Giardia cysts and Cryptosporidium oocysts detection in the intake and rapid filter system in a water purification plant

Water samples, taken from the intake and rapid filter system of a water purification plant, were analyzed using an immunofluorescence antibody method for detecting the presence of Giardia cysts and Cryptosporidium oocysts. Giardia cysts and Cryptosporidium oocysts were found in the intake water from zero to 38.7 cysts/100 l and 1.7–50.5 oocysts/100 l with averages of 9.6 cysts/100 l and 19.4 oocysts/100 l. Giardia cysts and Cryptosporidium oocysts were also detected in the samples taken from the rapid filtration unit with mean concentrations of zero to 2.3 cysts/100 l and 0–2.5 oocysts/100 l, respectively. The efficacy of the rapid filter in suspended material and (oo)cyst removal was significant. The removal late was 56–97% for suspended material and 69–100% for the (oo)cysts.     

Improvement of recoveries for the determination of protozoa Cryptosporidium and Giardia in water using method 1623

The U.S. Environmental Protection Agency has developed method 1623 for simultaneous detection of Cryptosporidium oocysts and Giardia cysts in water. Method 1623 includes four major steps: filtration, immunomagnetic separation (IMS), fluorescent antibody (FA) staining and microscopic examination. It was noted that the recovery levels following IMS-FA and FA staining were high, averaging more than 92.0% and 89.0% for C. parvum oocysts and G. lamblia cysts, respectively. In contrast, when the filtration step was incorporated, the recovery level of C. parvum oocysts declined significantly to 18.1% in seeded tap water, while a relatively high recovery level of 77.2% for G. lamblia cysts could still be achieved. Further study indicated that the recovery level of C. parvum oocysts could be enhanced significantly when an appropriate amount of silica particles was added to a water sample. The recovery level of C. parvum oocysts was affected by particle size and concentration. The optimal silica particle size was determined to be within the range of 5-40 microm, and the corresponding optimal silica concentration was 1.42 g for 10-l tap water. When both G. lamblia cysts and C. parvum oocysts were spiked into the tap water sample containing the optimum amount of silica particles, the average recovery levels of oocysts and cysts were 82.7% and 75.4%, respectively. The results obtained clearly suggested that addition of an appropriate amount of silica particles could improve the recovery level of C. parvum oocysts significantly and yet there was no noticeable deleterious effect on the recovery level of G. lamblia cysts. Further study indicated that the rotation time in the IMS procedure using the Dynal GC-Combo IMS kit (which was recommended in method 1623) was important for G. lamblia cyst detection. In contrast, the recovery level of C. parvum oocysts was not affected by the rotation time. Furthermore, it was found that the recovery levels of C. parvum oocysts using methods 1622 and 1623 were quite close although different IMS kits were used in the two methods.     

Adaptive Copper Tolerance in <Emphasis Type="Italic">Elsholtzia haichowensis</Emphasis> Involves Production of Cu-induced Thiol Peptides

Copper (Cu) accumulation and tolerance mechanisms in Elsholtzia haichowensis, an indicator plant of Cu mines, were investigated under hydroponics supplied with different concentrations (0.32, 50.0, 100.0 and 200.0 μM) of Cu for 8 days. Cu at 100 and 200 μM significantly decreased the root dry weight, but had no significant effect on shoot dry weight. The plants grown in the presence of 200 μM Cu accumulated 288 and 7626 μg g⁻¹ DW total Cu in the shoots and roots, respectively. A greater proportion of accumulated Cu was water-soluble accounting for 42–93% of the total Cu content in the shoots. The concentrations of reduced glutathione (GSH) and protein thiols were significantly enhanced under excess Cu supply. However, the concentrations of these compounds, particularly protein thiols, were much higher in the leaves than that in the roots. Three UV-absorbing peaks could be eluted out through gel filtration chromatography on Sephadex G-50. A large amount of Cu was detected in the UV-absorbing peaks in 40–50 and 70–90 ml elution fractions of the root extract, and in 40–50 and 120–140 ml elution fractions of the leaf extract. The results suggested that the adaptive Cu tolerance mechanism in E. haichowensis might involve the active participation of protein thiols which had a more important role in the leaves than in the roots.     

