Temperature-dependent protection against Ichthyophthirius multifiliis following immunisation of rainbow trout using live theronts

Rainbow trout Oncorhynchus mykiss Walbaum, 1792 fingerlings were vaccinated by intraperitoneal (i.p.) injection using live theronts of the skin parasitic ciliate Ichthyophthirius multifiliis Fouquet, 1876 at 2 temperatures (12 and 20 degrees C), and protection against challenge infections was subsequently evaluated by bath exposure to live theronts. Vaccination conferred a relative protection (evaluated as the decrease in the number of established theronts) at 12 degrees C and almost complete immunity at 20 degrees C. Significantly increased immobilisation titers (using plasma immobilisation of live theronts) were found in immunised fish at Week 2 and 4 post-vaccination. Lysozyme activity of plasma from vaccinated fish increased from Week 1 to 4. Both immobilisation titers and lysozyme activity were significantly higher at 20 degrees C. This study demonstrated that live theronts are good candidates for an antigen source for development of effective vaccines against white spot disease in this fish host, and further indicated that the protection of rainbow trout against I. multifiliis infection is highly temperature dependent and may be associated with both adaptive and innate response mechanisms.     

Effect of lectins on the invasion of Ichthyophthirius theront to channel catfish tissue.

This study determined the effects of lectin binding to theronts of Ichthyophthirius multifiliis on theront immobilization, invasion, trophont development and survival in channel catfish Ictalurus punctatus excised fins in vitro. Soybean agglutinin (SBA), lentil agglutinin (LCA), gorse agglutinin (UEA-I) and wheat germ agglutinin (WGA) were used to treat theronts. Percentages of theronts immobilized by 4 lectins ranged from 12.0 to 19.4% at a concentration of 1000 microg ml(-1). These lectins bound more than half of the theronts at a concentration of 50 microg ml(-1). More theronts were labeled by SBA and WGA than by lectin LCA at concentrations of 50 and 100 microg ml(-1), respectively. The binding of these lectins to theronts indicated that monosaccharides (D-galactose, L-fucose, D-mannose and D-glucose) and amino sugar derivatives (N-acetylgalactosamine and N-acetylglucosamine) were present on the surface of theronts. Invasion was reduced significantly for theronts treated with LCA, UEA-I and WGA. No difference in invasion was found between control and SBA bound theronts (p > 0.05). The binding of lectin LCA, UEA-I and WGA to theronts significantly reduced the development of trophonts (p < 0.05). The mean volumes of trophonts labeled with these 3 lectins were smaller than volumes in control trophonts from 8 to 48 h after exposure. Survival was lower in trophonts labeled with lectins than in control trophonts at 48 h after exposure.     

Optimum energy intake and gross efficiency of energy conversion for brown trout, Salmo trutta, feeding on invertebrates or fish

1. The chief objectives were to determine the daily optimum energy intake ( C OPT cal day−1) for growth and the gross efficiency ( K G%) for converting energy intake into growth for brown trout, Salmo trutta . Energy budgets for individual fish were obtained from experiments with 292 trout (initial live weight 1–318 g) bred from wild parents, and kept at five constant temperatures (5, 10, 13, 15, 18 °C) and 100% oxygen saturation. Most trout (252) were fed over a period of 42 days on a fixed ration of shrimps, Gammarus pulex , the ration levels varying between zero and maximum, but 40 of the larger trout were fed to satiation on freshly-killed sticklebacks ( Gasterosteus aculeatus ).
2. Energetics models developed in earlier studies on the same data were summarized briefly and were used to predict the relationship between the change in the total energy content of a trout ( C G cal day−1) and its energy intake ( C IN cal day−1), and hence to estimate C OPT. The models were also used to predict the relationship between K G and C IN. In both comparisons, there was good agreement between observed values from the experiments and expected mean values predicted from the models. For trout feeding on invertebrates, C OPT lay closer to the maximum, rather than the maintenance, energy intake. When the diet changed from invertebrates to fish, there was a marked increase in C IN, C G and K G.
3. For trout feeding on invertebrates, K G exceeded 30% within 7–11 °C, with a maximum K G of 31.8% at 8.9 °C. For piscivorous trout, K G exceeded 30% within 4–16 °C and 40% within 6.5–12 °C, with a maximum K G of 41.8% at 9.3 °C. These differences were discussed in relation to the results of previous workers, and the models used in the present study provided a method of exploring the limitations of the ' K -line' hypothesis for the relationship between K G and C IN.     

