Carbol Fuchsin Research Articles

Cryptosporidiosis: comparison of three diagnostic methods and effects of storage temperature on detectability of cryptosporidia in cattle faeces

Three diagnostic methods (a modified Ziehl-Neelsen staining technique (MZN), a negative staining with carbol fuchsine (CF) and a commercial enzyme immunoassay (EIA) kit, ProSpecT® Cryptosporidium Microplate Assay (Remel, Lenexa, KS, USA)) for detection of Cryptosporidium oocysts in cattle faeces were compared regarding sensitivity and suitability under routine laboratory conditions, with particular emphasis on sample storage. In the 103 faecal samples examined, cryptosporidia infections were detected significantly more often by EIA (p<0.05; n=76) than by MZN (n=65) if ten random fields were evaluated microscopically, but not if the whole coverslip was scanned. In contrast, sensitivities of EIA and CF (n=69) did not differ significantly. Results were obtained very rapidly by CF. However, the hands-on time of CF is comparable to EIA, while MZN is more time consuming. EIA is more expensive than CF and MZN but easy to perform and to evaluate and does not need considerably experienced staff in contrast to CF and MZN. Moreover, 45 faecal samples stored for up to 27 days at different temperatures (+6°C, +16°C, +30°C, +40°C) were examined. The sensitivity of microscopic detection of oocysts in stained smears (CF, MZN) decreased in a temperature and time-dependent manner, while EIA results were not influenced by sample storage at any temperature.

Karyotypes of 25 Populations of 15 Species in Allium Section Rhiziridium from China

Karyotypes of 25 populations of 15 species in Allium section Rhiziridium from China were analyzed to discuss the evolutionary mechanisms of Allium section Rhiziridium and cytogeography of some species. Root tips for the study of mitotic chromosomes were obtained from potted plants and pretreated with paradichlorobenzene at room temperature for 9 hours. After 12-24 hours fixation in 1∶3 acetic-alcohol, the root tips were macerated in 1 mol · L-1 HC1 for 9 minutes at 60℃ and then stained and squashed in carbol fuchsin. Karyotype asymmetry was assessed by As.K%. Results showed that taxa investigated were diploid or tetraploid. Furthermore, the karyotype types were 1A, 2A or 2B according to Stebbins’ karyotype classification. Karyotypes of A.tekesicola, A.tianschanicum, A.beesianum and A.spirale were reported for the first time. Combined with previous karyotype studies, we suggested that: (1) The basic chromosome number of Allium section Rhiziridium from China is x=8. (2) The karyotype types evolutionary trend of Allium section Rhiziridium from China is 1A→2A→2B→2C . (3) Satellite chromosomes of Allium section Rhiziridium from China are varied in numbers and shapes. (4) Polyploidy and chromosome structural rearrangement are two important evolution patterns in Allium section Rhiziridium from China. (5) A.cyaneum and A.nu- tans have exploited new niches through polyploidy and asexual reproduction.

New details indicated by different stainings during conjugation of ciliated protozoa Paramecium.

During conjugation of Paramecium caudatum, nuclear events occur in a scheduled program. Morphological studies on nuclear behavior during conjugation of P. caudatum have been performed since the end of the 19th century. Here we report on new details concerning the conjugation of P. caudatum through the staining of conjugating cells with protargol, carbol fuchsin solution, Hoechst 33342 and immunofluorescence labeling with monoclonal antibody of anti-α tubulin. 1) The crescent nucleus is a characteristic of the meiotic prophase of P. caudatum, has an unstained area. We stained this area with protargol, which was separated from the chromatin area and was not detected by the other stainings. 2) In regards to the four meiotic products, it has long been considered that only one product enters the paroral cone region (PC) and survives after meiosis. However, our protargol and immunofluorescence labeling results indicated that PC entrance of the meiotic product happened before the completion of meiosis instead of after. 3) In our previous study, protargol staining indicated the presence of a swollen structure around the central part of the "U" and "V" shaped spindles connecting the two types of prospective pronuclei. However, immunofluorescence labeling with anti-α tubulin antibodies gave a different image from protargol. All these observations form the basis for further studies of their molecular mechanisms.

