Cyst Suspension Research Articles

Reducing costs of Giardia spp. cyst enumeration using machine learning-based systems

Abstract Giardia spp. cyst enumeration is a laboratory procedure that is frequently required in water treatment-related research. Currently, detection conducted by experts using fluorescence microscopy on samples stained with specific markers for Giardia spp. cysts is still the standard method, despite its high costs limiting its usage worldwide and, ultimately, hindering waterborne analyses in low-income countries. We present an approach based on darkfield imaging and machine learning to reduce costs associated with Giardia spp. cyst enumeration and the lack of experts. Automated counts were compared to manual counts, achieving an average sensitivity (SE) rate of 88%, specificity (SP) of 100% and accuracy of 88% across a wide range of cyst concentrations. By using machine learning in conjunction with darkfield microscopy, a low-cost illumination technique that can be easily integrated into standard laboratory microscopes, we have significantly reduced the costs associated with Giardia spp. cyst detection, all while still maintaining the SE and SP of fluorescence microscopy. Based on the findings, the proposed system has the potential to be a useful tool to enumerate Giardia spp. cyst suspensions. It can be accessed by virtually any microbiology laboratory as it is consumable-free and expert-independent.

Exploring challenges in Giardia cyst visualisation by common microscopy methods

Abstract Giardia spp. is an intestinal parasite responsible for worldwide disease outbreaks. Guiding researchers and practitioners to choose among current methods for microscopy detection of the infectious forms may be directly beneficial to public health and the environment. This study provides an overall comparison of brightfield (BF), fluorescence and darkfield (DF) microscopies for detecting Giardia duodenalis and Giardia muris cysts, by illustrating micrographs of such protocols applied to purified samples, as well as discussing advantages and constraints based on secondary information and collected data. BF analysis included Lugol's iodine staining. In fluorescence microscopy, samples were processed by immunofluorescence assay (IFA) with DAPI and by standalone DAPI dye. Cyst suspensions were also analysed by DF microscopy using a recently developed low-cost system. The three techniques enabled detecting Giardia spp. cysts, although they did not provide species identification by morphology. The overview of each method points out some relevant aspects to consider when selecting common optical microscopy techniques, and includes challenges and advantages regarding each of them.

Label-free detection and enumeration of Giardia cysts in agitated suspensions using in situ microscopy

Laboratory procedures performed in water treatment studies frequently require the characterization of (oo)cyst suspensions. Standard methods commonly used are laborious, expensive and time-consuming, besides requiring well-trained personnel to prepare samples with fluorescent staining and perform analysis under fluorescence microscopy. In this study, an easy cost-effective in situ microscope was assessed to acquire images of Giardia cysts directly from agitated suspensions without using any chemical labels or sample preparation steps. An image analysis algorithm analyzes the acquired images, and automatically enumerates and provides morphological information of cysts within 10 min. The proposed system was evaluated at different cyst concentrations, achieving a limit of detection of ~30 cysts/mL. The proposed system overcomes cost, time and labor demands by standard methods and has the potential to be an alternative technique for the characterization of Giardia cyst suspensions in resource-limited facilities, since it is independent of experts and free of consumables.

Direct centrifugation for detecting Giardia spp. cysts in filter backwash water

Abstract Pathogenic protozoa endanger human health and challenge water treatment, especially during outbreaks in developing countries. For instance, Giardia spp. cysts can recirculate in the filter backwash water (FBW), reinforcing the relevance of their detection protocol in negligible matrices. This study aimed to detect Giardia spp. cysts in the FBW by direct centrifugation (DC) and immunomagnetic separation (IMS) with the addition of the detergent dispersion solution ICN 7X at 1.0%. To do this, cyst suspensions were inoculated into FBW samples (14 NTU), which was generated in bench-scale drinking-water treatment with high-turbidity study water (112 NTU). Furthermore, the DC+ICN 7X method was compared with the calcium carbonate flocculation. For instance, the DC+ICN 7X method provided cleaner microscope slides and minor damage to the cyst walls. The commercial suspension of Giardia lamblia had an adequate recovery rate (19.5%). However, the recovery rate of the EasySeed® suspension was 7.8%, which was below the required range by Method 1623.1 (above 8%). High costs and low efficiencies challenge several methods for detecting Giardia spp. cysts. Therefore, future studies should develop and improve detection protocols, especially for complex matrices. Detecting cysts in water treatment residues is crucial for addressing current sanitation issues in developing countries.