Extracellular Calcium Concentration Affects Infectivity of Ichthyophthirius (Ciliophora) Theronts

Tomonts and their theront offspring of the hymenostomatid fish parasite Ichthyophthirius multifiliis were exposed to calcium levels from 0 to 0.8 mM Ca^2+. The survival and reproductive rates of tomonts in the absence of extracellular calcium were not significantly different from rates of tomonts provided calcium. Theronts that developed in the absence of calcium, however, were not infective for Ictalurus punctatus even when the extracellular magnesium concentration was doubled. Theronts that developed in 0.10 mM Ca²⁺ were infective (0.77 trophonts/mm² of pectoral fin) to essentially the same extent as theronts provided 0.33 mM Ca^2+. Infectivity of those provided 0.8 mM Ca²⁺ was 1.79 trophonts/mm² of fin, similar to that of theront controls. Theronts deprived of extracellular calcium as they developed contained significantly fewer secretory mucocysts than did theronts provided 0.1 to 0.8 mM Ca²⁺ although no significant differences among groups occurred with respect to abundance of crystalline or differentiating mucocysts. Theronts deprived of extracellular calcium also had swollen or enlarged mitochondria and abnormal crystalline mucocysts.     

Critical Periods in Development of Ichthyophthirius multifiliis (Ciliophora) Populations1

ABSTRACT. Three periods in development that strongly influence population dynamics of Ichthyophthirius multifiliis were identified in experimental infections of channel catfish. The first occurred upon establishment within the host, 0 to 10 min postexposure (PE), when the parasite population that gained entrance declined 50%. Survival from 10 to 45 min PE, however, was constant. The second period identified came after I. multifiliis left the host and the free-living tomont encysted. The third occurred during reproduction. Although survival of encysting tomonts approached 100% among individuals departing after three to five days residence in the host, theront production varied significantly with parasite size, culture temperature during development, and length of residence by the trophont in the host. Theront production per tomont increased daily and on days three, four, and five PE was significantly higher for parasites developing at 24°C than for those at 21°C. At five days PE, mean production was 562 theronts/tomont and 240 theronts/tomont, respectively, and production by tomonts of equal size was greater for parasites maintained at 24°C.     

Development time and survival of Verrallina funerea (Theobald) (Diptera: Culicidae) immatures and other brackish water mosquito species in southeast Queensland, Australia

Abstract  Verrallina funerea (Theobald) is a brackish water mosquito that is recognised as an important pest and vector in southeast Queensland, Australia. Immature development time and survival of Ve. funerea was defined in the laboratory in response to a range of temperatures (17–34°C) and salinities (0–35 parts per thousand (p.p.t)). The expression of autogeny in this species was also assessed. Salinity only had a slight effect on mean development time from hatching to adult emergence (7.0–7.4 d at salinities of 0, 17.5 and 31.5 p.p.t) and survival was uniformly high (97.5–99.0%). Mean development times were shorter at 26, 29 and 32°C (7.0, 6.8 and 6.8 d, respectively) and longest at 17°C (12.2 d). The threshold temperature ( t ) was 5.8°C and the thermal constant ( K ) was 142.9 degree-days above t . Survival to adulthood decreased from >95% (at 17–29°C) to 78% (at 32°C) and 0% (at 34°C). No expression of autogeny was observed. Immature development times of Ve. funerea , Ochlerotatus vigilax (Skuse) and Oc. procax (Skuse) were then determined under field conditions at Maroochy Shire. Following tide and rain inundation, cohorts of newly hatched larvae were monitored daily by dipping, and time until pupation was noted. Tidal inundation triggered hatching of Ve. funerea and Oc. vigilax larvae whereas Oc. procax larvae were found only after rain inundation. Estimates of Ve. funerea and Oc. vigilax field development times were similar (8–9 d) while Oc. procax development time was slightly longer (9–10 d). Based on these survey results, control activities targeting Ve. funerea must be initiated 4 d (if using Bacillus thuringiensis var. israelensis de Barjac) or 5 d (if using s -methoprene) after inundation. However, Casuarina glauca Sieber canopy and branchlets covering breeding habitats may present a problem for the penetration of such treatments.     