Application of extracts of Henna (Lawsonia inamis) leaves as a counter stain

Aqueous (cold and hot) and ethanol extracts solutions of the Henna plant (Lawsonia inamis) leaves was adapted for the first time as a counter stain in Gram staining reaction. Different extracts of L. inamis leaves were formulated into various staining solutions of different concentrations and modified with hydrogen peroxide, ferric chloride, potassium alum and potassium permanganate. These staining solutions were used to stain both known Gram positive and Gram negative bacterial isolates using Gram staining technique. The experimental Henna plant extracts solutions were used with usual counter stains (neutral red, safranine and dilute carbol fuchsin) as positive controls. Phytochemical screening of the extracts revealed the presence of tannin (hennatonic acid or Lawsone) and saponin. The aqueous extracts of the Henna plant (cold and hot) oxidized with potassium permanganate (pH 7.00 to 7.16) gave a better staining reaction with Gram negative bacteria, while the ethanol extract oxidized with potassium permanganate (pH 6.55) had no staining reaction with Gram negative bacteria. Hence the aqueous Henna leaves extracts (cold or hot) when oxidized with potassium permanganate can be a substitute to the usual counter stains used in Gram staining procedure. The results of staining ability of the various henna leaves extracts solutions are discussed. Key words: Lawsonia inamis, extracts, bacteria, Gram staining, counter stain.

Standardization of laboratory tests for tuberculosis and their proficiency testing

Explanations of proper collection procedures are imperative for accurate laboratory analysis. The quality of specimens collected and the proper transport of those specimens to the laboratory are critical to successful isolation of etiological agents. Most mycobacteria grow at a relatively slow rate. Therefore, the acid-fast smear plays an important role in the early diagnosis of mycobacterial infection. There are several methods of determining the acid-fast nature of an organism. In the carbolfuchsin procedures (Ziehl-Neelsen, Kinyoun), acid-fast organisms appear red, and in the fluorochrome procedures (auramine O, auramine-rhodamine, acridine orange), the acid-fast organisms fluoresce yellow to orange. Fluorochrome-stained slides may be directly restained with any of the carbolfuchsin staining procedures. This may be done to confirm a positive fluorochrome slide and to study organism morphology. In the last 10 years, there were many advances in the culture examinations of mycobacteria. The newly developed Mycobacteria Growth Indicator Tube (MGIT), BacT/Alert, ESP Myco, MB Redox and KRD "Nichi B", biphasic Septi-Chek AFB and Myco-Acid, and radiometric BACTEC 460TB systems based on liquid media, proved to be significantly better than the egg-based solid media for the isolation of mycobacteria from clinical specimens. In addition to liquid-based medium, agar (Middlebrook 7H10 or 7H11)- or egg (Ogawa or Löwenstein-Jensen)-based media should be used in the primary isolation of mycobacteria. To identify mycobacteria, conventional biochemical tests are traditionally used. Key test can be used to identify species, or further preliminary grouping may be used. Other approaches to identifying some species of mycobacteria are available. They include niacin accumulation, p-nitrobenzoic acid and p-nitro-alpha-acetylamino-beta-hydroxypropiophenone tests for discrimination of the Mycobacterium tuberculosis complex from mycobacteria other than M. tuberculosis (MOTT); DNA probe methods for identification or confirmation of the M. tuberculosis complex, M. avium complex, M. kansasii, and M. gordonae; DNA-DNA hybridization method for identification of 22 Mycobacterium species; and gas-liquid chromatography or high performance liquid chromatography analyses for recognizing the patterns of the mycobacterial cell wall fatty acids or mycolic acids. The advantages of the last four methods are that they are capable of providing definitive identification within 2 to 5 h after adequate growth. Capilia TB is the newly developed immunochromatographic assay for rapid discrimination between the M. tuberculosis complex and MOTT bacilli. The kit can be easily used for rapid identification of the M. tuberculosis complex in combination with the culture systems based on liquid media. In addition, Capilia TB could correctly detect the M. tuberculosis complex from mixed cultures with the M. tuberculosis complex and MOTT bacilli. The WHO/IUATLD supranational reference laboratory (SRL) network was created in 1994, to ascertain the accuracy of the susceptibility test methods used in different laboratories across the world, and to allow comparability of the surveillance data gathered in countries participating in the Global Project on Anti-tuberculosis Drug Resistance Surveillance. Today, the network has evolved and 23 SRLs actively participate. Results of five rounds of proficiency testing in the SRL network suggest that performance of the network has improved substantially through the years. This progress has been particularly evident for streptomycin and ethambutol sensitivity, which was very low in the first rounds of proficiency testing. In 1998 sensitivity for these two drugs was higher than 95%. For isoniazid and rifampin, sensitivity has been consistently high since the beginning of the Global Project. This indeed reflects the enhanced efforts made by the SRLs to improve their individual performance.

