Inhibition of excystation and metacystic development of Entamoeba invadens by the dinitroaniline herbicide oryzalin

The effect of oryzalin on excystation and metacystic development of Entamoeba invadens strain IP-1 was examined by transfer of cysts to a growth medium containing the drug. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by oryzalin in a concentration-dependent manner. Metacystic development, which was determined by the number of nuclei in metacystic amoebae, was also inhibited by oryzalin because the percentage of 4-nucleate amoebae at day 1 remained unchanged at day 3. The addition of oryzalin after the induction of excystation decreased the number of metacystic amoebae, compared with control cultures. When cysts were incubated for 1 day in growth medium plus oryzalin, little increase in the number of metacystic amoebae was observed after removal of the drug. Excystation and metacystic development were further inhibited when the cysts were incubated for 30 min in encystation medium containing oryzalin before transfer to growth medium with the drug. When cysts were incubated for 30 min in encystation medium before transfer to growth medium without the drug, metacystic amoebae decreased in number. Pretreatment of cysts with oryzalin for 30 min in phosphate-buffered saline markedly reduced viability and prevented excystation in growth medium with or without the drug. The results indicate that oryzalin inhibits excystation and metacystic development of E. invadens, suggesting that it may be an inhibitor of Entamoeba infection.     

Viability of the encysted metacercariae of Echinostoma caproni judged by light microscopy versus chemical excystation

The purpose of this study was to determine the viability of encysted metacercariae of Echinostoma caproni stored in Locke's solution 1:1 at 4 C for 1-24 wk. Viability was judged by light microscopy (LM) based on morphological characteristics of the encysted metacercariae versus chemical excystation of the cysts in a trypsin-bile salts excystation medium. The percent viability was very similar under both methods of assessment at 4, 8, and 16 wk poststorage. At 1 and 24 wk poststorage, viability was found to be about 2x greater based on excystation than using LM. We concluded that LM alone underestimated the viability of cysts and that determination of cyst viability was more accurate under assessment by chemical excystation.     

Ozone inactivation of Cryptosporidium parvum in demand-free phosphate buffer determined by in vitro excystation and animal infectivity.

Inactivation of Cryptosporidium parvum oocysts by ozone was performed in ozone demand-free 0.05 M phosphate buffer (pH 6.9) in bench-scale batch reactors at 7 and 22 degrees C. Ozone was added to each trial from a concentrated stock solution for contact times ranging from 5 to 15 min. The viability of the control and treated oocysts was determined by using in vitro excystation and infection in neonatal CD-1 mice. It was found that excystation consistently underestimated inactivation when compared with animal infectivity (P < or = 0.05). As inactivations increased, the difference between excystation and infectivity also increased. The inactivation kinetics of C. parvum by ozone deviated from the simple first-order Chick-Watson model and was better described by a nonlinear Hom model. The use of the Hom model for predicting inactivation resulted in a family of unique concentration and time values for each inactivation level rather than the simple CT product of the Chick-Watson model.     

Microsomal monooxygenase system in Morris hepatoma: purification and characterization of cytochromes P-450 from Morris hepatoma 5123D of 3-methylcholanthrene-treated rats

Two forms of cytochrome P-450 (hepatoma P-450MCI and P-450MCII) were purified from hepatoma 5123D microsomes of tumor-bearing rats treated with 3-methylcholanthrene. Hepatoma P-450MCI had a specific content of 18.4 nmol/mg protein and showed a main protein band with a minimum molecular weight of 56,000 on sodium dodecyl sulfate-polyacrylamide gel. Hepatoma P-450MCII had a specific content of 7.38 nmol/mg protein and a minimum molecular weight of 50,000. The carbon monoxide-reduced difference spectral peak of hepatoma P-450MCI was at 446.5 nm, whereas the peak of hepatoma P-450MCII was at 451 nm. In the reconstituted system, hepatoma P-450MCI catalyzed 3-hydroxylation of benzo[a]pyrene and O-deethylation of 7-ethoxycoumarin, but showed low activities for N-demethylation of benzphetamine and aminopyrine, O-demethylation of p-nitroanisole, and p-hydroxylation of aniline. On the other hand, hepatoma P-450MCII did not catalyze hydroxylation of any of the substrates tested. By Ouchterlony double-diffusion analysis, hepatoma P-450MCI was immunologically indistinguishable from rat liver cytochrome P-450c, but hepatoma P-450MCII was distinct from hepatoma P-450MCI and rat liver cytochrome P-450c. Peptide maps of hepatoma P-450MCI and rat liver cytochrome P-450c after proteolysis with Staphylococcus aureus V8 protease demonstrated the similarity of the two cytochromes P-450.     

