbonate treatment.

Effects of solution pH and bicarbonate on the growth and nodulation of a range of grain legume species

Defining plant adaptation to soil conditions is critical for the successful introduction of grain legume species into farming systems. This nutrient solution study examined the effects of pH (4, 5, 6, 7 and 8) and bicarbonate (5 mM KHCO3) on the growth and nodulation of 14 grain legume species supplied with N or reliant on N2-fixation. Species includedPisum sativum L.,Cicer arietinum L.,Lens culinaris Med., and a range ofLupinus, Vicia andLathyrus species. Species differed greatly in response to solution pH. For both N-fertilized and N2-fixing plants, shoot growth ofL. culinaris was very sensitive to low pH (pH < 7), whereas shoot growth ofLupinus angustifolius L. andLupinus albus L. was sensitive to higher pH (pH ≥ 6). Other species had a broader optimal pH range for growth when supplied with N, but were generally sensitive to low pH (pH < 7 forC. arietinum andVicia sativa L., pH < 6 forP. sativum, Vicia faba L.,Lathyrus sativus L. andLathyrus cicera L., and pH < 5 forVicia benghalensis L. andVicia narbonensis L.) when reliant on N2-fixation. For these other species, symbiotic N2-fixation appeared to be more sensitive than host plant growth to low pH. This finding was supported by lower nodule numbers and mass, and lower N concentrations in shoots of sensitive species at low pH relative to higher pH. ForL. culinaris, nodule numbers and mass were relatively unaffected by pH 5–8, N concentrations in shoots were high at low pH and plants developed symptoms relating to H+ toxicity at pH as high as 7. These results indicate that host plant growth ofL. culinaris is more sensitive to low pH than theRhizobium symbiosis. ForL. albus andL. angustifolius, both host plant growth and symbiotic N2-fixation appeared to be equally sensitive to pH ≥ 6.Lupinus pilosus Murr. was more tolerant of high pH than the otherLupinus species. At pH 4, two genotypes ofC. arietinum had better early nodulation than other species.Vicia ervilia L. nodulated poorly at all levels of solution pH, indicating that the commercial Group E inoculum (Rhizobium leguminosurum bv.viceae SU303) may not be effective for this species in solution culture. Addition of bicarbonate decreased shoot growth, nodulation and N concentrations in shoots of most species. Early nodulation (nodule number) ofLathyrus ochrus (L.) DC was not affected by the bicarbonate treatment.

Combined chemical defenses against an insect-fungal complex

This study considered how host plant allelochemicals may contribute to defense against insects and fungi that jointly colonize the subcortical tissues of trees, the relative roles of constitutive and inducible chemistry in these defenses, and how the actions of two different feeding guilds might be interrelated. Our model consisted of the coniferous treePinus resinosa, the root- and lower stem-colonizing beetlesHylastes porculus andDendroctonus valens, and their associated fungiLeptographium procerum andL. terebrantis, and the stem-colonizing bark beetleIps pini and its associated fungusOphiostoma ips. In a novel bioassay, extracts from reaction tissue elicted by wound inoculation withL. terebrantis were more repellent to beetles than were similar extracts from constitutive or mechanically wounded tissue. The effect on beetle behavior was more pronounced in nonpolar extracts, which contain mostly monoterpenes, than in polar extracts, which contain mostly phenolics. Synthetic monoterpenes at concentrations present in the various tissues exerted similar effects and were likewise repellent in dose-response experiments. Growth ofL. procerum andL. terebrantis was inhibited by polar extracts from constitutive and reaction tissue. Inhibition was higher in wounded than control tissue, but the inhibition response did not vary with the type of wounding. Synthetic monoterpenes strongly inhibited spore germination and mycelial growth of both fungi. Colonization of red pine roots byLeptographium spp. altered the subsequent effects of extracts of stem phloem tissue onI. pini. These effects varied with host condition. Beetles preferred extracts from constitutive stem phloem tissue of healthy trees to that of root-diseased trees. However, extracts from reaction tissues of healthy trees were more repellent toI. pini than were the reaction tissues of root-diseased trees. The implications of these results to plant defense against insect-fungal complexes and interactions among different feeding guilds are discussed.

The effect of acriflavine and nalidixic acid on the growth of Listeriaspp. in enrichment media

The use of acriflavine in enrichment media forListeria spp. has both direct and indirect effects on the isolation ofListeria monocytogenes . Increasing acriflavine concentrations affect both lag time and generation time ofL. monocytogenes , whereas hardly any effect is observed onListeria innocua . Because acriflavine binds to protein in the samples, a decrease in acriflavine activity results. This lesser activity may result in a better growth ofL. monocytogenes . At low pH-values (pH<5.8) more acriflavine is bound, but growth promoting effects are limited because growth of this pathogen is restricted at low pH. On account of this, one may expect that enrichment protocols employing low acriflavine concentrations with an adequate buffer, favour the isolation ofL. monocytogenes . Because comparative studies have paid no attention to the ratio ofL. monocytogenes to otherListeria spp., virtually nothing is known about the inferior detection ofL. monocytogenes . Previous comparative studies combined with the results of this work indicate strongly that during enrichment procedures, other listeriae or competitive micro-organisms may mask the presence of this pathogen. For that reason, in enrichment protocols for the detection ofL. monocytogenes , it is worthwhile introducing an isolation medium that facilitates identification ofL. monocytogenes in the presence of high numbers of other listeriae.

