Synergistic interactions between artocarpin-rich extract,lawsone methyl ether and ampicillin on anti-MRSA and their antibiofilm formation

Artocarpin-rich extract (ARE) was prepared using a green technology and standardized to contain 49·6% w/w artocarpin, while lawsone methyl ether was prepared using a green semi-synthesis. ARE, LME and ampicillin exhibited weak anti-MRSA activity with the MICs of 31·2–62·5 µg/ml. Based on the checkerboard assay, the synergistic interaction between ARE (0·03 µg/ml) and LME (0·49 µg/ml) against four MRSA isolates were observed with the fractional inhibitory concentration index (FICI) value of 0·008, while those of ARE (1·95–7·81 µg/ml) and ampicillin (0·49 µg/ml) as well as LME (0·49–1·95 µg/ml) and ampicillin (0·49 µg/ml) were 0·016–0·257. The time kill confirmed the synergistic interactions against MRSA with different degrees. The combination of ARE and LME as well as its combinations with ampicillin altered the membrane permeability of MRSA, which led to release of the intracellular materials. In addition, each compound inhibited the biofilm formation of standard MRSA (DMST 20654) and the clinical isolate (MRSA 1096). These findings suggested that cocktails containing ARE and LME might be used to overcome problems associated with MRSA. Additionally, the results implied that combination of either ARE or LME with available conventional antibiotic agents might be effective in countering these perilous pathogens.     

Cadmium‐induced cell death in BY‐2 cell culture starts with vacuolization of cytoplasm and terminates with necrosis

Cadmium is a potent inducer of programmed cell death (PCD) in plants but the morphological changes in cells exposed to cadmium are poorly characterized. Using light and transmission electron microscopy (TEM) we have investigated the changes in ultrastructure of tobacco BY‐2 cells treated with 50 µM CdSO₄. The cadmium‐induced alterations in cell morphology occurred gradually over a period of 3–4 days and the first stages of the response resembled vacuolar type of cell death. The initial formation of numerous small cytoplasmic vacuoles and dilation of endoplasmic reticulum was followed first by fusion of smaller vacuoles with each other and with big vacuoles, and then by the appearance of autophagic vacuoles containing autophagic bodies. The final stages of cell death were accompanied by necrotic features including loss of plasmalemma integrity, shrinkage of the protoplast and unprocessed cellular components. In addition, we observed a gradual degradation of nuclear material. Our results demonstrate that cadmium‐induced plant cell death is a slow process featuring elements of vacuolar cell death and terminating with necrosis.     

Validation of daily microincrement deposition in otoliths of juvenile and adult Peruvian anchovy Engraulis ringens

Wild adult specimens of the Peruvian anchovy Engraulis ringens were captured and reared to validate the daily periodicity of otolith microincrement formation. The postcapture stress generated spontaneous spawning, making it possible to conduct a rearing trial on larvae first in an artificial nutrient‐enriched system (ANES) for 52 days followed by an artificial feeding regime in a culture tank until day 115 post‐hatch. Microincrements of the sagittal otoliths of sacrificed juveniles [mean ± s.d. total length (L_T) = 5·13 ± 0·37 cm, range 5–6 cm; c.v. = 7·5%] showed very distinct light and dark zones. The slope of the relationship between the total number of increments after the hatch check and days elapsed after hatching was not significantly different from 1. The transfer from ANES to the artificial feeding regime induced a mark in the sagittal otoliths. The number of microincrements after this induced mark coincided with the number of days elapsed after the transfer date. In parallel experiments, adult E. ringens (mean ± s.d. L_T = 14·92 ± 0·55 cm, range 13–16 cm) were exposed to one of two fluorescent marking immersion treatments with either alizarin red S (ARS; 25 mg l^?1 per 6 h) or oxytetracycline hydrochloride (OTC; 200 mg l^?1 per 10 h). The microincrements between fluorescent bands were distinct, ranging from 0·89 to 2·75 µm (mean ± s.d. =1·43 ± 0·28 µm; c.v. = 32%) and from 0·71 to 2·89 µm (1·53 ± 0·27 µm; c.v. = 35%) for ARS and OTC, respectively. The relationship between the number of microincrements between marks and the number of elapsed days for ARS and OCT treatments indicated that there was a significant correspondence between the number of increases observed and the number of days. Hence, daily microincrements of otoliths of E. ringens are likely to be formed in juveniles and adults under natural conditions.     