Immune-relevant genes expressed in rainbow trout following immunisation with a live vaccine against Ichthyophthirius multifiliis

Rainbow trout Oncorhynchus mykiss were immunised by intra-peritoneal injection using a live vaccine based on Ichthyophthirius multifiliis (Ich) theronts, which previously has shown protection against white spot disease. Samples were taken pre-vaccination and on Day 1, 7, 21 and 28 post-immunisation (p.i.). Expression of immune relevant genes in the liver, spleen and head kidney was monitored by qPCR. To describe the immune reaction following this immunisation, a series of genes encoding cytokines, complement factors, immunoglobulins and acute phase reactants were studied. Genes encoding acute phase reactants in the liver were up-regulated with serum amyloid A (SAA) as the most pronounced with a 2299-fold increase at 24 h p.i. Hepcidin and pre-cerebellin were also up-regulated in the liver 24 h p.i., by 7- and 4-fold, respectively. Complement factors C3, C5 and factor B (Bf) were up-regulated in the spleen and the head kidney 24 h and 28 d p.i. Genes encoding immunoglobulins were not up-regulated, but a specific low titer IgM response (titer 25) against parasite antigens was detected by a modified ELISA 4 wk p.i.     

Cross-immunity and antibody responses to different immobilisation serotypes of Ichthyophthirius multifiliis

Vaccination of channel catfish with either of two serotypes of the parasitic ciliate Ichthyophthirius multifiliis conferred protection against challenge infection by either serotype. Fish were vaccinated by intracoelomic injection with live theronts of isolate G5 (serotype D) or isolate G12 (a new serotype), which express different surface immobilisation antigens. Vaccination with live G12 theronts conferred complete protection against subsequent challenge by both serotypes while vaccination with G5 theronts elicited only partial protection against both serotypes. Vaccination with trophont lysates did not protect against challenge infection. Sera from vaccinated fish were tested in immobilisation assays, ELISAs, and Western blots. Serum antibodies recognised only immobilisation antigens of the serotype used for vaccination in immobilisation assays or on Western blots. No antigens common to both serotypes were identified by Western blots. In contrast, serum antibodies bound antigens in cell lysates from both serotypes by ELISA, demonstrating that antibodies recognising both serotypes are produced in response to infection, which presumably confer observed cross-serotype protection.     

Effects of environmental factors on virulence of the entomopathogenic fungus, Nomuraea rileyi, against the corn earworm, Helicoverpa armigera (Lep., Noctuidae)

The effects of environmental factors on infection of the entomopathogenic fungus, Nomuraea rileyi , isolated from the corn earworm, Helicoverpa armigera , in Taiwan, to its host insect were studied in the laboratory. The fungus caused higher larval mortality at 20°C than at 30°C when 5 × 10⁶ conidia/ml were sprayed on the fourth instar. However, mortality of the fifth instar injected with 1 × 10³ conidia/larva was not significantly different when the inoculated larvae were incubated from 15 to 30°C. The fungal development in inoculated larvae was best at 20 and 25°C after shifting from 20°C to either lower or higher temperatures. The germination rate was higher at 20 and 25°C than at 30 or 35°C. Conidial germination was better on the wash-off of insect cuticle than on Sabouraud maltose agar with yeast extract. Sporulation on chill-dried cadavers was maximal at 95 or 100% relative humidity than at lower levels of relative humidity. The time required for sporulation was 2 days less at 100% than at 95% relative humidity. Although photoperiod did not affect fifth instar mortality caused by N. rileyi , the median lethal time (LT₅₀) values were shorter upon incubating under light than in darkness. Incubation of infected cadavers under 12 or 24 h light resulted in 20-fold more conidial production than under full darkness. Therefore, illumination is necessary for development of this isolate on insect cadavers.     

Efficacy of Beauveria bassiana (Bals.) Vuill. against the spruce bark beetle, Ips typographus L., in the laboratory under various conditions