Encystment Processes and the “Matrioshka-like Stage” in a Moss-dwelling and in a Limnic Species of Eutardigrades (Tardigrada)

Tardigrades have two forms of dormancy, namely cryptobiosis and encystment. The encystment is a form of diapause known for a limited number of species of tardigrades and still little studied. To increase the knowledge on encystment, two species of eutardigrades from Italy have been considered: the moss-dwelling Amphibolus volubilis (Eohypsibiidae), and the limnic Dactylobiotus parthenogeneticus (Murrayidae). Cysts have been collected in nature, or induced under laboratory conditions. In the latter case, it was possible to follow the several steps of encystment processes. Two different types of cyst (“type 1” and “type 2”) have been found in A. volubilis, while in D. parthenogeneticus only one type has been found. In general, the ovoid-shaped cysts are constituted by a series of cuticles surrounding the animals and resemble an onion or a Matrioshka Russian doll. In all three types of cyst, the encystment processes show both common and peculiar traits. Encystment begins with the discharging of the sclerified parts of the buccal-pharyngeal apparatus, as in the molting process, but without the loss of the old cuticle. Then, two or three new cuticles are serially synthesized, according to cyst type. In A. volubilis, the ultrastructure of these new cuticular involucra is similar to that of non-encysted animal cuticles, while in D. parthenogeneticus the ultrastructure of the new cuticular involucra differs from that of non-encysted animal cuticle. A modified buccal-pharyngeal apparatus has been observed both in “type 2” cyst of A. volubilis and in the D. parthenogeneticus cyst.     

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies ¹². The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used^1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocystsand Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously^{1-3,5-8,10,11}. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters⁴. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices^9,13 .This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.     

In vitro shoot-tip grafting improves recovery of cotton plants from culture

A rapid in vitro shoot-tip grafting (STG) technique was adapted to increase recovery of intact cotton plants from shoots developed in culture. Induction of root organogenesis in cotton shoots is genotype dependent and unreliable. The resulting loss of regeneration potential due to failure to form roots can vary from 30 to 80% according to genotype and represents a significant bottleneck in the overall recovery of plants from culture. If the non-rooting shoots are transgenic, the loss in regenerated plant material can be substantial. In vitro grafting of cotton shoots to seedling rootstock proved to be a simple and reliable method allowing 90–100% recovery of non-rooting shoots from culture. Success of any given graft was directly related to scion size (0.8–1.0 cm) and age (14–35 days) of the seedling rootstock. The method appeared to be genotype independent, and varietal differences between rootstock and scion did not effect the rate of plant recovery from culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chlorophyll fluorescence imaging of photosynthetic activity in sun and shade leaves of trees

The differences in pigment levels, photosynthetic activity and the chlorophyll fluorescence decrease ratio R _Fd (as indicator of photosynthetic rates) of green sun and shade leaves of three broadleaf trees (Platanus acerifolia Willd., Populus alba L., Tilia cordata Mill.) were compared. Sun leaves were characterized by higher levels of total chlorophylls a + b and total carotenoids x + c as well as higher values for the weight ratio chlorophyll (Chl) a/b (sun leaves 3.23–3.45; shade leaves: 2.74–2.81), and lower values for the ratio chlorophylls to carotenoids (a + b)/(x + c) (with 4.44–4.70 in sun leaves and 5.04–5.72 in shade leaves). Sun leaves exhibited higher photosynthetic rates P _N on a leaf area basis (mean of 9.1–10.1 μmol CO₂ m⁻² s⁻¹) and Chl basis, which correlated well with the higher values of stomatal conductance G _s (range 105–180 mmol m⁻² s⁻¹), as compared to shade leaves (G _s range 25–77 mmol m⁻² s⁻¹; P _N: 3.2–3.7 μmol CO₂ m⁻² s⁻¹). The higher photosynthetic rates could also be detected via imaging the Chl fluorescence decrease ratio R _Fd, which possessed higher values in sun leaves (2.8–3.0) as compared to shade leaves (1.4–1.8). In addition, via R _Fd images it was shown that the photosynthetic activity of the leaves of all trees exhibits a large heterogeneity across the leaf area, and in general to a higher extent in sun leaves than in shade leaves.     