In Vitro Excystation of Spironucleus muris

ABSTRACT. In vitro excystation of Spironucleus muris cysts, purified by sequential sucrose and Percoll gradients from mouse feces, was studied. Three in vitro excystation procedures, used for Giardia , were assessed to determine the most useful method. Excystation was monitored by light microscopy and subsequently characterized by transmission and scanning electron microscopy. Spironucleus muris excysted routinely at a level greater than 90% when induced in Hanks' balanced salt solution containing sodium bicarbonate at pH 2.0 and transferred to Tyrodes' salt solution as an excystation medium. Similarly, high rates of excystation were recorded after induction of S. muris cysts in 0.1 M potassium phosphate buffer (pH 7.0) with sodium bicarbonate and excystation in trypticase-yeast extract-iron medium (TYI medium) or phosphate-buffered saline. A lower rate and percentage of excystation were observed after induction of S. muris cysts in an aqueous hydrochloric acid solution (pH 2.0) followed by excystation in TYI medium. All excystation methods produced extremely active S. muris trophozoites with normal morphology. Nonexcysting S. muris cysts have a wall composed of an outer fibrous and an inner membranous portion. Following induction, numerous vesicles appeared in the peritrophic space. Excystation began by the cyst wall opening at one pole, and the anterior part of the trophozoite protruding from the cyst wall. The trophozoite emerged progressively from the cyst wall and the empty cyst wall appeared to collapse. Excysted trophozoites exhibited normal morphological features of S. muris trophozoites isolated from the mouse intestine.     

Changes of deep cervical lymph flow following the infusion of isotonic, hypotonic, and hypertonic NaCl in rabbit.

Isotonic, hypotonic, or hypertonic saline was infused in anesthetized rabbits in order to test the effects of osmolality in cerebral vessels on lymph flow. The jugular lymph trunk was cannulated by PE tubing in a headward direction. Either a hypo-(100 mosmol), iso-(310 mosmol), or hypertonic (605 mosmol) NaCl solution was infused into the internal carotid artery (ICA) or the right lateral ventricle (RIV). Lymph was continuously collected at slight negative pressure, and measured over a 90 min preinfusion period, as well as during saline infusion and intermittent recovery periods. Mean peak flow rates for the first 30 min infusion of hypertonic saline via ICA and RLV were 5.1 +/- 1.2 and 6.7 +/- 1.6 microliters/min, respectively, or a significant increase of 38% and 40% over those of isotonic saline (3.7 +/- 0.9 microliters/min via ICA; 4.8 +/- 1.0 microliters/min via RLV). Conversely, for hypotonic saline, lymph flow rates were significantly reduced by 19% (2.9 +/- 0.6 microliters/min) and 23% (3.7 +/- 0.7 microliters/min) for the first 30 min infusion via ICA and RLV, respectively. Increases in arterial and intracranial pressures, as well as an enhancement of respiratory movements following hypertonic saline infusion, augmented lymph formation. The results suggest that the observed changes in jugular lymph flow following saline infusion can be correlated to the resulting increase in intracranial pressure and respiratory movements, and changes in the osmolality and blood pressure of cerebral vessels.     