Timecourse of oocyte development in saithe Pollachius virens

Wild caught North Sea saithe Pollachius virens were monitored for growth, sex steroid profiles and oocyte development pre‐spawning and measured for egg size and group fecundity during the spawning season in the laboratory. Vitellogenesis commenced in late October–early November, at a leading cohort size (C_L) of c. 250 µm, after which oocytes grew rapidly in size until spawning started in February. Notably, a distinct cortical alveoli stage was virtually absent with yolk granules observed in developing oocytes at the very beginning of vitellogenesis. Little atresia was observed pre‐spawning, but atretic re‐absorption of remnant oocytes containing yolk granules was found in all females immediately post‐spawning. As expected, concentrations of sex steroids, oestradiol‐17β (females), testosterone (both sexes) and 11‐ketotestosterone (both sexes), increased pre‐spawning before dropping post‐spawning. The present experiment provides the first validation of sex steroid levels in P. virens. Post‐ovulatory follicles were visible in histological sections from female gonads 9–11 months post‐spawning, but then disappeared. Spawning commenced around a C_L of c. 750 µm (700–800 µm). Hydrated oocytes (eggs) measured between 1·04 and 1·31 mm (mean = 1·18 mm) with decreasing sizes towards the end of spawning. The average estimated realized fecundity was c. 0·84 million eggs (median female total length, L_T = 60 cm). Spawning lasted from 13 February to 29 March.     

Size and growth rate differences of larval Baltic sprat Sprattus sprattus collected with bongo and MIK nets

The effect of sampling with bongo (0·6 m diameter frame with 500 µm mesh) and Methot Isaac Kidd (MIK) (2 m diameter frame with 2 mm mesh finished with 500 µm codend) nets on standard length (L_S) range and growth rate differences was tested for larval Sprattus sprattus (n = 906, L_S range: 7·0–34·5 mm) collected during four cruises in the summer months of 2006, 2007, 2009 and 2010 in the southern Baltic Sea. Although the minimum size of larvae collected with the bongo and MIK nets was similar in each cruise (from c. 7 to 9 mm), the MIK nets permitted collecting larger specimens (up to c. 34 mm) than the bongo nets did (up to c. 27 mm). The growth rates of larvae collected with the bongo and MIK nets (specimens of the same size range were compared for three cruises) were not statistically different (mean = 0·55 mm day^?1, n = 788, L_S range: 7·0–27·4 mm), which means the material collected with these two nets can be combined and growth rate results between them were compared.     

Structural and ultrastructural studies of male reproductive tract and spermatozoa in Xylocopa frontalis (Hymenoptera,Apidae)

Fiorillo, B. S., Zama, U., Lino‐Neto, J. and Báo, S. N. 2010. Structural and ultrastructural studies of male reproductive tract and spermatozoa in Xylocopa frontalis (Hymenoptera, Apidae). —Acta Zoologica (Stockholm) 91 : 176–183. In Xylocopa frontalis the reproductive tract is composed of testes, deferent ducts, seminal vesicles, accessory glands and an ejaculatory duct. Each testis comprises four testicular tubules in which multiple cysts are present containing approximately 64 spermatozoa per cyst. The seminal vesicle consists of an epithelium, a thick basement lamina and a muscular external sheet. In the luminal region some vesicles can be observed; however, the epithelial cells of the seminal vesicle do not display morphological features associated with secretory functions. The spermatozoa, measuring approximately 260 µm long, are similar to the hymenopteran pattern. The head region consists of an acrosome with an inner perforatorium that penetrates an asymmetrical nuclear tip. The nucleus is linear, electron‐dense and its posterior tip projects into the beginning of the axoneme. The centriolar adjunct is asymmetric with many electron‐lucent lacunae interspersed throughout. The axoneme has the 9 + 9 + 2 pattern of microtubules and in the posterior region the central microtubules finish first, followed by the doublets and finally the accessory microtubules. The mitochondrial derivatives are asymmetric in both length and diameter with paracrystalline material present only in the larger one. These features may be useful characters for taxonomy and phylogenetic studies.     