Effect of water activity adjusted with different solutes on growth and lactic acid production byLactobacillus helveticus

Effect of water activity (a w) adjusted with NaCl or glycerol on the growth and metabolism ofLactobacillus helveticus var.pragensis at 40°C was demonstrated by growth curves (generation time) and changes in the degree of acidity of a milk medium expressed as lactic acid content. NaCl-regulateda w of 0.970 inhibited growth and acid production completely. The same, glycerol-regulateda w of 0.970 increased the generation time only from 33 min (a w=0.994) to 67 min which was less than the generation time at the highera w of 0.982 adjusted with NaCl (103 min). Glycerol-regulateda w of 0.970 did not decrease the acid production (2.6% lactic acid). Ata w of 0.951, the acid production was decreased by 39% compared with the values found in milk media with the original, unadjusteda w of 0.994, after the same time of incubation. Media witha w adjusted to the same values with NaCl or glycerol do not influence the growth and acid production ofL. helveticus in the same way. In contrast to glycerol, NaCl has a strong effect.

The potential for diazotrophy in iron-and sulfur-oxidizing acidophilic bacteria

Acetylene reduction was observed with ferrousiron-oxidizingThiobacillus ferrooxidans, as expected from previous studies with this bacterium. Acetylene reduction was also found during the growth ofT. ferrooxidans on tetrathionate. OnlyLeptospirillum ferrooxidans, one of several other phylogenetically diverse, ferrous-iron-and/or sulfur-oxidizing acidophilic microorganisms, also reduced acetylene. A reduction of the oxygen concentration in the culture atmosphere was necessary to alleviate inhibition of nitrogenase activity. DNA sequences homologous tonif structural genes were found in both organisms. Diazotrophic growth ofL. ferrooxidans was inferred from an increase in iron oxidation in ammonium-free medium when the oxygen concentration was limited and from apparent inhibition by acetylene under these conditions.

Investigation of an Iron-Oxidizing Microbial Mat Community Located near Aarhus, Denmark: Field Studies

We investigated the microbial community that developed at an iron seep where anoxic groundwater containing up to 250 muM Fe flowed out of a rock wall and dense, mat-like aggregations of ferric hydroxides formed at the oxic-anoxic interface. In situ analysis with oxygen microelectrodes revealed that the oxygen concentrations in the mat were rarely more than 50% of air saturation and that the oxygen penetration depth was quite variable, ranging from <0.05 cm to several centimeters. The bulk pH of the mat ranged from 7.1 to 7.6. There appeared to be a correlation between the flow rates at different subsites of the mat and the morphotypes of the microorganisms and Fe oxides that developed. In subsites with low flow rates (<2 ml/s), the iron-encrusted sheaths of Leptothrix ochracea predominated. Miniature cores revealed that the top few millimeters of the mat consisted primarily of L. ochracea sheaths, only about 7% of which contained filaments of cells. Deeper in the mat, large particulate oxides developed, which were often heavily colonized by unicellular bacteria that were made visible by staining with acridine orange. Direct cell counts revealed that the number of bacteria increased from approximately 10 to 10 cells per cm and the total iron concentration increased from approximately 0.5 to 3 mmol/cm with depth in the mat. Primarily because of the growth of L. ochracea, the mat could accrete at rates of up to 3.1 mm/day at these subsites. The iron-encrusted stalks of Gallionella spp. prevailed in localized zones of the same low-flow-rate subsites, usually close to where the source water emanated from the wall. These latter zones had the lowest O(2) concentrations (<10% of the ambient concentration), confirming the microaerobic nature of Gallionella spp. In subsites with high flow rates (>6 ml/s) particulate Fe oxides were dominant; direct counts revealed that up to 10 cells of primarily unicellular bacteria per cm were associated with these particulate oxides. These zones exhibited little vertical stratification in either the number of cells or iron concentration. Finally, mat samples incubated anaerobically in the presence of acetate or succinate exhibited significant potential for iron reduction, suggesting the possibility that a localized iron cycle could occur within the mat community.

Kinetics and Modeling of Lactic Acid Production by Lactobacillus plantarum

An unstructured model was developed to describe bacterial growth, substrate utilization, and lactic acid production by Lactobacillus plantarum in cucumber juice. Significant lactic acid production occurred during growth, as well as stationary phases. The percentage of acid produced after growth ceased was a function of the medium composition. Up to 51% of the lactic acid was produced after growth ceased when NaCl was not present in the medium, whereas not more than 18% of the total lactic acid was produced after the growth ceased in presence of NaCl, probably because of an increase in the cell death rate. An equation relating the specific death rate and NaCl concentration was developed. With the kinetic model proposed by R. Luedeking and E. L. Piret (J. Biochem. Microbiol. Technol. Eng. 1:393-412, 1958) for lactic acid production rate, the growth-associated and non-growth-associated coefficients were determined as 51.9 (+/-4.2) mmol/g of cells and 7.2 (+/-0.9) mmol/g of cells h respectively. The model was demonstrated for batch growth of L. plantarum in cucumber juice. Mathematical simulations were used to predict the influence of variations in death rate, proton concentration when growth ceased, and buffer capacity of the juice on the overall fermentation process.