Effects of various physical and chemical factors on excystation of the encysted metacercariae of Echinostoma caproni

Various physical and chemical factors were studied to determine their effects on the viability of encysted metacercariae of Echinostoma caproni. Viability was equated with chemical excystation in an alkaline trypsin-bile salts (TB) medium. Control cysts showed excystation percentages of > 90% in TB. Excystation proved to be a more reliable criterion of cyst viability than observations by light microscopy. Isolated cysts and cysts left in the snail (in situ cysts) were studied. Generally, in situ cysts proved more resistant to various physical and chemical treatments than did isolated cysts. Cysts stored for 7 days at 28 C in a Locke's 1:1 solution showed 97% excystation, suggesting that cysts of this species would survive postal delays during shipment. Of numerous marinades tested, the one that was most harmful to isolated and in situ cysts was vinegar. Isolated and in situ cysts were killed by boiling (100 C) for 1 or 3 min, but freezing at -10 C did not kill all isolated or in situ cysts after 24 hr. Concentrations of potassium permanganate ranging from 300 to 1,200 mg/L killed most isolated cysts within 5 min, but in situ cysts survived these concentrations for 24 hr. Concentrated solutions of NaCl and sucrose had no effect on the viability of isolated and in situ cysts, suggesting that their use in food preparations for molluscs would not be effective in killing echinostomatid cysts in tainted snail tissues.     

Excystation of Giardia muris induced by a phosphate-bicarbonate medium: localization of acid phosphatase

Giardia muris cysts were incubated briefly in an aqueous induction medium of 0.1 M potassium phosphate with 0.1, 0.2, or 0.3 M sodium bicarbonate. High rates of excystation (91.1-96.7%) were recorded within 5 min after the cysts were placed in trypticase-yeast extract-iron-serum (TYI-S) medium. Substitution of phosphate-buffered saline for TYI-S as the excystation medium resulted in high rates (95.9%) of excystation but required an incubation of 15 min. Excystation was inhibited by the presence of 4-4'-diisothiocyanatostilbene-2-2'-disulfonic acid (DIDS), a specific inhibitor of vacuolar and lysosomal acidification. Microscopic observation showed the loss of the peritrophic space and a change in the refractile nature of the cyst wall prior to excystation. Histochemical studies demonstrated a reaction product of acid phosphatase activity in the lysosomelike peripheral vacuoles in induced cysts and in the peritrophic space of cysts placed in excystation medium. Staining with acridine orange suggested that the peripheral vacuoles become acidified during induction. This staining was inhibited also by DIDS. These studies show that in vitro excystation can be produced at high rates by easily prepared media without exogenous enzymes, low pH, reducing agents, or complex components. The data also suggest that excystation may be stimulated by the bicarbonate-phosphate medium accompanied by acidification of the peripheral vacuoles and the release of their contents into the peritrophic space.     

Secretion of endogenous regulators of Na K ATPase by isolated rat duodenum in osmotic stimulation of its mucous membrane]

The experiments in vitro have shown that perfusion of the isolated duodenum cavity in rats by hypotonic (20 mosmol/l H2O) or hypertonic (500 mosmol/l H2O) solutions of NaCl and mannitol during 10 min stimulates secretion of endogenic factors (activators or inhibitors of enterocytes of Na+, K(+)-ATPase) into the serous incubation solution. Contact of the duodenum mucose surface with isotonic (300 mosmol/l H2O) solutions does not stimulate the secretion of this enzyme regulators. Endogenic substance are thermostable: they retain activity relative to Na+, K+ ATPase of enterocytes after heating (100 degrees C, 10 min duration) of serous incubation solutions.     

Giardia sp.: physical factors of excystation in vitro, and excystation vs eosin exclusion as determinants of viability.

The effects of several factors on Giardia sp. excystation in vitro were investigated. Temperature, pH, time, and incubation medium were shown to affect the levels of excystation achieved. In general, those conditions most closely approximating the organism's in vivo environment induced the highest levels of excystation. The viability of Giardia sp. cyst suspensions was compared by eosin exclusion and excystation. Eosin exclusion consistently indicated higher cyst viability than could be demonstrated by in vitro excystation. Using excystation as the criterion of viability, the effect of storage at ?13, 8, 21, and 37 C and of exposure to boiling water on Giardia sp. cyst survival was studied. Storage at 8 C permitted longest cyst survival, 77 days, at which time the cyst suspension was exhausted. Cysts stored at 21 C retained their viability for 5 to 24 days, while those at 37 C never survived longer than 4 days. Freezing and thawing cysts resulted in an almost complete loss of viability although a low level of viability (< 1%) persisted for at least 14 days. Cysts exposed to boiling water were immediately incapable of excystation.     