Abscisic acid introduced into the transpiration stream accumulates in the guard-cell apoplast and causes stomatal closure

Petioles of water‐sufficient intact Vicia faba L. plants were infused with 1 µm abscisic acid (ABA) to simulate the import of root‐source ABA. This protocol permitted quantitative ABA delivery, up to 300 pmol ABA over 60 min, to the leaf without ambiguities associated with perturbations in plant–water status. The ABA concentrations in whole‐leaf samples and in apoplastic sap increased with the amount infused; ABA degradation was not detected. The ABA concentration in apoplastic sap was consistent with uptake of imported ABA into the leaf symplast, but this interpretation is qualified. Our focus was quantitative cellular compartmentation of imported ABA in guard cells. Unlike when leaves are stressed, the guard‐cell symplast ABA content did not increase because of ABA infusion (P = 0·48; 3·0 ± 0·5 versus 4·0 ± 1·2 fg guard‐cell‐pair^?1). However, the guard‐cell apoplast ABA content increased linearly (R² = 0·98) from ?0·2 ± 0·5 to 3·1 ± 1·3 fg guard‐cell‐pair^?1 (≈ 3·1 µm ) and was inversely related to leaf conductance (R² = 0·82). Apparently, xylem ABA accumulates in the guard‐cell wall as a result of evaporation of the apoplast solution. This mechanism provides for integrating transpiration rate and ABA concentration in the xylem solution.     

Cryopreservation of Atlantic salmon Salmo salar sperm: effects on sperm physiology

The objective of this study was to determine the effect of freezing on the function in Atlantic salmon Salmo salar spermatozoa. The semen was frozen in Cortland's medium + 1.3M dimethyl sulphoxide + 0.3M glucose + 2% bovine serum albumin (final concentration) in a ratio of 1:3 (semen:cryoprotectant) as the treatment (T) and fresh semen as the control (F). Straws of 0·5 ml of sperm suspension were frozen in 4 cm of N₂L. They were thawed in a thermoregulated bath (40° C). After thawing, the percentage of spermatozoa with fragmented DNA [transferase dUTP (deoxyuridine triphosphate) nick‐end labelling (TUNEL)], plasma membrane integrity (SYBR‐14/PI) and mitochondrial membrane potential (ΔΨMMit, JC‐1) were evaluated by flow cytometry and motility was evaluated by optical microscope under stroboscopic light. The fertilization rates of the control and treatment semen were tested at a sperm density of 1·5 × 10⁷ spermatozoa oocyte^?1, by observation of the first cleavages after 16 h incubation at 10° C. In the cryopreserved semen (T), the mean ± s.d . DNA fragmentation was 4·8 ± 2·5%; plasma membrane integrity 75·2 ± 6·3%; mitochondrial membrane potential 51·7 ± 3·6%; motility 58·5 ± 5·3%; curved line velocity (V_CL) 61·2 ± 17·4 µm s^?1; average‐path velocity (V_AP) 50·1 ± 17·3 µm s^?1; straight‐line velocity (V_SL) 59·1 ± 18·4 µm s^?1; fertilization rate 81·6 ± 1·9%. There were significant differences in the plasma membrane integrity, mitochondrial membrane potential, motility, fertilization rate, V_CL, V_AP and V_SL compared with the controls (P < 0·05). Also the mitochondrial membrane potential correlated with motility, fertilization rate, V_CL and V_SL (r = 0·75; r = 0·59; r = 0·77 and r = 0·79, respectively; P < 0·05); and the fertilization rate correlated with V_CL and V_SL (r = 0·59 and r = 0·55, respectively).     

Effects of ozone on membrane permeability and ultrastructure in Pseudomonas aeruginosa

Aims: To examine the mechanism of ozone‐induced damage to cytoplasmic membrane and cell ultrastructure of Pseudomonas aeruginosa ATCC27853. Methods and Results: Cell suspensions of Ps. aeruginosa ATCC27853 were treated with ozonated water. The leakages of cellular potassium (K⁺), magnesium (Mg²⁺) and adenosine triphosphate (ATP), determined by inductively coupled plasma/mass spectrometry (ICP/MS) and a commercial bioluminescence assay kit, were to assess ozone‐induced damage to the cytoplasmic membrane. Maximum leakages of K⁺ and Mg²⁺ were attained, respectively, at 0·53 mg l^?1 ozone after 0·5 and 2 min with >99% inactivation of culturable bacteria, while that of ATP was achieved at 0·67 mg l^?1 ozone after 1 min. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed that treated cells retained intact shapes and cytoplasm agglutinations and vacuoles occurred. Conclusions: Ozone inactivates Ps. aeruginosa ATCC27853 by the combined results of increased cytoplasmic membrane permeability and cytoplasm coagulation, rather than by severe membrane disruption and cell lysis. Significance and Impact of the Study: Pseudomonas aeruginosa is a common water‐related pathogen. These insights into the leakage of cytoplasmic components and ultrastructural changes provide evidence for the mechanisms of ozone‐mediated inactivation.     