Influence of Plumbing Materials on Biofilm Formation and Growth of Legionella pneumophila in Potable Water Systems

A two-stage chemostat model of a plumbing system was developed, with tap water as the sole nutrient source. The model system was populated with a naturally occurring inoculum derived from an outbreak of Legionnaires' disease and containing Legionella pneumophila along with associated bacteria and protozoa. The model system was used to develop biofilms on the surfaces of a range of eight plumbing materials under controlled, reproducible conditions. The materials varied in their abilities to support biofilm development and the growth of L. pneumophila. Elastomeric surfaces had the most abundant biofilms supporting the highest numbers of L. pneumophila CFU; this was attributed to the leaching of nutrients for bacterial growth from the materials. No direct relationship existed between total biofouling and the numbers of L. pneumophila CFU.

Some cultural characteristics of mycelia of a mycorrhizal fungus, Lyophyllum shimeji

Cultural characteristics of 45 strains ofLyophyllum shimeji and 10 strains of three related species were determined. The average optimum temperature for mycelial growth ofL. shimeji on a medium consisting of rye grains was 24.9°C, slightly higher than those forL. fumosum andL. decastes. The average mycelial growth rate of theL. shimeji strains, each at its optimum temperature, was 2.0 mm/day, almost the same as that ofL. decastes and 2 times greater than those ofL. semitale andL. fumosum. All strains ofL. shimeji could grow on beech and pine sawdust, but none could significantly decompose beech wood blocks. Of the 45 strains ofL. shimeji, 3 strains had ability to form primordia on the rye grain medium without the host plant.

Legionella pneumophila virulence conserved after multiple single-colony passage on agar

Multiple passage ofLegionella pneumophila on either supplemented Mueller-Hinton or charcoal yeast extract agar by the conventional batch passing technique results in loss of virulence. In this studyL. pneumophila virulence was maintained after multiple passage on buffered charcoal yeast extract (BCYE α) agar by a single-colony transfer technique. Virulence was determined by assessing the growth ofL. pneumophila for thioglycolate-induced susceptible A/J mouse macrophages in vitro and infectivity for susceptible A/J mice in vivo. Passage of the virulentL. pneumophila, 30 times on BCYE α agar by the single-colony transfer procedure still resulted in virulence, as compared with the nonpassaged parental bacteria, both in vitro and in vivo. Lethality for susceptible, A/J mice by systemic infection was comparable for the 30th colony-passaged bacteria and the parentalL. pneumophila. These results show that theL. pneumophila phenotype associated with intracellular growth in macrophages or infectivity for susceptible mice is stable following passage by the single-colony transfer procedure on BCYE α agar.

Characterization of the Heat Shock Response in Lactococcus lactis subsp. lactis

The heat shock response in Lactococcus lactis subsp. lactis was characterized with respect to synthesis of a unique set of proteins induced by thermal stress. A shift in temperature from 30 to 42 degrees C was sufficient to arrest the growth of L. lactis subsp. lactis, but growth resumed after a shift back to 30 degrees C. Heat shock at 50 degrees C reduced the viable cell population by 10; however, pretreatment of the cells at 42 degrees C made them more thermoresistant to exposure at 50 degrees C. The enhanced synthesis of approximately 13 proteins was observed in cells labeled with S upon heat shock at 42 degrees C. Of these heat shock-induced proteins, two appeared to be homologs of GroEL and DnaK, based on their molecular weights and reactivity with antiserum against the corresponding Escherichia coli proteins. Therefore, we conclude that L. lactis subsp. lactis displays a heat shock response similar to that observed in other mesophilic bacteria.

Acid Tolerance of Leuconostoc mesenteroides and Lactobacillus plantarum

In this study, we determined the internal cellular pH response of Leuconostoc mesenteroides and Lactobacillus plantarum to the external pH created by the microorganisms themselves or by lactic or acetic acids and their salts added to the growth medium. Growth of Leuconostoc mesenteroides stopped when its internal pH reached 5.4 to 5.7, and growth of L. plantarum stopped when its internal pH reached 4.6 to 4.8. Variation in growth medium composition or pH did not alter the growth-limiting internal pH reached by these microorganisms. L. plantarum maintained its pH gradient in the presence of either 160 mM sodium acetate or sodium lactate down to an external pH of 3.0 with either acid. In contrast, the DeltapH of Leuconostoc mesenteroides was zero at pH 4.0 with acetate and 5.0 with lactate. No differences were found between d-(-)- and l-(+)-lactic acid for the limiting internal pH for growth of either microorganism. The comparatively low growth-limiting internal pH and ability to maintain a pH gradient at high organic acid concentration may contribute to the ability of L. plantarum to terminate vegetable fermentations.

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.