Large-scale production of Anogeissus pendula and A. Latifolia by micropropagation

Summary Success has been achieved in developing a complete protocol for mass propagation of Anogeissus pendula and A. latifolia, two important forest species found in India. Seeds cultured on plant growth regulator-free, semisolid Murashige and Skoog (MS) medium germinated within 5–6 wk and formed 4–6-cm long shoots. The shoots multiplied on MS+4.4 μM benzyladenine (BA)+5.7 μM indoleacetic acid (IAA) + casein hydrolysate (100 mgl⁻¹) + ascorbic acid (50 mgl⁻¹) + sucrose (3%) + agar (0.8%). A majority of the genotypes rooted with more than 90% efficiency when 5–6 cm individual shoots were cultured on 1/2MS (only major salts reduced to half strength)+2.3 μM IAA+2.5 μM indolebutyric acid (IBA) + sucrose (3%)+agar (0.8%) for 15 d. Those 10% (approx.) genotypes that did not root well on the above medium could be rooted with ease by increasing the concentration of IAA in the rooting media from 2.3 to 5.7 μM. The in vitro-raised plants were successfully transferred to the soil with a success rate of over 85%. Using this protocol, over 560 000 tissue-cultured plants of these two species have been produced and dispatched to various state forest departments for field trials and routine plantations.     

Cryopreservation of chestnut by vitrification of <Emphasis Type="Italic">in vitro</Emphasis>-grown shoot tips

Summary Plants of European chestnut (Castanea sativa) have been consistently recovered from cryopreserved in vitro-grown shoot apices by using the vitrification procedure. Factors found to influence the success of cryopreservation include the source of the shoot tips (terminal buds or axillary buds), their size, the duration of exposure to the cryoprotectant solution, and the composition of the post-cryostorage recovery medium. The most efficient protocol for shoot regrowth employed 0.5–1.0 mm shoot tips isolated from 1 cm-long terminal buds that had been excised from 3–5-wk shoot cultures and cold hardened at 4°C for 2 wk. The isolated shoot tips were precultured for 2d at 4°C on solidified Gresshoff and Doy medium (GD) supplemented with 0.2M sucrose, and were then treated for 20 min at room temperature with a loading solution (2M glycerol+0.4M sucrose) and for 120 min at 0°C with a modified PVS2 solution before rapid immersion in liquid nitrogen (LN). After 1 d in LN, rapid rewarming and unloading in 1.2M sucrose solution for 20 min, the shoot tips were plated on recovery medium consisting of GD supplemented with 2.2 μM benzyladenine, 2.9 μM 3-indoleacetic acid, and 0.9 μM zeatin. This protocol achieved 38–54% shoot recovery rates among five chestnut clones (three of juvenile origin and two of mature origin), and in all cases plant regeneration was also obtained.     

Method for the quantification of the blue-green pigment “marennine” synthesized by the marine diatom <Emphasis Type="Italic">Haslea ostrearia</Emphasis> (Gaillon/Bory) Simonsen using HPLC gel-filtration and photodiode-array detection

This paper describes a new approach for quantifying marennine, a blue-green pigment synthesized by the marine diatom Haslea ostrearia, which is known to be responsible for the greening of cultured oysters in French coastal areas. The method uses gel-filtration HPLC interfaced with a photodiode-array detector (PDA). Under the chromatographic conditions applied, the peak of marennine is identified on the dextran pattern at 674 nm by its elution time (ET = 21–22 min) and its UV-Visible absorption spectrum. After calibration with pure marennine sample as external standard, consisting in plotting peak area as a function of marennine concentration, one can back-calculate concentrations of unknown samples. Results for quantitative determination of marennine using this procedure are validated and discussed in relation to those obtained by the spectrophotometric methods, the most often employed until now.     

Rapid recovery of an insect–plant interaction following habitat loss and experimental wetland restoration