Analogues of the frog skin peptide alyteserin‐2a with enhanced antimicrobial activities against Gram‐negative bacteria

The emergence of strains of multidrug‐resistant Gram‐negative bacteria mandates a search for new types of antimicrobial agents. Alyteserin‐2a (ILGKLLSTAAGLLSNL.NH₂) is a cationic, α‐helical peptide, first isolated from skin secretions of the midwife toad, Alytes obstetricans, which displays relatively weak antimicrobial and haemolytic activities. Increasing the cationicity of alyteserin‐2a while maintaining amphipathicity by the substitution Gly¹¹→ Lys enhanced the potency against both Gram‐negative and Gram‐positive bacteria by between fourfold and 16‐fold but concomitantly increased cytotoxic activity against human erythrocytes by sixfold (mean concentration of peptide producing 50% cell death; LC₅₀ = 24 µm ). Antimicrobial potency was increased further by the additional substitution Ser⁷→Lys, but the resulting analogue remained cytotoxic to erythrocytes (LC₅₀ = 38 µm ). However, the peptide containing d ‐lysine at positions 7 and 11 showed high potency against a range of Gram‐negative bacteria, including multidrug‐resistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (minimum inhibitory concentration = 8 µm ) but appreciably lower haemolytic activity (LC₅₀ = 185 µm ) and cytotoxicity against A549 human alveolar basal epithelial cells (LC₅₀ = 65 µm ). The analogue shows potential for treatment of nosocomial pulmonary infections caused by bacteria that have developed resistance to commonly used antibiotics. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Subcellular Distribution of Inorganic Phosphate, and Levels of Nucleoside Triphosphate, in Mature Maize Roots at Low External Phosphate Concentrations: Measurements with 31P-NMR

Maize plants were grown in nutrient solution without phosphate,or in which inorganic phosphate (Pi) was maintained at nearlyconstant concentrations of 1 µM, 10µM or 0·5mM. In vivo ³¹P-NMR measurements showed that there was no discernibledifference in the cytoplasmic Pi content (µmol cm^–3root volume) of the mature roots of plants exposed to 1 µM,10µM or 0·5 mM external phosphate for up to 12d. However, the vacuolar Pi content of the mature roots variedabout 10-fold between these three groups. The cytoplasmic Pi content of roots receiving no external phosphatedecreased significantly after about 7 d total growth, and atabout this time the vacuolar pool of Pi became too small foraccurate measurement. The presence of 1 µM Pi in the nutrientsolution completely prevented this decline in cytoplasmic Pi,and there was some evidence that it also raised the Pi contentof the root vacuoles above the almost undetectable level foundin the totally P-starved roots. During the first 7–9 d of growth, the nucleoside triphosphatecontent of the mature roots was unaffected by the concentrationof phosphate in the nutrient solution. The results highlight the close control of cytoplasmic concentrationsof certain important phosphorus metabolites in roots growingin soil of normal agricultural fertility. Key words: Vacuole, cytoplasm, intracellular compartmentation, NTP, P-nutrition     

Two New Species of Hartmannella Amebae Infecting Freshwater Mollusks

SYNOPSIS. Two new species of small amebae, Hartmannella biparia n. sp. and Hartmannella quadriparia n. sp., were 1st observed in the freshwater mollusks Bulinus globosus and Biomphalaria pallida, respectively. The amebae multiplied in cytoplasmic vacuoles in host cells, particularly in foci in the mantle collar, foot, and intestinal wall. Both amebae had functional contractile vacuoles while within host cells. H. biparia emerged from intracytoplasmic vacuoles in pairs, H. quadriparia in fours, suggesting characteristic reproductive stages.
H. biparia had limax-type motility by smooth lobopodia, was 22 μ long; with vesicular nucleus 3 μ and central endosome 1.5 μ, multiplied by binary fission and formed spherical, smooth-walled cysts 11 μ in diameter. H. quadriparia had limax-type motility by lobopodia with fine, acute projections; was 10 μ long, with vesicular nucleus 2 μ and central endosome 0.75 μ, multiplied and formed spherical, smooth-walled cysts 5 μ in diameter. Neither species multiplied in cysts outside the host.
H. biparia was found infecting 12 species and experimentally transferred to 4 more species of freshwater snails; H. quadriparia was found in one species and experimentally transferred to 6 other species.     