Evaluation of sputum smear examination--from the cases of Nepal, Yemen and the Philippines

Evaluation of sputum smear examination was carried out with smear slides stained by the Ziehl-Neelsen method, which were collected from Nepal, Yemen and the Philippines. The smear slides were checked macroscopically and microscopically according to the following points: 1) smear area size, 2) thickness of smear, 3) evenness of smear, 4) decolorizing condition by Ziehl-Neelsen stain, 5) smear cleanliness, 6) presence of cells in sputum and 7) smear reading accuracy by cross-checking. As the result of smear slide evaluation, it was concluded that proper sputum specimens have been smeared but smears were generally too thin and contaminated with too many dirt . Agreement rate of the slide reading in Nepal, Yemen and the Philippines were 73%, 90% and 88% respectively. Disagreement cases were concentrated in the subtle number of acid-fast bacilli or (+/-) and (+) results. The cause of disagreement might be attributed to contamination with debris, deposit etc., which hindered reading or were misread as acid-fast bacilli. Some improvement should be considered to eliminate the dirt , such as filtration of carbol fuchsin solution, preparation of proper quantity of carbol fuchsin solution to be consumed within three months, mixing the fuchsin stock solution and 5% carbol solution just before use, rinsing the mouth before sputum collection, usage of clean sputum container and slides, and smearing and drying of the slides within clean environment. However, in many developing countries there are many difficulties for facilities, equipments, system and management. Some of these difficulties may be solved by the efforts of laboratory side but some are beyond the limits of their efforts.(ABSTRACT TRUNCATED AT 250 WORDS)

Genome Size Estimates and Chromosome Numbers of Callicarpa L. (Lamiaceae)

Genome size estimates and chromosome number information can be useful for studying the evolution or taxonomy of a group and also can be useful for plant breeders in predicting cross-compatibility. Callicarpa L. is a group of ≈140 species with nearly worldwide distribution. There are no estimates of genome size in the literature and the information on chromosome numbers is limited. Genome size estimates based on flow cytometry are reported here for 16 accessions of Callicarpa comprising 14 species in addition to chromosome counts on six species. Chromosome counts were conducted by staining meristematic cells of roots tips using modified carbol fuchsin. Holoploid genome size estimates ranged from 1.34 pg to 3.48 pg with a mean of 1.74 pg. Two tetraploids (2n = 4x = 68; C. salicifolia P'ei &amp; W. Z. Fang and C. macrophylla Vahl GEN09-0081) were identified based on holoploid genome size and confirmed by chromosome counts. There was little variation among species for monoploid genome size. 1Cx-values ranged from 0.67 pg to 0.88 pg with a mean of 0.77 pg. Chromosome counts for six species revealed a base chromosome number of x = 17. Callicarpa chejuensis Y. H. Chung &amp; H. Kim, C. japonica Thunb. ‘Leucocarpa’, C. longissima Merr., and C. rubella Lindl. were confirmed as diploids (2n = 2x = 34). Cytology supported flow cytometry data that C. salicifolia and C. macrophylla GEN09-0081 were tetraploids. The two accessions of C. macrophylla included in the study were found to be of different ploidy levels. The presence of two ploidy levels among and within species indicates that polyploidization events have occurred in the genus.