Abstract:  In laboratory bioassays, the efficacy of the entomopathogenic fungus Beauveria bassiana against the spruce bark beetle, Ips typographus , was tested under various conditions. Four of the tested isolates and the commercial product Boverol^® caused 99–100% mortality when tested at a concentration of 1.0 × 10⁷ conidia/ml at 25°C. Using B. bassiana isolate 138 at a concentration of 1.0 × 10⁶, the median survival time (MST) was 6.1 d and significantly longer compared with the MST of 4.2 and 4.0 d at 1.0 × 10⁷ and 1.0 × 10⁸ conidia/ml, respectively. In the next experiment, the beetles were maintained on spruce bark, filter paper or artificial diet during the bioassay with Boverol^®, and significant differences in the MST of 3.6, 2.5 and 5.3 d, respectively, were noticed. The experiment with Boverol^® at different temperatures showed that the beetles lived significantly longer at 15°C (MST 8.7 d) than at 20, 25, 30 and 35°C. At 25°C, the beetles died most rapidly (MST 3.5 d). At different relative humidities (RH) of 40, 70 and 100%, nearly all beetles were dead after treatment with a suspension of Boverol^® at 1.0 × 10⁷ conidia/ml. At 40% RH, 49% of the untreated beetles died after 7 d. The best effects were achieved with the following bioassay: beetles were fed for three days on artificial diet, then dipped into a solution of 1.0 × 10⁷ conidia/ml and transferred on a piece of spruce bark in Petri dishes at 25°C and 70% RH.     

Comparing tolerance of Ichthyophthirius multifiliis and Tetrahymena thermophila for new cryopreservation methods

Ichthyophthirius multifiliis is an obligate protozoan parasite of freshwater fishes that has a complex developmental cycle. It has not been successfully cryopreserved, so management studies are restricted to parasites obtained during outbreaks or perpetuated by passage in live fishes. To overcome this serious limitation, free-swimming I. multifiliis parasites were tested in a cryopreservation protocol routinely used for a related ciliate, Tetrahymena. In this protocol, I. multifiliis theronts retained infectivity for 3 days, although the protocol itself was ultimately lethal. Exposure of I. multifiliis and Tetrahymena thermophila to a battery of media and cryopreservative reagents showed that I. multifiliis was less hardy than T. thermophila and likely had significant biological and cytoskeletal differences. No combination of reagents, media, freezing rates, or dilution media permitted cryopreservation of I. multifiliis parasites that could then undergo development or infect fish. However, a vitrification protocol was formulated using Ficoll, 1,2-propanediol, and N,N-dimethylacetamide from which intact cryopreserved theronts with some motility were recovered. Understanding the effects of these reagents may lead to both a cryopreservation method for I. multifiliis and to improved understanding of the biology of ciliates.     

Prolonged in vitro cultivation of Ichthyophthirius multifiliis using an EPC cell line as substrate

The ciliate Ichthyophthirius multifiliis, which normally requires a fish host to develop from the theront stage to the trophont stage, was cultivated in vitro for part of its life cycle. Experiments were conducted using a laboratory strain of the parasite originally isolated from rainbow trout Oncorhynchus mykiss in a Danish trout farm. Theronts escaping from tomontocysts were kept in water, cell culture media (E-MEM or L-15), or cultures of EPC (Epithelioma Papulosum Cyprini) cells in plastic tissue culture dishes (Nunc multidish plates). In addition, a 2-compartment system, with water separated from tissue culture media by a monolayer of EPC cells on an Anopore Tissue Culture Insert (mimicking the fish epidermis) was tested as an experimental habitat for the parasite. Theronts transformed into trophonts in all treatments except in water alone. However, development was accelerated in wells containing EPC cells, and survival and growth of trophonts were significantly increased compared to water or tissue culture media alone. Further, the 2-compartment system allowed superior performance of the parasites (attachment of parasites to cells and growth from 36 to 46 microm). In all experiments it was found that the presence of host factors (mucus and serum) stimulated parasite development.     

Reproductive migration of brown trout in a small Norwegian river studied by telemetry

The movement of 34 large (39–73 cm standard length) brown trout Salmo trutta was monitored using radio telemetry for up to 74 days in Brumunda, a small Norwegian river (mean annual discharge 3·3 m³ s⁻¹) flowing into the large Lake Mjøsa. The maximum range of movement in the river was 20 km. No clear relationships existed between individual movement and water discharge, temperature and barometric pressure. Brown trout migrated at all levels of water discharge. At low discharge (<2 m³ s⁻¹) movements were nocturnal. A weir 5·3 km from the outlet restricted ascending brown trout at low ( c . 6° C), but not at high ( c . 8° C) water temperatures. Spawning occurred in September to October and tagged individuals spent 2–51 days at the spawning sites. Mean migration speed from tagging to when the fish reached the spawning area, and from when they left the spawning areas and reached the lake was 1·0 and 2·3 km day⁻¹, respectively. All tagged brown trout that survived spawning returned to the lake after spawning.     