Observations on Chaetoceros salsugineus (Chaetocerotales,Bacillariophyceae): first record of this bloom-forming diatom in a European estuary

Examination of material obtained from the Urdaibai estuary (North Spain) during a 2 year phytoplankton sampling programme revealed a rare species of the genus Chaetoceros, C. salsugineus Takano. The morphology of this small (4·0×5·5?µm) member of the subgenus Hyalochaetae has been described using both light and electron microscopy. Regarding the original material, two new features have been observed: longer chains up to 24 cells and a new type of aperture, bisected by the fusion of central protuberances of adjacent valves. This bloom-forming diatom reached high densities in the poly-euhaline zone of the estuary closely related with high temperatures, denoting its neritic and summer preferences. Maximal cell densities (10⁶–10⁷ cells l^?1) were reached in August and September when the water column was completely mixed and the salinity and temperature were about 31 psu and 20°C, respectively. This is the first report of C. salsugineus in Europe and contributes to the knowledge of the morphological and ecological features of this species. Relationships with other small species of the subgenus Hyalochaetae are also discussed.     

Performance of polymyxin B agar-based tests among carbapenem-resistant Enterobacterales

Therapeutic options for infections caused by Carbapenem-resistant Enterobacterales (CRE) are restricted and include polymyxins-centred schemes. Evaluation of in vitro susceptibility is difficult and time consuming. Agar-based methodologies are an alternative to broth microdilution (BMD) and we aimed to evaluate the accuracy of those methods among Enterobacterales. A total of 137 non-duplicated CRE were subjected to polymyxin B BMD, agar screening test (Mueller Hinton plates containing 3 µg ml⁻¹ of polymyxin B) and agar dilution (antibiotic serially diluted 0·25–64 µg ml⁻¹). CRE of 42·3% were resistant to polymyxin B (MICs range: 0·25–>64 µg ml⁻¹) and 16·8% presented borderline MICs. Sensitivity, specificity, PPV and NPV were 86·2, 98·7, 98 and 90·7% for screening test and 86·2, 97·5, 96·1 and 90·6% for agar dilution. ME was 0·73 and 1·5% for screening and agar dilution respectively; VME was 5·8% for both techniques. In general, agar-based methods had a good performance. As far as we know, this is the first study to propose an agar screening test using polymyxin B instead of colistin.     

The ability of Pichia kudriavzevii to tolerate multiple stresses makes it promising for developing improved bioethanol production processes

Thermotolerant ethanol fermenting yeasts have been extensively used in industrial bioethanol production. However, little is known about yeast physiology under stress during bioethanol processing. This study investigated the physiological characteristics of the thermotolerant yeast Pichia kudriavzevii, strains NUNS-4, NUNS-5 and NUNS-6, under the multiple stresses of heat, ethanol and sodium chloride. Results showed that NUNS-4, NUNS-5 and NUNS-6 displayed higher growth rates under each stress condition than the reference strain, Saccharomyces cerevisiae TISTR5606. Maximum specific growth rates under stresses of heat (45°C), 15% v/v ethanol and 1·0 M sodium chloride were 0·23 ± 0·04 (NUNS-4), 0·11 ± 0·01 (NUNS-5) and 0·15 ± 0·01 h^–1 (NUNS-5), respectively. Morphological features of all yeast studied changed distinctly with the production of granules and vacuoles when exposed to ethanol, and cells were elongated under increased sodium chloride concentration. This study suggests that the three P. kudriavzevii strains are potential candidates to use in industrial–scale fermentation due to a high specific growth rate under multiple stress conditions. Multiple stress-tolerant P. kudriavzevii NUNS strains have received much attention not only for improving large-scale fuel ethanol production, but also for utilizing these strains in other biotechnological industries.     

Fine Structure of the Adrenocortical Homolog and the Corpuscles of Stannius of Amia calva L.