Does each bead count? A reduced-cost approach for recovering waterborne protozoa from challenge water using immunomagnetic separation.

Giardia duodenalis and Cryptosporidium spp. are two of the most prominent aetiological agents of waterborne diseases. Therefore, efficient and affordable methodologies for identifying and quantifying these parasites in water are increasingly necessary. USEPA Method 1623.1 is a widely used and validated protocol for detecting these parasites in water samples. It consists of a concentration step, followed by parasite purification and visualization by immunofluorescence microscopy. Although efficient, this method has a high cost particularly due to the immunomagnetic separation (IMS) step, which is most needed with complex and highly contaminated samples. Based on this, the present study aimed to determine whether it is possible to maintain the efficiency of Method 1623.1 while reducing the amount of beads per reaction, using as a matrix the challenge water recommended by the World Health Organization. As for Giardia cysts, a satisfactory recovery efficiency (RE) was obtained using 50% less IMS beads. This was evaluated both with a commercial cyst suspension (56.1% recovery) and an analytical quality assessment (47.5% recovery). Although RE rates obtained for Cryptosporidium parvum did not meet Method 1623.1 criteria in any of the experimental conditions tested, results presented in this paper indicated the relevance of the described adaptations, even in challenge water.

Label-Free Darkfield-Based Technique to Assist in the Detection of Giardia Cysts

Giardia is a pathogenic protozoan that has caused giardiasis outbreaks worldwide, and this water-borne disease may occur due to faults in water supply and treatment systems. To support surveillance and control programs, the monitoring of this parasite is essential in water samples. Although Giardia cyst detection is usually based on standard light microscopy, the resulting low contrasted cysts together with a wide array of particles of comparable size to the target protozoan demands a high level of observer skill and experience, as well as a long time to process the individual samples. The visualization of this protozoan can be significantly improved by using staining procedures, e.g., Lugol’s iodine in brightfield or fluorescence-based methods such as fluorescence-isothiocyanate (FITC) and 4′,6-diamidino-2-phenylindole (DAPI). However, the significant costs make fluorescence microscopy difficult to be performed in less-developed countries. Accordingly, the present study evaluated the effectiveness of inexpensive darkfield (DF) setups to acquire higher contrasted images of unstained cyst samples as a direct and rapid method for monitoring protozoa. Two low-cost custom-made DF illumination modes, transmitted and reflected, were evaluated on a standard light microscope. Examination of purified Giardia cyst suspensions with both DF setups revealed a direct correlation between morphological appearance and uptake of DAPI. Images captured under transmitted illumination showed higher contrast and sharpness when compared to the reflected images. DF microscopy might provide a simple, direct, and inexpensive method for observing Giardia cysts, which shows basic aspects of their intracellular structure, although the applicability of the method to raw water concentrates remains to be demonstrated.

Cryptosporidium oocysts and Giardia cysts—A practical and sensitive method for counting and manipulating small numbers

A rapid and inexpensive method is described for accurate and reproducible counting and manipulating small numbers of Cryptosporidium oocysts and Giardia cysts. From a suspension of oocysts or cysts at concentration from 1000 to 5000/mL (1–5/μL), replicate 5μL droplets are micro pipetted onto the edge of a microscope slide. Unstained oocysts or cysts in each droplet can be counted in a few minutes and replicated for statistical strength. The concentration of the suspension can then be verified by pipetting desired volumes containing approximately desired numbers onto confined 13mm membranes for IFA staining and counting with replication as desired. Requiring only a micropipette, analytical balance, and a microscope, this provides a useful tool accessible to virtually any microbiology laboratory.

Anti-giardial therapeutic potential of dichloromethane extracts of Zingiber officinale and Curcuma longa in vitro and in vivo.