Structural and qualitative evaluation of microscopy and directly observed treatment centers under revised national tuberculosis control programme in Nanded city of Maharashtra

Structural and qualitative evaluation of Revised National Tuberculosis Control Programme (RNTCP) is important to determine actual status of the programme in the field settings and to uncover the concealed gaps. The present cross-sectional study assessed the infrastructural facilities and quality of services provided through microscopy and directly observed treatment (DOT) centers at Nanded city of Maharashtra. The investigator made on spot observation on the activities at microscopy and DOT centers and assessed the infrastructural facilities using an observational checklist. Expert microbiologist cross checked the microscopy report done by the laboratory technicians. It revealed that retrieval mechanism was not functioning in more than half of the DOT centers. Only 5 DOT providers were trained in RNTCP. Stock of sputum containers, methylene blue, and carbol fuchsin was found to be inadequate at some microscopy centers. Half of the laboratory technicians reported high false positive result in spite of being trained. Improvement of infrastructural and logistic support along with the refreshing training for the workers are needed for effective implementation of RNTCP.

Quantitative comparison of different purification and detection methods for Cryptosporidium parvum oocysts

Cryptosporidium parvum ( C. parvum) is the causal agent of cryptosporidiosis in many animals, mainly cattle, and possesses a high zoonotic potential. It occurs worldwide and ubiquitously. Detection of C. parvum is mainly performed directly but purification of the oocysts is useful to increase sensitivity and to obtain oocyst material for further use. The study was designed to compare (a) three different direct diagnostic methods, namely modified Ziehl–Neelsen staining, carbol fuchsin staining and conventional PCR, and (b) three routine oocyst purification methods, in particular flotation with saturated sodium chloride solution, Sheather's sucrose solution and a Percoll ® gradient. During comparison of purification methods, special regard was paid to the ability to separate morphologically intact oocysts from the morphologically degenerated fraction or viable from non-viable oocysts, respectively. Results: (a) Diagnostic methods: Most effective in C. parvum oocysts detection in calf faeces was PCR; carbol fuchsin and modified Ziehl–Neelsen stainings achieved comparable results. (b) Purification methods: Oocyst flotation using sodium chloride solution showed to be superior to Percoll ® gradient centrifugation and sugar flotation in terms of purification quality, recovery efficacy (yield) and reduction of the proportion of degenerated or non-viable oocysts.

Morphological apoptotic characteristics of the post-meiotic micronuclei in Paramecium caudatum

In a previous study, the apoptotic degeneration of meiotic products outside the paroral region of Paramecium caudatum was indirectly demonstrated by means of “apofluor” staining. In this experiment, conjugating pairs and exconjugants of P. caudatum were stained with either “apofluor” or carbol fuchsin or both to find some direct evidence to demonstrate the apoptotic characteristics of this process. As a result, asynchronous meiotic nuclear degeneration was observed. Furthermore, a number of additional meiotic nuclei were found. Disintegrating/dividing meiotic nuclei outside the paroral region were observed, which might be the origin of these additional meiotic nuclei. Condensed chromatin and disintegrated chromatin attached to the nuclear membrane were also observed in degenerating nuclei, which are the typical morphological characteristics of apoptosis. Comparison of the cells stained by the above two methods indicated that “apofluor”-stained meiotic nuclei could not be detected by carbol fuchsin in some cells, which suggests a time lag between meiotic nuclear DNA degradation and their eventual disappearance. In this study, some direct evidence was found to show that the meiotic nuclear degeneration in P. caudatum is of apoptotic nature, which further confirmed our previous study ( Yang et al. 2007) and indicated that morphological apoptotic characteristics discovered in multicellular organisms do exist in unicellular eukaryotic ciliate protozoa.