Characteristics of the spawning migrations of brown trout, Salmo trutta L., and rainbow trout, S. gairdneri Richardson, in Great Lake, Tasmania

Upstream spawning migrations of mature brown trout, S. trutta , and rainbow trout, S. gairdneri , were studied in Liawenee Canal, Great Lake from 1949 to 1985. Brown trout migrations normally occurred from early April to mid-May and rainbow trout from late August to early November. In 1983, 16 425 brown trout and 1338 rainbow trout passed through a fixed upstream diversion trap. Brown trout spawning migrations occurred predominantly over the temperature range 6–10° C, while rainbow trout migrated predominantly over the range 5–11° C. Migrations peaked at water temperatures of 7.6°C (males) and 7.8°C (females) for brown trout, and 8.3°C (males) and 9.6°C (females) for rainbow trout. Rainbow trout migrations occurred at high flow conditions and were positively correlated with canal flow increases, while brown trout migrated under low canal flow. Mean length, weight and condition of rainbow trout of both sexes decreased significantly during migrations. Female brown trout decreased in weight and condition but not in length; male brown trout did not change in condition despite decreases in both length and weight during migrations. Overall sex ratio was 2:1 (female:male) for both species, with the relative proportion of male fish decreasing as migrations progressed. Age composition changed during migrations; dominant age classes were 3 < 4 < 5 + years for both species. Comparison of length, weight, condition and age revealed minor changes during the 37-year period 1949–1985.     

Partial cross protection against Ichthyophthirius multifiliis in Gyrodactylus derjavini immunized rainbow trout.

Partial cross protection against a skin-parasitic ciliate has been recorded in rainbow trout previously immunized with an ectoparasitic platyhelminth. The susceptibility to infection by Ichthyophthirius multifiliis differed significantly between naive and Gyrodactylus derjavini immunized rainbow trout. Fish partly immune to the ectoparasitic monogenean G. derjavini became less infected and experienced lower mortality than naive fish when exposed to I. multifiliis infections. In vitro studies on immobilization of theronts using decomplemented (heat-inactivated) serum from G. derjavini immune or non-immune hosts showed no immobilization. However, untreated serum from both immune and non-immune fish containing intact complement immobilized theronts (titre 128-256). In addition, non-specific priming of the host response with interleukin (IL-1), bacterial lipopolysaccharide (LPS), concanavalin A (Con A) or mannan did confer a partial resistance to I. multifiliis infection. This will suggest that non-specific factors including complement could be partly responsible for the host response against infections with this ciliate.     

Differential expression of cysteine proteases in developmental stages of the parasitic ciliate Ichthyophthirius multifiliis

An expressed sequence tag database of the freshwater fish parasite, Ichthyophthirius multifiliis (Ciliophora) was analyzed to seek for proteases potentially involved in the invasion and degradation of host tissues during infection. The translation of the database revealed two cathepsin L cysteine proteases (Icp1 and Icp2) of the C1A peptidase subfamily. The analysis of Icp1 and Icp2 sequences suggested that both proteases would be synthesized as preproproteins, with a mature domain of 27.9 and 22.8 kDa, respectively. Their expression level was determined in the trophont parasitic stage, in the tomont reproductive stage, and in the theront infective stage by real-time RT-PCR. ICP1 and ICP2 were significantly upregulated in trophont and theront stages in comparison with the tomont stage. Mature peptides of Icp1 and Icp2 were identified in crude extracts of I. multifiliis trophonts by LC-MS/MS. Zymograms showed three to seven activity bands at the optimum pH of cathepsin L cysteine proteases. Two bands displaying cysteine protease activity were identified by inhibition with E-64. They represented the major proteolytic activity of the trophont stage at pH 5-7, suggesting that cysteine proteases play an important role in the infection process.     

Protective immunity of Nile tilapia against Ichthyophthirius multifiliis post-immunization with live theronts and sonicated trophonts

Two immunization trials were conducted to evaluate host protection of Nile tilapia, Oreochromis niloticus against Ichthyophthirius multifiliis (Ich). Immunizations were done with live theronts or sonicated trophonts by bath immersion and intraperitoneal (IP) injection. The immunized fish were challenged with theronts 21 days post-immunization in trial I and 180 days post-immunization in trial II. The serum anti-Ich antibody and cumulative mortalities of tilapia were determined after theront challenge. Serum anti-Ich antibody was significantly higher (P<0.05) in tilapia immunized with live theronts by immersion or IP injection or with sonicated trophonts administered by IP injection than tilapia immunized with sonicated trophonts by immersion, with bovine serum albumin by IP injection, or non-immunized controls. Host protection was acquired in fish immunized with live theronts by immersion or IP injection. Tilapia immunized with sonicated trophonts by IP injection were partially protected with a 57-77% survival in both trials. At 180 days post-immunization, serum antibody titers had declined in immunized fish yet they were still able to survive challenge. The protection appears not to be solely depending on serum antibody response against Ich.     