Giardiosis is one of the common parasitic diarrhoea in humans, especially in children, worldwide. Many drugs are used for its treatment, but there is evidence of drug resistance, insufficient efficacy and unpleasant side effects. Natural products are good candidates for discovering more effective anti-giardial compounds. This study evaluated the activity of extracts of Zingiber officinale (ginger) and Curcuma longa (curcumin) against Giardia lamblia in vitro and in vivo. Giardia cyst suspension was prepared from children faecal specimens. For the in vitro experiment, 1, 10 and 50mg⁄mL dichloromethane extracts of ginger and curcumin separately were incubated with Giardia cysts for 5, 10, 30 and 60min. The viability was distinguished by 0.1% eosin and a haemocytometer. For the in vivo experiments, Balb/c mice were infected with Giardia cyst suspension containing 10,000cysts/mL. Infected mice were administered 10 and 20mgkg(-1)day(-1) ginger and curcumin extracts separately for 7days post-infection. The effectiveness of the extracts was evaluated by faecal cyst and intestinal trophozoite counts and histopathological examination of the small intestine. In vitro ginger extract had a higher significant effect on cyst viability than curcumin, in a dose- and time-dependent manner. In vivo ginger (more effective) and curcumin extracts significantly treated infected mice, and this was evidenced by the faecal cyst and intestinal trophozoite counts reduction, in addition to evident improvement of intestinal mucosal damages induced by Giardia infection. Z. officinale and C. longa extracts may represent effective and natural therapeutic alternatives with low side effects and without drug resistance in the treatment of giardiosis.

Simplified protocol for DNA extraction and amplification of 2 molecular markers to detect and type Giardia duodenalis

We evaluated the ability of 3 kits: QIAmp® DNA stool mini kit (Qiagen, Hilden, Germany), PureLink PCR Purification®, and PureLink™ Genomic DNA® (Invitrogen, Carlsbad, CA, USA) for DNA extraction, and of 2 molecular markers (heat shock protein [HSP] and β-giardin genes) for detection and genotyping of Giardia duodenalis stool samples. The detection and typing limits of the markers were determined by the DNA concentration of trophozoites and cysts and were tested in 26 clinical samples. Of the 3 kits tested, the PureLink PCR Purification gave the best results when tested with clinical samples with low, intermediate, and high numbers of cysts. The DNA extracted from trophozoites and cysts was diluted successively in 1:2 ratios until it was no longer possible to observe the amplified product in polyacrylamide gel. Similarly, a suspension of cysts was diluted until no cysts were observed, and then the DNA was extracted. The amount of DNA of trophozoites and cysts for the typing of the parasite was smaller for the HSP marker than for β-giardin. Combined use of both markers allowed us to detect DNA of Giardia in parasitologically positive samples in a higher percentage (75%) than the results obtained for each marker and in 1 parasitologically negative sample, indicating that this combination increased the potential to accurately detect and genotype this parasite. We also concluded that the HSP marker has a higher limit of detection and typing than the β-giardin marker and that the DNA extraction method tested for G. duodenalis is simpler and more efficient than those that are currently in use and can be applied on a large scale.

Application of Hybrid Process of Coagulation/Flocculation and Membrane Filtration for the Removal of Protozoan Parasites from Water