Characterization of a chloride current in the larval epidermis of the beetle Tenebrio molitor

Voltage-clamp analysis of single cuticle-attached epidermal cells dissected from the newly-ecdysed mealworm revealed the presence of a large inwardly-rectifying anion (i.e. outwardly-going) current. In many cells this current formed spontaneously on breaking into the cell with the patch pipette when the bath solution was isoosmotic with the pipette solution (415 mosmol/l). The current was evoked rapidly by electrical stimulation or by bathing the cells in hyposmotic saline (335 mosmol/l). The reversal potential of the activated current shifted in agreement with the Nernst prediction for Cl(-) when the transmembrane chloride gradient was altered by partially substituting bath or patch pipette Cl(-) with gluconate(-). Substitution of Na(+) with choline(+) or K(+) with TEA and Ba(+) in the bath or pipette solutions did not alter the reversal potential. Addition of 200 &mgr;mol/l cyclic AMP or 1 mmol/l cyclic GMP to the pipette solution increased the initial current strength and reduced the time taken to reach half peak amplitude from 117 sec to 49 sec and 41 sec, respectively. Cyclic AMP also raised the threshold at which the current developed under hyperosmotic conditions by about 20 mosmol/l. Addition of the Cl(-) channel blockers diphenylamine-2-carboxylic acid (200 &mgr;mmol/l) and diisothiocyanostilbene-2,2'-disulphonic acid (250 &mgr;mol/l) to the bath solution reduced the inwardly-rectifying anion current by 50%. This current was barely detectable in cells prepared from the mid-instar integument. This non-constitutive pattern of expression suggests that cellular Cl(-) efflux (and that of other anions) may be required during moult-cycle specific processes such as moulting fluid formation and cell volume regulation. As the strength of the epidermal anion current could be raised by the exogenous application of cytosolic cyclic nucleotides, the activity of the anion channels responsible for this current may normally be regulated by yet-to-be-identified hormone(s) or neuropeptide(s) acting on this tissue.     

Development of a Defined Minimal Medium for the Growth of Edwardsiella ictaluri

In this report, a complete defined medium and a minimally defined medium are described for Edwardsiella ictaluri. The complete defined medium consists of 46 individual components, including a basal salt solution, glucose, magnesium sulfate, iron sulfate, six trace metals, four nucleotides, 10 vitamins, and 19 amino acids. This medium supports growth in broth and on solid media. Optimal growth at 30(deg)C was obtained at pH 7.0, and at an osmolality of 390 mosmol/kg of H(inf2)O, with a glucose concentration of 4 g/liter. The defined minimal medium reduces the 46 components of the complete medium to eight essential components, including the basal salt solution, glucose, magnesium sulfate, pantothenic acid, and niacinamide. In addition, specific amino acids that depend on the specific requirements of the individual strains of E. ictaluri are added.     

The spermatozoon of the African catfish: fine structure, motility, viability and its behaviour in seminal vesicle secretion

The spermatozoon of the African catfish Clarias gariepinus is a simple organized aquasperm although it reveals very unique characteristics: the cytoplasmic channel is lacking, the mitochondria form a complex structure and the arrangement of the centriolar complex is species specific. Semen has high initial motility rates ( c. 70–90%) and swimming velocities ( c. 120–140 μm s⁻¹), the main swimming type is linear. Motility duration in water is 30 s and is prolonged only to 40 s in NaCl solutions or more complex bu ered motility activating saline solutions. A pH between 7.0 and 9.0 has no e ect on the sperm motility parameters. Motility is completely and reversibly suppressed in electrolyte and non-electrolyte solutions with an osmolality of 200 mosmol kg⁻¹. During immotile storage the sperm viability is influenced by the osmolality and the potassium levels of the storage medium, by the temperature and by the dilution. At optimal conditions (bu ered sperm motility inhibiting saline solution: 150 mmol l⁻¹ NaCl, 2.5 mmol l⁻¹ KCl, 1 mmol l⁻¹ CaCl₂, 1 mmol l⁻¹ MgSO₄, 20 mmol l⁻¹ Tris solution, pH 8.5; dilution rate 1: 5; storage temperature, 4°C) sperm viability persists for >7 days. High viscosity of the pure seminal vesicle secretion completely inhibits the sperm motility. When the seminal vesicle secretion is diluted in water the viscosity decreases and the motility suppressing e ect is neutralized. When semen is mixed with seminal vesicle secretion the sperm viability decreases to zero within 10 min.     