This study examined the impact of wetland habitat loss and isolation on an insect–plant interaction, and the subsequent rate of recovery of the interaction following experimental habitat restoration. We compared herbivore colonisation rates and herbivory damage by ‘Batrachedra’ sp. (Lepidoptera: Coleophoridae) on experimentally placed potted Sporadanthus ferrugineus (Restionaceae) plants at increasing distances (up to 800 m) from an intact habitat (the source population). These tests showed that even a moderate degree of isolation (i.e. greater than 400 m) from the intact wetland habitat caused an almost complete collapse of the insect–plant interaction, at least in the short term. The number of eggs and larvae of colonising ‘Batrachedra’ sp., as well as average larval size and the proportion of S. ferrugineus stems damaged, all decreased logarithmically with increasing distance from the intact habitat, presumably due to dispersal limitation of the herbivore. Subsequently, to test whether the interaction can recover following habitat restoration, we surveyed herbivore colonisation rates and herbivory damage on naturally regenerated S. ferrugineus plants on experimentally restored ‘islands’ at increasing distances (up to 800 m) from an intact habitat. The rate of recovery of the interaction was surprisingly rapid (i.e. between 196 and 308 weeks). The degree of difference in the density of eggs and larvae, and in the proportion of stems damaged with increasing isolation from the intact wetland, gradually diminished over 196 weeks. After 308 weeks there was no significant difference in the insect–plant interaction between the intact wetland sites and any of the experimentally restored sites up to 800 m away. These results suggest that some insect–plant interactions can recover rapidly from habitat loss with restoration management.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Recovery of Cryptosporidium oocysts and Giardia cysts from source water concentrates using immunomagnetic separation

Immunomagnetic separation (IMS) procedures for the simultaneous isolation of Cryptosporidium oocysts and Giardia cysts have recently become available. We validated Dynal's GC-Combo IMS kit using source water at three turbidity levels (5000, 500 and 50 nephelometric turbidity units [ntu]) obtained from different geographical locations and spiked with approximately 9--11 (oo)cysts per ml. Mean recoveries of Cryptosporidium oocysts and Giardia cysts in deionized water were 62% and 69%, respectively. In turbid water matrices, mean recoveries of Cryptosporidium oocysts were between 55.9% and 83.1% while mean recoveries of cysts were between 61.1% and 89.6%. Marginally higher recoveries of the heat inactivated (oo)cysts were observed (119.4% Cryptosporidium oocysts and 90.9% Giardia cysts) in deionized water when compared with recoveries of viable (oo)cysts (69.7% Cryptosporidium oocysts and 79% Giardia cysts). Age of (oo)cysts on recoveries using the GC-Combo IMS kit demonstrated no effects up to 20 months old. Recovery of Giardia cysts was consistent for isolates aged up to 8 months (81.4%), however, a significant reduction in recoveries was noted at 20 months age. Recoveries of low levels (5 and 10 (oo)cysts) of Cryptosporidium oocysts and Giardia cysts in deionized water using IMS ranged from 51.3% to 78% and from 47.6% to 90.0%, respectively. Results of this study indicate that Dynal's GC-Combo IMS kit is an efficient technique to separate Cryptosporidium/Giardia from turbid matrices and yields consistent, reproducible recoveries. The use of fresh (recently voided and purified) (oo)cysts, aged (oo)cysts, viable and heat-inactivated (oo)cysts indicated that these parameters do not influence IMS performance.     

Effect of sucrose,inorganic salts,inositol, and thiamine on protease excretion during pineapple culture in temporary immersion bioreactors

Summary Although pineapple plants have been found to produce proteases ex vitro, most of the biotechnological investigations of this crop have been focused on propagation. The procedure involving the use of temporary immersion bioreactors is one of the most outstanding because of its high multiplication rate. We previously recorded specific protease activity in the culture medium during the pre-elongation step of this protocol. Therefore, we decided to modify the culture medium composition of this phase looking for an increase in protease excretion. Four independent experiments were performed to evaluate the effects of different levels of sucrose (0–350.4 mM), inorganic salts [0–200% Murashige and Skoog (MS) salt strength], inositol (0–2.20 mM), and thiamine (0–1.2μM). The following indicators were recorded: shoot fresh mass per bioreactor; and protein concentration, proteolytic activity, and specific protease activity in culture media. Specific protease activity, the most important indicator recorded, was highest with 262.8 mM sucrose, 100% MS salt strength, 0.3 μM thiamine and no inositol. Results shown here demonstrate that conditions adequate for propagation purposes (87.6 mM sucrose, 100% MS salt strength, 0.55 mM inositol, 0.3 μM thiamine) are not always adequate for protease excretion.     