Occurrence and Thermoresistance of Spores of Psychrophilic and Psychrotrophic Aerobic Sporeformers in Soil and Foods

Spores of psychrotrophic (able to grow at 5°C) aerobic sporeformers occurred in soil in high numbers (2 × 10³-5 × 10⁶/g), whereas psychrophilic (able to grow at 0°C) spores were present at significantly lower levels (500–10⁵/g). Psychrotrophic spores were absent in herbs and spices: in pasteurized meals prepared industrially their numbers varied from <10 to 1000/g. For spores harvested from Trypticase Soy Agar (TSA), the heat resistance of the cold-tolerant sporeformers was low with D _90°C-values from 1–11 min. The recovery of heated psychrophilic spores on this medium at 5°C was equal to their recovery at 20°C. However, the recovery of heated psychrotrophic spores was lower at 5°C than at 20°C, whereas unheated spores gave the same counts at both temperatures. The heat resistance of naturally occurring spores of cold-tolerant sporeformers washed from soil was comparable with the resistance of spores formed on TSA.     

Temperature effects on immune response and hematological parameters of channel catfish Ictalurus punctatus vaccinated with live theronts of Ichthyophthirius multifiliis

This study evaluated the influence of temperature on the immune responses and hematological parameters in channel catfish Ictalurus punctatus immunized via intraperitoneal injection with live theronts of Ichthyophthirius multifiliis. Fish were distributed in 18 aquaria and received 9 treatments: 4 groups of fish were vaccinated with live theronts and maintained at constant temperature 15 °C, 20 °C, 25 °C and 30 °C; 3 groups of fish vaccinated and subjected to cycling temperature regime from 15-25 °C, 20-25 °C and 20-30 °C, changed 5 °C each day; 2 groups of fish were not vaccinated and served as controls at 25 °C, one with Ich challenge and the other without challenge. Non vaccinated fish and those vaccinated at 15 °C or 15-25 °C did not show anti-Ich antibodies in the serum 14 and 21 days post-immunization. The antibody levels were significantly higher from fish vaccinated at 25 °C, 30 °C, 20-25 °C and 20-30 °C compared to fish at 15 °C, 20 °C and 15-25 °C both 14 and 21 days post-immunization. At constant water temperature, fish vaccinated at 15 °C showed significantly higher mortality rate (67.8%, P < 0.05) than those vaccinated at 20 °C, 25 °C, and 30 °C (0-10.7% mortalities). At cycling water temperature, fish vaccinated at 15-25 °C showed significantly higher mortality rate (67.8%) than those vaccinated at 20-25 °C and 20-30 °C (P < 0.05). Twenty days after immunization fish vaccinated at 30 °C and 20-30 °C showed significant increase in the red blood cells, white blood cells, thrombocytes and monocytes. Six days after challenge with I. multifiliis theronts the fish showed decreased white blood cells, thrombocytes and monocytes. This study suggests that vaccinated catfish were severely impacted by low temperature, either at 15 °C constant temperature or at 15-25 °C cycling temperature. The fish showed no anti-Ich antibodies and suffered high mortality similar to non vaccinated control fish.     

Effects of temperature and heat activation on germination of individual spores of Bacillus subtilis

Phase intensity changes of individual germinating spores of Bacillus subtilis were determined by phase-contrast light microscopy and image analysis. Two germination phases were investigated. The length of the time period before a change in phase brightness was evident and the duration of the phase intensity change until a constant greylevel was maintained. The incubation temperature (37 and 20 °C) and heat activation (10 min at 65 °C) had a distinct effect on both phases. At 37 °C, spores of B. subtilis 604 started to show a decrease in brightness in l -alanine buffer after 3–39 min and needed 10–39 min to complete the phase change. At 20 °C, lag times of 10–100 min were observed and the spores needed 30–100 min to reach a constant greylevel. Heat activation and subsequently exposure to l -alanine buffer at 20 °C reduced the lag phase to 6–90 min and the phase change was finished after 30–60 min. Our results indicate enzymatic involvement before and during the phase intensity change of germinating spores.