Among the waterborne diseases, enteric diseases are most frequent. Approximately 19% of waterborne gastroenteritis outbreaks in the United States are attributed to parasitic protozoans, particularly Giardia and Cryptosporidium species, due to their wide distribution in the environment, high incidence in the population, and resistance to conventional water treatment. These microorganisms and impurities present in water do not approach each other, it is necessary to add a coagulant. Chemical coagulants are the most used and among them the most common is aluminum sulfate, since it is cheap and easily obtained. However, chemical coagulants have certain disadvantages, as they require tight control over their residual concentration in treated water for human consumption as well as in industrial food production. Then, the objective of the present work was to evaluate the efficiency of Moringa oleifera seeds as natural coagulant in removing apparent color, turbidity, Giardia spp. cysts and Cryptosporidium spp. oocysts from water in association with microfiltration. The surface water used in the tests was collected at the Pirapó river basin, which supplies the city of Maringá, Paraná, Brazil. Samples of high and low turbidity were mixed to obtain water with different initial turbidities. Water samples with initial turbidity of 50, 150, 250, 350, and 450 NTU were used in this study. Prepared samples were artificially contaminated with 106 cysts/L of Giardia and 106 oocysts/L of Cryptosporidium obtained from the positive control (suspension of cysts and oocysts) present in the commercial kit Merifluor (Meridian Bioscience, Cincinnati, OH, USA). After being prepared, these samples were subjected to the processes of (1) coagulation/flocculation with moringa seeds (CFM), (2) coagulation/flocculation with aluminum sulfate; (3) microfiltration (MF), and (4) combined coagulation/flocculation with moringa seeds or aluminum sulfate followed by microfiltration (CFM-MF or CFM-MF). Coagulation/flocculation tests with moringa were performed in a jar test device. Each sample, with initial turbidity from 50 to 450 NTU, was subjected to 12 different concentrations.of moringa coagulant solution, ranging from 25 to 350 mg/L. Polyimide membrane with porosity of 0.40 μm was used for the MF process with an operating pressure of 1 bar. Moringa solution concentrations above 150 mg/L showed the best removal efficiency of turbidity – which varied from 3% to 97% – and color, ranging from 6.7% to 73.5%. The CFM process achieved 1.2 log removal for Giardia and 1.0 log removal for Cryptosporidium. To membrane process, color and turbidity removal ranged from 80 to 100%, and the best results were obtained with the combined CFM-MF. It was also observed that the pretreatment with moringa reduced membrane fouling (which was between 7.48 and 40.9%) and increased permeate flux (between 157 and 226 L/hm) when compared with the MF (membrane fouling from 6.13 to 56.32% and permeate flow from 157 to 187 L/hm2). It can be concluded that coagulation with moringa showed satisfactory results, reducing the number of (oo)cysts of protozoan parasites in accordance with WHO recommendations for the process of coagulation/flocculation, as well as reducing color and turbidity. Combined with MF, the proposed process removed nearly 100% of Giardia and Cryptosporidium (oo)cysts and achieved high levels of color and turbidity removal from water samples, presenting the advantage of improving the quality of filtered water and the characteristics of the MF process (reduction of fouling and increase of permeate flux).

Optimization of a flow cytometry protocol for detection and viability assessment of Giardia lamblia

Giardia lamblia is responsible for causing diarrhoeal diseases in humans. Infection occurs by fecal-oral route and is considered an important water pathogen, since many water surfaces are infected by cysts. Most studies involve cyst concentration procedures, followed by conventional microscopy, but are often tedious and influenced by fatigue. Our main objective was to optimize a specific flow cytometric (FC) protocol for detection of G. lamblia, to establish its sensibility limit and also the cyst viability. G. lamblia cysts (Waterborne, Inc., USA) were used for protocol optimization. FC analysis was performed using cyst suspensions stained with serial concentrations of a fluorescein-labelled mouse monoclonal antibody (Giardia-a-Glo, Waterborne). Serial concentrations (2 x 10(5), 1 x 10(5), 2 x 10(4), 1 x 10(4), 2 x 10(3), 1 x 10(3), 2 x 10(2) and 1 x 10(2)cysts/ml) were stained with the optimized antibody concentration and analysed by FC. Specificity and sensibility limit of the method were established using both prokaryotic (Escherichia coli, Staphylococcus aureus) and eukaryotic microorganisms (Candida albicans, Cryptosporidium parvum oocysts). Dead cysts were stained with 5.0 microg/ml of propidium iodide (PI, Sigma), with and without the specific fluorescent antibody. As the antibody concentration decreased, a decline of peak intensity was registered; 1.5 microg/ml was considered as the optimal antibody concentration, yielding a histogram clearly separated. We established a threshold of detection of 2 x 10(2)cysts/ml. Below threshold limit fluorescence was not enough to allow the discrimination of cysts. The staining procedure was shown to be specific, no cross-reaction occurring with bacteria, fungi or parasites. When using both antibody and PI, we could distinguish the viable cyst. With the use of specific antibodies, a distinct cellular population corresponding to cysts could be represented in the FC histogram.