Media effects on the growth ofThiobacillus novellus

The growth ofThiobacillus novellus (ATCC 8093) and of a laboratory strain was observed in a mineral salts medium (MSM) and a medium containing a trace metal solution (TMM). The generation time during growth on MSM was found to be almost half the value for growth on TMM (134 min vs 252 min), with overall yields in the latter being about one-third those on MSM. Cultures maintained for 5 weeks in trace metal media exhibited decreased generation times, but no change in overall yields. An increase in glucose concentration resulted in an increased yield but had no effect on generation times. The addition of biotin to either medium resulted in minor decrease in glucose utilization. The omission of the trace metal constituents, individually, usually resulted in shorter generation times and increased growth yields. It is concluded that differences in growth conditions have been responsible for discrepancies in data from some previous studies.     

Metabolic and circulatory responses to the ingestion of glucose polymer and glucose/electrolyte solutions during exercise in man

Six men exercised on a cycle ergometer for 60 min on two occasions one week apart, at 68 +/- 3% of VO2max. On one occasion, a dilute glucose/electrolyte solution (E: osmolality 310 mosmol X kg-1, glucose content 200 mmol X l-1) was given orally at a rate of 100 ml every 10 min, beginning immediately prior to exercise. On the other occasion, a glucose polymer solution (P: osmolality 630 mosmol X kg-1, glucose content equivalent to 916 mmol X l-1) was given at the same rate. Blood samples were obtained from a superficial forearm vein immediately prior to exercise and at 15-min intervals during exercise; further samples were obtained at 15-min intervals for 60 min at rest following exercise. Heart rate and rectal temperature were measured at 5-min intervals during exercise. Blood glucose concentration was not different between the two tests during exercise, but rose to a peak of 8.7 +/- 1.2 mmol X l-1 (mean +/- SD) at 30-min post-exercise when P was drunk. Blood glucose remained unchanged during and after exercise when E was drunk. Plasma insulin levels were unchanged during exercise and were the same on both trials, but again a sharp rise in plasma insulin concentration was seen after exercise when P was drunk. The rate of carbohydrate oxidation during exercise, as calculated from VO2 and the respiratory exchange ratio, was not different between the two tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ion transport and regulation of respiratory tract fluid output in dogs.

To investigate the regulation of respiratory tract fluid output (RTFO), we collected the RTFO in an anesthetized canine model after a series of pharmacological interventions (inhibition of Na(+)-K(+)-ATPase or Na(+)-K(+)-2Cl(-) cotransporter, 250 microl) and physiological challenges (ionic and/or osmotic perturbation in airway lumen, 250 microl). Whereas 250 microl of aerosolized 0.9% saline caused a transient increase in RTFO, a 250-microl bumetanide-induced increase in RTFO was evident for 18 min and a 250-microl acetylstrophanthidin-induced increase in RTFO persisted for at least 30 min. Dry air ventilation decreased the responses of RTFO to the saline (sham) and acetylstrophanthidin intervention but not the bumetanide intervention. Delivery of 250 mosmol/kgH(2)O ion-free mannitol (250 microl) caused marked increases in RTFO that were little affected by the administration of acetylstrophanthidin or bumetanide 30 min before these challenges. A 250-microl 550 mosmol/kgH(2)O ion-free mannitol challenge caused a more marked and prolonged increase in RTFO. Thus aerosol delivery of a low dose of a cardiac glycoside or a near-isosmotic, ion-free, impermeant osmolyte solution may be therapeutically useful by increasing the clearance of secretions from the tracheobronchial airways.     