Induction of pathogenesis-related proteins during biocontrol of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> with <Emphasis Type="Italic">Pseudomonas aureofaciens</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.) plants

To investigate the biocontrol effectiveness of the antibiotic producing bacterium, Pseudomonas aureofaciens 63–28 against the phytopathogen Rhizoctonia solani AG-4 on Petri plates and in soybean roots, growth response and induction of PR-proteins were estimated after inoculation with P. aureofaciens 63–28 (P), with R. solani AG-4 (R), or with P. aureofaciens 63–28 + R. solani AG-4 (P + R). P. aureofaciens 63–28 showed strong antifungal activity against R. solani AG-4 pathogens in Petri plates. Treatment with P. aureofaciens 63–28 alone increased the emergence rate, shoot fresh weight, shoot dry weight and root fresh weight at 7 days after inoculation, when compared to R. solani AG-4; P + R treatment showed similar effects. Peroxidase (POD) and β-1,3-glucanase activity of P. aureofaciens 63–28 treated roots increased by 41.1 and 49.9%, respectively, compared to control roots. POD was 26% greater in P + R treated roots than R. solani treated roots. Two POD isozymes (59 and 27 kDa) were strongly induced in P + R treated roots. The apparent molecular weight of chitinase from treated roots, as determined through SDS-PAGE separation and comparison with standards, was about 29 kDa. Five β-1,3-glucanase isozymes (80, 70, 50, 46 and 19 kDa) were observed in all treatments. These results suggest that inoculation of soybean plants with P. aureofaciens 63–28 elevates plant growth inhibition by R. solani AG-4 and activates PR-proteins, potentially through induction of systemic resistance mechanisms.     

Bioenergetic Investigation of the Effects of La(III) and Ca(II) on the Metabolic Activity of Tetrahymena thermophila BF5

The heat flux of Tetrahymena thermophila BF5 during growth and the effects of La³⁺ and Ca²⁺ on them were investigated with microcalorimetry; simultaneously, morphological changes of T. thermophila were obtained by light microscope. La³⁺ in low concentration (0–5.0 × 10^–4 mol/l) remarkably stimulated T. thermophila metabolism, but high dose of La³⁺ (5.8–8.6 × 10^–4 mol/l) restrained it in a linear manner with IC₅₀ being 7.2 × 10^–4 mol/l. In contrast, low concentration of Ca²⁺ did not manifest obvious stimulation on T. thermophila metabolism; moreover, the IC₅₀ of Ca²⁺ was much higher than that of La³⁺. Low concentration of La³⁺ did not lead to changes in appearance of T. thermophila, but low dose of Ca²⁺ clearly promoted the cell proliferation. In addition, the morphological changes of T. thermophila evoked by high concentrations of La³⁺ and Ca²⁺ were consistent with relevant microcalorimetric results. It is concluded that La and Ca influence T. thermophila via different pathways, and La represents toxic action rather than Ca analogy.     

Influence of light,DMSO and glycerol on the hatchability ofThamnocephalus platyurus Packard cysts

Effects of two light intensities and different concentration of dimethyl sulfoxide (DMSO) and glycerol on hydrated cyst hatching inThamnocephalus platyurus were studied. A maximum of 65±6% hatching was recorded within seven days at 2500 lux continuous light regime. Hatching was at a minimum during the first two days, peaked between the third and fourth days, decreased thereafter. Hatching success was a function of duration of light exposure. Eight percent of cysts hatched in the dark, while cysts exposed to 24h light and subsequently incubated in the dark showed 27±2% hatching. Hatchability was significantly increased (23%) in 0.0375% DMSO and 0.0125% glycerol. Concentration above 0.05% DMSO and 0.025% glycerol had no or a negative influence on hatching. Since low concentrations of DMSO were non-toxic, apolar compounds like Ca²⁺ ionophore can be dissolved in DMSO to study the role of Calcium in cyst hatching.     

Separation and purification of useful proteins using hydrogel ultrafiltration

The hydrogel process is a different form of ultrafiltration and has been used to separate biological molecules. In this study, the gel pore size was predicted by pulse NMR technique and neural network using a data base obtained from gel filtration chromatography and diffusion experiment. Recombinant alkaline phosphatase expressed in insect cells was concentrated 1.5 times by hydrogel ultrafiltration by swelling at 20°C and collapsing at 35°C at 53–65% separation efficiency and 78–83% enzyme recovery. Wild and recombinantAutographa californica nuclear polyhedrosis viruses (AcNPV) were also concentrated 1.4 and 1.6 times of the feed solution at 48.5 and 60.0% separation efficiency, respectively. Hydrogel ultrafiltration appears to be an attractive alternative for the concentration of AcNPV and recombinant proteins from insect cells.     