Contact Lens Solution Efficacy Against Acanthamoeba castellani

Acanthamoeba castellani, ATCC 30234, cysts, and trophozoites after a 6-hour exposure. Trophozoite cultures were prepared at Bio-Concept Laboratories in vented tissue culture flasks containing peptone yeast glucose broth by incubation (35 degrees C +/- 1 degrees C for 11 days). Cyst suspensions were prepared by incubation of trophozoites in phosphate-buffered saline plus heat-killed yeast on Page's saline agar plates (35 degrees C +/- 1 degrees C for 14 days). The solutions were inoculated in triplicate in respective lens cases. After the 6-hour exposure, aliquots of challenged solutions were transferred to Dey-Engley neutralizing broth and further diluted in peptone yeast glucose broth in tissue culture plates to the -7 dilution. Flasks and plates were incubated for 14 days at 35 degrees C +/- 1 degrees C and were examined with an inverted light microscope at day 14 for the presence of viable trophozoites. The most probable number method was used for approximate enumeration of the number of survivors. Mean log reductions for cysts were 1.8 for Clear Care/AOSEPT Plus, 2.0 for BLUE Vision/BLUE SEPT, 0.7 for Oxysept 1 Step, 0.5 for OPTI-FREE Express with Aldox, and 0.2 for easyvision one step+. Mean log reductions for trophozoites were 2.2 for Clear Care/AOSEPT Plus, 2.7 for BLUE Vision/BLUE SEPT, 2.5 for Oxysept 1 Step, 2.5 for OPTI-FREE Express with Aldox, and 1.8 for easyvision one step+. Only Clear Care/AOSEPT Plus and BLUE Vision/BLUE SEPT showed high levels of antimicrobial activity against the cyst form of A. castellani. Oxysept 1 Step showed mild activity against the cysts and easyvision one step+ and OPTI-FREE Express with Aldox showed virtually no antiacanthamoeba activity against the cyst form after 6 hours of exposure.

Photosensitized Inactivation of Cyst Forms of the Freshwater Amoeba Vahlkampfia hartmanni by Aluminium (III) Phthalocyanine Tetrasulfonate

In this work we attained a successful cysticidal effect against Vahlkampfia hartmanni cysts using mild to moderate experimental conditions, regarding light energy delivered and concentrations of aluminium phthalocyanine tetrasulfonate (AlPCS4). The dark and phototoxic activity of AlPCS4 towards the cyst forms of Vahlkampfiahartmanni, a freshwater amoeba previously isolated from keratitic patients, were tested. The photosensitized deactivation of the cyst form of this amoeba depends mainly on the concentration of AlPCS4 and the light fluence rate as well as the total fluence delivered during the irradiation process. Upon irradiation of the cyst suspension at 100 mW/cm2 for 10 minutes in the presence of 3 μ M AlPCS4, a complete photodamaging effect on the cysts was induced. The photodamaging effect on Vahlkampfia cysts induces a state of inability of the cysts to excyst, vacuolation of the inner contents as well as a significant damage of the outer cyst wall upon microscopic examination.

Rapid detection of Pneumocystis carinii in bronchoalveolar lavage specimens from human immunodeficiency virus-infected patients: use of a simple DNA extraction procedure and nested PCR.