Evaluation of the GenoType Mycobacteria Direct assay for direct detection of the Mycobacterium tuberculosis complex obtained from sputum samples

An increase in the prevalence of tuberculosis (TB) in recent years has accelerated the search for novel tools for the rapid diagnosis of TB infection. This study evaluated the GenoType Mycobacteria Direct (GTMD) assay (Hain Lifescience) for direct detection of the Mycobacterium tuberculosis complex (MTBC) from sputum samples and compared it with conventional methods. The GTMD test is a commercial assay produced using strip techniques and works based on a nucleic acid sequence-based amplification technique. This test allows 23S rRNA amplification-based detection of MTBC, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii and Mycobacterium malmoense directly from decontaminated clinical samples within 6 h. In the present study, 115 sputum samples were processed to detect acid-fast bacilli (AFB) using two microscopy methods (carbol fuchsin and fluorescent staining), two culture methods [Löwenstein-Jensen (LJ) and BACTEC 12B media] and the GTMD test. The results showed that 86 of the samples were positive by direct microscopy, 84 were positive by BACTEC 12B culture, 73 were positive by LJ culture and 95 were positive by the GTMD test. All of the isolates turned out to be MTBC. Moreover, the sensitivity and specificity of the GTMD test for MTBC in patients were 97 and 58 %, respectively, taking the culture combination as the gold standard. When the test was compared with culture of samples from anti-TB-treated patients, the sensitivity and specificity for the test were 100 and 15 %, respectively. Low specificity in treated people might arise from depressed proliferation of AFB. As the two methods target the same living bacilli, the difference is obviously notable. When the culture results and clinical findings of the patients were evaluated together (true-positive specimens), the sensitivity and specificity values of the GTMD test for all patients were 97 and 90 %, respectively. However, both of these values increased to 100 % for the patients receiving anti-TB treatment. These results implied that, to determine whether the patient's sputum contains living AFB, more sensitive techniques should be employed during the follow-up of the patients. These observations suggest that the GTMD method can be useful for early diagnosis of clinically and radiologically suspicious TB cases where smears are negative for Mycobacterium. In addition, the use of a GTMD test in smear-positive cases is helpful and practical in order to identify MTBC quickly. This allows more rapid treatment decisions and infection control precautions.

Extraction and detection of Mycobacterium leprae DNA from ZNCF-stained skin smear slides for better identification of negative skin smears

Identification of Mycobacterium leprae, which causes leprosy, is done by Ziehl Neelsen Carbol Fuchsin (ZNCF) stained slit skin smear microscopy that aids in the diagnosis and quantification of approximate bacterial load carried by the patient. We attempted M. leprae DNA extraction from 46 stained slit skin smear negative slides, using Proteinase K and SDS lysis, followed by ethanol precipitation. M. leprae specific primers (16SrRNA) were used for PCR-based amplification of DNA. We could detect M. leprae DNA in 15 (32.6%) samples. The method can be useful in the diagnosis of apparently slit skin smear negative leprosy cases.

Detection of Cryptosporidium spp. in Humans and Calves Through Nested PCR and Carbol Fuchsin Staining Methods in Ankara, Turkey

In this study, a total of 130 fecal samples, obtained from 98 humans and 32 calves from different hospitals and dairy farms in Ankara, respectively were analyzed with carbol fuchsin staining and nested PCR to determine the presence of Cryptosporidium spp. Nested PCR revealed a total of 13 (10.0%) positive amplification results; 12 (37.5%) were from calves and 1 (1.02%) from humans. Oocysts were observed in 8 (6.15%) fecal samples belonging to 7 (21.88%) calves and 1 (1.02%) human by carbol fuchsin staining. Concerning the detection technics, it was concluded that nested PCR can be successfully used in diagnosis of cryptosporidiosis, and although carbol fuchsin staining method has sufficient specificity it is less sensitive, especially in cases characterized with excretion of fewer oocysts.