Improved purification of Candida boidinii formate dehydrogenase

Formate dehydrogenase (FDH, EC 1.2.1.2) from Candida boidinii was purified to homogeneity. The two step procedure comprised anion exchange chromatography (2.9-fold purification, 85% step yield, elution with 35 mM KCl), followed by dye-ligand affinity chromatography on immobilized Cibacron Blue 3GA (1.4-fold purification, 75% step yield, elution with 0.15 mM NAD⁺/2 mM Na₂SO₃). The procedure afforded FDH at 63.8% overall yield and a specific activity of 7.2 units/mg. The purity of the final FDH preparation was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), high performance gel filtration liquid chromatography (gfHPLC) and N-terminal amino acid sequencing. The analytical techniques showed the presence of a single polypeptide chain that corresponds to the molecular weight of 41 kDa (as determined by SDS-PAGE) and 81 kDa (as determined by gfHPLC).     

Atypical Ca<Superscript>2+</Superscript>-independent,raw-starch hydrolysing α-amylase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. GRE1: characterization and gene isolation

Bacillus sp. GRE1 isolated from an Ethiopian hyperthermal spring produced raw-starch digesting, Ca²⁺-independent thermostable α-amylase. Enzyme production in shake flask experiments using optimum nutrient supplements and environmental conditions was 2,360 U l⁻¹. Gel filtration chromatography yielded a purification factor of 33.6-fold and a recovery of 46.5%. The apparent molecular weight of the enzyme was 55 kDa as determined by SDS-PAGE. Presence or absence of Ca²⁺ produced similar temperature optima of 65–70°C. The optimum pH was in the range of 5.5–6.0. The enzyme maintained 50% of its original activity after 45 min of incubation at 80°C and was stable at a pH range of 5.0–9.0. The V _max and K _m values for soluble starch were 42 mg reducing sugar min⁻¹ and 4.98 mg starch ml⁻¹, respectively. Strong inhibitors of enzyme activity included Cu²⁺, Zn²⁺ and Fe²⁺. The enzyme coding gene and the deduced protein translation revealed a characteristic but markedly atypical homology to Bacillus species α-amylase sequences. The enzyme hydrolyzed wheat, corn and tapioca starch granules efficiently below their gelatinization temperatures. Rather than the higher oligosaccharides normally produced by Bacillus α-amylases operating at high temperatures, maltose was the major hydrolysis product with the present enzyme.     

Development and evaluation of an off‐the‐slide genotyping technique for identifying Giardia cysts and Cryptosporidium oocysts directly from US EPA Method 1623 slides

Aims

This study developed and systematically evaluated performance and limit of detection of an off‐the‐slide genotyping procedure for both Cryptosporidium oocysts and Giardia cysts.

Methods and Results

Slide standards containing flow‐sorted (oo)cysts were used to evaluate the off‐the‐slide genotyping procedure by microscopy and PCR. Results show approximately 20% of cysts and oocysts are lost during staining. Although transfer efficiency from the slide to the PCR tube could not be determined by microscopy, it was observed that the transfer process aided in the physical lysis of the (oo)cysts likely releasing DNA. PCR detection rates for a single event on a slide were 44% for Giardia and 27% for Cryptosporidium, and a minimum of five cysts and 20 oocysts are required to achieve a 90% PCR detection rate. A Poisson distribution analysis estimated the relative PCR target densities and limits of detection, it showed that 18 Cryptosporidium and five Giardia replicates are required for a 95% probability of detecting a single (oo)cyst on a slide.

Conclusions

This study successfully developed and evaluated recovery rates and limits of detection of an off‐the‐slide genotyping procedure for both Cryptosporidium and Giardia (oo)cysts from the same slide.

Significance and Impact of the Study

This off‐the‐slide genotyping technique is a simple and low cost tool that expands the applications of US EPA Method 1623 results by identifying the genotypes and assemblages of the enumerated Cryptosporidium and Giardia. This additional information will be useful for microbial risk assessment models and watershed management decisions.