A comparison is made between the fine structure of yellow corpuscles and white corpuscles located within the kidneys of the holostean fish, Amia calva L. The yellow corpuscles are composed of epithelial cells possessing all the features of steroid-producing tissues, namely an abundance of vacuoles, tubular smooth endoplasmic reticulum, and mitochondria with tubular cristae. The Golgi apparatus is also a conspicuous component of their cytoplasm. These cells are homologous to adrenocortical cells of higher vertebrates and they have cytoplasmic projections which extend into the lumina of surrounding sinusoids. The white corpuscles possess epithelial cells of variable appearance but all cells contain secretory granules and an extensive rough endoplasmic reticulum. The secretory granules appear to originate at the Golgi apparatus and occasionally are observed intact in the intercellular space. However the method of release of these granules was not clearly defined. These corpuscles are similar to the corpuscles of Stannius which have been described in modern bony fish. The presence of multivesicular bodies and smooth endoplasmic reticulum in some cells may reflect the origin of the corpuscles of Stannius from the tubular nephron. A. calva appears to be a suitable organism for comparative studies into the function of the adrenocortical homolog and corpuscles of Stannius in “primitive” fish.     

Morphological and morphometric aspects of early life stages of piabanha Brycon gouldingi (Characidae)

Adult specimens of piabanha Brycon gouldingi were collected from Rio das Mortes (Mato Grosso, Brazil), adapted to captivity and induced to spawn at Buriti Fisheries (Nova Mutum, MT, Brazil). The early developmental stages of B. gouldingi were then characterized. Samples were collected at pre‐determined times from oocyte extrusion to total yolk absorption. Oocyte diameter, total larval length (L_T) and yolk‐sac volume were measured. The mean ± s.d . duration of embryo developmental of B. gouldingi was 13·90 ± 0·06 h at 26·40 ± 1·13° C. The mean ± s.d . oocyte diameter was 1·13 ± 0·06 mm with 54% of oocytes ranging from 1·11 to 1·20 mm. Seven stages characterized the early developmental phase of this species: zygote, cleavage, morula, blastula, gastrula, histogenesis–organogenesis and hatching, with unique features related to each stage. At hatching, the larvae measured 3·40 ± 0·07 mm, presented an elongated shape with yolk‐sac volume of 0·46 ± 0·08 µl, non‐pigmented eyes and exhibited swimming ability. When the yolk was completely absorbed at 55 h post‐hatch, mean ± larval L_T was 6·68 ± 0·65 mm, the eyes were highly pigmented and the teeth were visible. These are the first reported findings on the initial developmental stages of B. gouldingi and could be used to improve captive breeding management and conservation practices.     

Analysis of broth-cultured Bacillus atrophaeus and Bacillus cereus spores

Aims: To compare physical properties of spores that were produced in broth sporulation media at greater than 10⁸ spores ml⁻¹. Methods and Results: Bacillus atrophaeus reproducibly sporulated in nutrient broth (NB) and sporulation salts. Microscopy measurements showed that the spores were 0·68 ± 0·11 μm wide and 1·21 ± 0·18 μm long. Coulter Multisizer (CM3) measurements revealed the spore volumes and volume-equivalent spherical diameters, which were 0·48 ± 0·38 μm³ and 0·97 ± 0·07 μm, respectively. Bacillus cereus reproducibly sporulated in NB, sporulation salts, 200 mmol l⁻¹ glutamate and antifoam. Spores were 0·95 ± 0·11 μm wide and 1·31 ± 0·17 μm long. Spore volumes were 0·78 ± 0·61 μm³ and volume-equivalent spherical diameters were 1·14 ± 0·11 μm. Bacillus atrophaeus spores were hydrophilic and B. cereus spores were hydrophobic. However, spore hydrophobicity was significantly altered after treatment with pH-adjusted bleach. Conclusions: The utility of a CM3 for both quantifying Bacillus spores and measuring spore sizes was demonstrated, although the volume between spore exosporium and spore coat was not measured. This study showed fundamental differences between spores from a Bacillus subtilis- and B. cereus-group species. Significance and Impact of the Study: This is useful for developing standard methods for broth spore production and physical characterization of both living and decontaminated spores.     

Blood platelets of the cheetah (Acinonyx jubatus)

The blood platelet count and platelet morphology of 22 adult cheetahs was investigated. The platelet counts of the animals displayed a normal distribution, with a mean count of 344×10⁹/l and a mean platelet volume of 11 fl. Morphological and ultrastructural features of the cheetah platelets revealed the typical platelet morphology of anuclear cells, with granules scattered throughout the cytoplasm. The characteristic surface canalicular system and microtubules were present. True cross‐sections of the platelets had a mean area of 2.146 µm², circumference of 6.805 µm, and mean minimum and maximum projections of 1.000 µm and 2.933 µm, respectively. Zoo Biol 23:263–271, 2004. © 2004 Wiley‐Liss, Inc.