An improved method for karyotype analyses of marine algae

Modified carbol fuchsin staining method was successfully introduced into the karyotype analyses of marine algae, including Porphyra, Undaria pinnatifida and Laminaria japonica. Haploid chromosomes were numbered clearly in the vegetative, spermatangial and conchosporangial cells of P. haitanensis and P. yezoensis. Diploid chromosomes were observed and numbered in immature conchosporangial cells of P. haitanensis and P. yezoensis. Pit-connections of Porphyra were also clearly demonstrated. Prophase chromosomes of conchocelis cells were also clearly stained with modified carbol fuchsin. One molar per liter hydrochloric hydrolysis at 60°C for 7–8 min is necessary for getting transparent cytoplasm for conchosporangial karyotype analysis of Porphyra. Staining effects of the three methods using iron alum acetocarmine, aceto-iron-haematoxylin-chloral hydrate and modified carbol fuchsin were compared on the vegetative, spermatangial and conchosporangial cells of Porphyra and the gametophytes of U. pinnatifida and L. japonica. Among the three methods, the modified carbol fuchsin method gave the best result of deep staining and good contrast between nucleus and cytoplasm.

Microscopy of 4 Pathogenic Enteric Protozoan Parasites: A Review

Nonimmunological microscopy procedures for Giardia lamblia, Cryptosporidium species, Cyclospora cayetanensis, and microsporidia are reviewed. In addition to the unstained wet preparation and the routine trichrome stain of the “routine” parasitology, the Gram stain, various modifications of the trichrome stain, the modified carbol fuchsin acid-fast stain, and several fluorescent variations of the acid-fast stain are also covered. The now-extensive literature on the newer techniques of the heated safranin stain, the use of autofluorescence, and the use of fluorescent brighteners, such as calcofluor white and related compounds, are reviewed. Emphasis is placed on the importance of the microscopist being aware of reported variations in both the staining protocols and their results. With increasing use being made of fluorescence microscopy in examination of specimens for the oocysts of cyclospora and the spores of microsporidium, an understanding of the effect of both excitation and barrier filters on the observed results of the procedures is emphasized.

Presence of thermophilic Campylobacter species in Broilers and pigs at certain abattoirs in Republic of Serbia

Examinations were carried out during the period from January 2006 until March 2007 on a total of 449 samples of the cecum of broilers and the cecum and the colon of pigs. These samples included 251 samples originating from broilers and 198 samples of pig cecums and colons. All the listed samples were obtained by scraping the surface of these parts of the digestive system of broilers and pigs. At the site of sampling, the diluted material was sown on a medium (Karmali agar), in order to get individual colonies. After sowing, the bases were placed in anaerobic jars in which microaerophilic conditions were achieved using Campy Pak, BBL bags. On arrival at the laboratory, the jars containing the sown bases were placed in a thermostat, at a temperature of 42oC for 48 hours for the purpose of incubation. Following incubation, the grown colonies were examined macroscopically, and then microscopic preparations were made from them, which were stained with 2% carbol fuchsin after drying and fixation. Those isolates which were in the form of a comma, the letter S, or gull's wings in the microscopic preparations were considered Campylobacter species (Figures 1 and 2). The isolated thermophilic campylobacteria were identified using conventional and commercial biochemical tests API Campy, manufactured by Bio M?rieux, France. With the application of these microbiological methods, thermophilic Campylobacter species were isolated from 203 (80.88%) of the 251 samples of broiler cecums. Furthermore, thermophilic campylobacteria were isolated from 153 (77.27%) of the 198 samples from the cecum and colon of pigs taken within these investigations. The obtained results indicate that there is a somewhat greater prevalence of these bacteria among the broilers. However, such a high percentage of both broilers and pigs colonized by thermophilic Campylobacter species could pose a serious problem, in particular when it is known that infections of humans caused by the consumption of insufficiently thermally processed meat of broilers or pigs are not infrequent.

Catalytic Features and Eradication Ability of Antibody Light-chain UA15-L against Helicobacter pylori

We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.