We report on the development of a rapid nested PCR protocol for the detection of Pneumocystis carinii DNA in bronchoalveolar lavage (BAL) specimens in which the protocol included the use of a commercially available DNA extraction kit (GeneReleaser). GeneReleaser enabled us to obtain amplification-ready DNA within 20 min without requiring the purification of the DNA. The nested PCR was performed with the primers pAZ102-E, pAZ102-H, and pAZ102-L2 (A. E. Wakefield, F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moxon, and J. M. Hopkin, Lancet 336:451-453, 1990.). Results were obtained in about 4 h with the adoption of denaturation, annealing, and extension steps shortened to 20 seconds. The sensitivity of the nested PCR was tested with a P. carinii cyst suspension and was found to be less than one cyst (one to eight nuclei). The detection limit was the same with the use of GeneReleaser or proteinase K-phenol chloroform for DNA extraction. The nested PCR assay was prospectively compared with staining with Giemsa and methenamine silver stains for the detection of P. carinii in 127 BAL samples from 105 human immunodeficiency virus-infected patients investigated for acute respiratory illness. Twenty-five BAL specimens (20%) were positive by staining and the nested PCR and 25 (20%) were negative by staining and positive by the nested PCR. These 25 BAL specimens with conflicting results were obtained from 23 patients, 82% of whom were receiving prophylactic therapy against P. carinii pneumonia (PCP). Only two patients were diagnosed with possible PCP. The final diagnosis was not PCP for 20 patients who were considered to be colonized or to have a low level of infection. This colonization is not of clinical importance but is of epidemiological importance. Our rapid, simple, and sensitive amplification protocol may be performed in clinical laboratories for the routine diagnosis of PCP with BAL specimens.

Evidence for the growth of Plasmodiophora brassicae in vitro

Plasmodiophora brassicae is a root-invading pathogen that causes club root disease of brassica crops. Although generally regarded as an obligate biotroph, a number of reports over the last century have suggested that the organism may have a free living saprotrophic amoebal stage. Here we present new evidence to support the contention that P. brassicae is capable of at least a shortlived period of saprotrophic amoebal growth. Spores extracted from surface sterilized blocks of club root tissue were incubated at 15°C on 1/4 strength Ringer's agar spread previously with a suspension of Escherichia coli. After a period of 5–10 d, outgrowth of amoebae commonly appeared around the inoculum. Amoebae were subcultured on to fresh Ringer's-E. coli plates, where they continued to grow and eventually encysted. An amoebal cyst suspension was prepared from these plates and used to inoculate Chinese cabbage (Brassica campestris var. chinensis) and Savoy cabbage (B. oleracea) seedlings. Of 27 amoebal isolates tested, nine were found which induced typical club root disease symptoms. However, when these putative positive P. brassicae amoebal isolates were subcultured, the subsequent generation of amoebal cysts failed to induce disease symptoms. A random genomic library was constructed in pUCl8 using BamH 1-digested DNA extracted from P. brassicae resting spores. The inserts from nine individual clones were then used to probe DNA extracted from 12 amoebal cultures; three showed positive hybridization to the P. brassicae probes but this hybridization signal was lost after subculture of the amoebae. These results are discussed in relation to the ability to culture P. brassicae in vitro.

Effects of calcium on germination and further zoospore release from zoospore cysts of Phytophthora parasitica

Cysts of Phytophthora parasitica, induced by vortex treatment of zoospore suspensions, germinated poorly when held in Eppendorf tubes but germinated well when transferred to glass slides within the first few minutes of encystment. The responsiveness to a surface, or to other treatments where tested, was lost after 12-15 min in the Eppendorf tubes. Slides pretreated with silicone (hydrophobic) were as effective as untreated slides in promoting germination. Dilution of cyst suspensions with distilled water or with the liquid into which zoospores were released caused a consistent decrease in germination. Dilution with CaCl2 (50 m m ) or with supernatant from centrifuged cysts gave germination equivalent to control (undiluted) cysts. Lower concentrations of CaCl2 were less effective, and there was no response to calcium by older (20 min) cysts. Dilution with zoospore lysate (caused by filtering zoospore suspensions through a Nucleopore filter) or with water passed through glass microfibre filters gave significant stimulation of cyst germination, above the undiluted control levels. Cysts that did not germinate within 2 h in response to treatments could release a further zoospore (diplanetism) by 8 h, but calcium suppressed this in a concentration-dependent manner. The findings support a previous model that encysting zoospores release a substance (probably calcium) that must be reabsorbed for germination, and that this reabsorption is facilitated when cysts are on a surface. However, other factors provide the germination trigger, because calcium added to diluted cyst suspensions only restored germination to the initial (undiluted) level. The loss of germination responsiveness within a few minutes of encystment is suggested to involve a loss of signal transduction, consistent with the reported microtubular rearrangements in young cysts. Release of a further zoospore from non-germinated cysts is a default mechanism that requires no specific trigger, but it is suppressible by external calcium, with potential for control of P. parasitica in recirculating irrigation systems in glasshouses.