Encystation of Entamoeba invadens IP-1 is induced by lowering the osmotic pressure and depletion of nutrients from the medium

Trophozoites of Entamoeba invadens IP-1 can be induced to encyst in simple solutions composed of semipermeable constituents (buffer, salts, or sugars) provided that their osmotic pressure is in the range of 60-160 mosmol/kg. Optimal yield of mature cysts was obtained when the osmotic pressure of the medium was 110 mosmol/kg. Encystation could be obtained in the absence of serum although higher yields were obtained in its presence. No difference in the yield of mature cysts was found when either dialyzed or full serum was used. High yields of encystation were obtained (greater than 70%) in the presence of 5% serum in solutions of NaCl, KCl, or MgSO4, suggesting that the mechanism of encystation is not induced via sodium or potassium channels. Cysts were obtained in the presence of 72 mM glucose, indicating that depletion of a carbon source is not the only requirement for encystation. A rapid change in the density of the Entamoeba cells was observed upon transfer of trophozoites (density 1.061-1.073 g/ml) from growth medium to the low osmotic pressure encystation solutions. Within the first 2 min their density decreased (to 1.050 g/ml), but it soon increased, reaching within 30 min a density higher than 1.120 g/ml. As the encystation process continued to completion, the density of the cells gradually decreased, the mature cysts reaching a density of 1.049-1.061 g/ml.     

Factors controlling the intracellular concentration of calcium and the spontaneous activity of the ureter

The role of the electrogenic Na(+)-Ca(2+)-exchange mechanism in regulating the spike activity of the ureter was studied. The ureter cells were shown to be capable of generating action potentials (AP) in sodium-free Krebs solution. The time during which the spikes are generated is in exponential dependence on the concentration of calcium ions in the medium, [Ca2+]o within 2.5 to 15 mmol/l. Simultaneously with the generation of the spikes, accumulation of calcium in the muscles is observed, proportional to the increase of [Ca2+]o. The addition of as little as 20 mmol/l Na+ or Li+ ions into the solution restores the prolonged electrical activity of the ureter. Under these conditions, the decrease of intracellular Ca2+ within 5 min was more than two times larger as compared with that in sodium-free medium. Upon substituting Ba2+ ions for Ca2+ ions in Krebs solution AP are generated within an interval which was the longer the higher the Ba2+ concentration in the medium. Li+ ions can replace Na+ ions in maintaining AP and in extruding calcium from the cell. It is supposed that the generation of the stable spike activity of the ureter depends on the functioning of Na(+)-Ca(2+)-exchange mechanism.     

Efficacy of two peroxygen-based disinfectants for inactivation of Cryptosporidium parvum oocysts

Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate Cryptosporidium parvum oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4',6'-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of C. parvum oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting C. parvum oocyst viability). These findings indicate the potential efficacy of both disinfectants for C. parvum oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection.     

In Vitro Excystation of Caryospora simplex (Apicomplexa: Eimeriorina)1

In vitro excystation of sporozoites of the heteroxenous coccidian Caryospora simplex Léger, 1904 (Apicomplexa: Eimeriorina) is described. Sporocysts freed mechanically from oocysts released a maximum of 51% of their sporozoites within 45 min at 25°C and a maximum of 74% within 20 min at 37°C when incubated in a 0.25% (w/v) trypsin–0.75% (w/v) sodium taurocholate (bile salt) excystation solution. At emergence from sporocysts, sporozoites were weakly motile then became highly active after about 2 min in excystation solution. Sporozoites within sporocysts exposed to bile salt only became highly motile within 25 min at 25°C and within 15 min at 37°C but did not excyst. When exposed only to trypsin at the above temperatures, the Stieda body dissolved; the substieda body remained intact, and the sporozoites exhibited only limited motility within sporocysts; only a few excysted. Intact, sporulated oocysts incubated at 25° or 37°C in 0.02 M cysteine-HC1 and a 50% CO₂ atmosphere for 18 h had no morphologic changes in the oocyst wall. Further incubation of these intact oocysts in excystation solution for 30 min at 37°C caused neither motility of sporozoites within sporocysts nor excystation. Grinding oocysts for 30 sec in a motor-driven, teflon-coated tissue grinder caused motility of some sporozoites within sporocysts but did not result in excystation.