Efficacy of nitazoxanide against experimental cryptosporidiosis in goat neonates

Preliminary results obtained in mice, rats and piglets experimentally infected with Cryptosporidium sp have indicated a partial prophylactic or curative efficacy of nitazoxanide when administered between 50 and 250 mg/kg BW. In this study, the efficacy of nitazoxanide was evaluated in goat neonates experimentally infected with Cryptosporidium sp oocysts. Forty-seven 2- to 4-day-old kids were experimentally infected once on day 0 with 10(6) Cryptosporidium oocysts isolated from a dead kid, and allocated to three groups. Group 1 acted as control untreated group, group 2 received nitazoxanide for 8 days from day -1 to day 6 at a daily dose rate of 200 mg/kg BW, group 3 received nitazoxanide for 7 days from day 2 to day 8 at 100 mg/kg BW. Individual oocyst shedding was monitored by daily examination of faecal smears stained by carbol fuchsin and scored semi-quantitatively (zero to four). The other criteria included for nitazoxanide evaluation were weight gain and mortality. In the control group, oocyst shedding that started 3 days post-inoculation (PI) was maximal 6-7 days PI (mean scores ranging from 1.69 to 1.94) and became undetectable from day 16 PI. In group 2, oocyst shedding started 1 day later, peaked 9-11 days PI (1.33 to 1.5) and vanished day 18 PI. In group 3, results were similar to those of group 1 except for the mean scores ranging from 1.0 to 1.58. No significant difference was seen for weight gains between groups. Five kids died in group 1 as well as in group 3, whereas seven kids died in group 2. An acute toxicity of nitazoxanide was suspected as soon as the first 2 days of treatment.

An improved method for staining chromosomes of marine algae

AbstractAn improved staining method using carbol fuchsin was successfully adopted for chromosomes of marine algae, includingPorphyra,Undaria pinnatifidaandSaccharina japonica. The haploid numbers ofP. haitanensisandP. yezoensis(observed clearly in vegetative, spermatangial and conchosporangial cells) were five and three, respectively. The diploid numbers ofP. haitanensisandP. yezoensis(in immature conchosporangial cells) were ten and six, respectively. Pit connections between cells of conchosporangia and cell mashes were clearly differentiated. Chromosomes in conchocelis cells in prophase were clearly stained with the improved carbol fuchsin method. The chromosome number of gametophytes ofS. japonicawas 31, and that ofU. pinnatifidawas 30. Hydrolysis with 1 M HCl at 60°C for 7–8 min to produce a very transparent cytoplasm was necessary for staining chromosomes in conchosporangial branches ofPorphyra. Iron alum acetocarmine, aceto-iron-hematoxylin-chloral hydrate and carbol fuchsin were used to stain the chromosomes of the vegetative cells, spermatangial cells and conchosporangial cells ofPorphyraand the gametophytes ofU. pinnatifidaandS. japonica. Among the three methods, the carbol fuchsin method was simple and rapid, yielding deep staining of chromosomes and good contrast between the nucleus and cytoplasm.

Nuclear division of the vegetative cells, conchosporangial cells and conchospores of Porphyra yezoensis (Bangiales, Rhodophyta)

SUMMARYTo confirm the position and timing of meiosis in Porphyra yezoensis Ueda, the nuclear division of vegetative cells, conchosporangial cells and conchospores was observed. An improved staining method using modified carbol fuchsin was introduced to stain the chromosomes of Porphyra. Pit‐connections between conchosporangial cells also stained well with this method. Leptotene, zygotene, pachytene, diplotene, diakinesis, metaphase, anaphase and telophase were observed in the conchosporangial cells. During the germination of conchospores, no characteristics of meiosis I were found. No difference between the nuclear division of vegetative cells and that of conchospores was observed, and 2–3 days were needed for the first cell division both in vegetative cells and conchospores. Therefore, the cell division that occurs during conchospore germination is not meiosis I. Our results indicate that the prophase of meiosis I begins during the formation of conchosporangial branches, and metaphase I, anaphase I and telophase I take place during the maturation of conchosporangial branches. Then the three‐bivalent nucleate sporangia complete cell division to form two individual conchospores, each with one three‐univalent nucleus. The conchospores released from the sporangia are at meiotic interphase. Meiosis II occurs at the first nuclear division during conchospore germination, which is a possible explanation for the observation of mosaic thalli in mutant germlings of P. yezoensis. The mosaic thalli might also arise from gene conversion/post meiotic segregation events, comparable to those in Sordaria fimicola (Roberge ex Desm.) Ces. &amp; De Not. and Neurospora crassa Shear &amp; B.O. Dodge.