Ultrastructure ofBlastocystis hominis cysts

Blastocystis hominis cysts were concentrated from human faeces by repeated washing in distilled water and centrifugation on Ficoll-Paque. This procedure gave a concentrated suspension of cysts without yielding any non-cystic forms. The cysts were examined by both transmission (TEM) and scanning electron microscopy (SEM). Cysts were surrounded by a fibrillar layer that appeared porous in SEM. Many naked cysts without the fibrillar layer were also visible in SEM. They were variable in shape but mostly circular to oval. The diameter of the cyst without the fibrillar layer was 3.5 microns in both TEM and SEM. The nuclear structure was typical of Blastocystis and exhibited multiple mitochondria with poorly developed cristae. Glycogen was present in small to large clumps in the cytoplasm. The cyst wall was 5-100 nm thick and was bounded internally by an electron-dense plasmalemma.

Changes in ion fluxes and the energy demand during spore development inPhytophthora infestans zoospores

Cell-free supernatant of pelleted zoospores was found to be more suitable for maintaining viable zoospores and developed cysts than the supernatant of mature cysts. Conductivity and pH measurements indicated quantitative changes in the ionic composition of a suspension ofP. infestans zoospores during their conversion into cysts. An increase in conductivity in the incipient cyst suspension was followed by a decrease of conductivity in the maturing cyst suspension. The conductivity changes correlated closely with K+ fluxes which, in turn, coincided with the reverse, but stoichiometrically smaller, H+ fluxes. Zoospores treated with 1.5 μmol/L DCCD (an inhibitor of plasma membrane H+-ATPase) or 100 mmol/L Li+ (an inhibitor of cell motility) released predominantly K+ and other cations and their O2 consumption decreased. The H+/K+ exchange is therefore very probably associated with an operation of the plasma membrane H+-ATPase. The differential decrease in respiration caused by DCCD and Li+ was used to estimate the energy demand for cell motility and spore development.

Elution mode of Pneumocystis carinii cysts in gravitational field-flow fractionation

The simplest field-flow fractionation technique, i.e. gravitational, was used in an attempt to purify a Pneumocystis carinii cyst suspension. This parasite is an opportunistic invader in immunocompromised patients, especially those suffering from AIDS. The cyst stage is spherical and 5 μm in diameter. Unexpected retention times, not systematically related to the size and the density of the parasite, were obtained under various experimental conditions. When silicone-coated walls were used, Pneumocystis carinii cysts were eluted in the void volume, whereas when uncoated walls were used with a sodium dodecyl sulphate-enriched carrier phase, retention was observed. These phenomena are probably related to the high degree of hydrophobicity of these micrometre-sized biological particles; this degree can be easily determined. The use of the gravitational field-flow fractionation technique can be of a great interest for the development of new methods for diagnostic purposes. Particle—wall interactions and their modifications due to the carrier phase or to the wall treatment can be employed in the search for new bronchoalveolar lavage solutions.

A simple method for excystation ofGiardia lambliacysts

Fresh Giardia lamblia trophozoites were isolated by in vitro excystation of purified cysts from the faeces of Saudi patients. The method consisted of mixing one part of the cyst suspension with nine parts of 1 N hydrochloric acid, at pH 2, then culturing the excysted trophozoites in modified TYI-S-33 medium in sealed glass vials. During the excystation process most cysts developed a space between the trophozoite and the cyst wall, and after several steps of enlargement the trophozoite emerged at the posterior end of the cyst. Keeping the cysts at 4 degrees C for more than 10 days did not affect the excystation process. Trophozoites obtained by this method can be cryopreserved and recultured without significant loss of activity. This is a simple and rapid method of obtaining G. lamblia trophozoites from cysts in stools or from other sources.