Hanging Drop Preparations Research Articles

ВОЗРАСТНЫЕ ОСОБЕННОСТИ ФОРМИРОВАНИЯ ВНЕКЛЕТОЧНЫХ ЛОВУШЕК НЕЙТРОФИЛОВ У ЗДОРОВЫХ ЛИЦ И БОЛЬНЫХ ТУБЕРКУЛЁЗОМ

Abstracts. Introduction. Forming a neutrophil extracellular trap (NET) from DNA (NETosis) is considered as a potentially significant marker in the diagnosis of tuberculosis. However, there is practically a lack of data regarding the age-related differences in forming extracellular traps in research publications. Aim. To determine the age-related differences of NETosis in healthy individuals and in tuberculosis (TB) patients. Materials and Methods. The ability of neutrophils to form extracellular traps was investigated in 4 groups: 1) 4-7-year-old children diagnosed with the primary tuberculosis complex, who fell ill within Omsk region; 2) healthy and uninfected children at the same age; 3) adults with tuberculosis, and 4) adults without tuberculosis. For this purpose, isolated fractions of neutrophils from peripheral blood of the groups under research were stimulated with a mixture of bacteria (probiotic) for 30 minutes. In the hanging-drop preparation, we analyzed the proportion of neutrophil extracellular traps and cells in the state of various motility degrees. Results and Discussion. Age-related differences in the ability of neutrophils to form extracellular traps were characterized by a more pronounced response of neutrophils to the NETosis stimulant in children in the control group in comparison with neutrophils isolated from blood in the group of healthy adult volunteers: Neutrophils of children (Me=16.1; Q1=12.8; Q3=18.3) were statistically significantly more intense (p=0.0002) than those of adults (Me=8.6; Q1=7.1; Q3=10.5) and formed filamentous NETs. In the groups of pediatric and adult pulmonary TB patients, the response of neutrophils to the NETosis stimulant was diametrically opposite. Neutrophils of children with primary tuberculosis complex, as of those in the control group, predominantly formed filamentous NETs, but in greater amounts, while neutrophils of adults with infiltrative or focal form and with at most 2 segments involved mainly formed cloudy NETs, whereas filamentous NETs prevailed for the adults in the control group after stimulation. At the same time, proportion of cloudy NETs did not exceed 5% in healthy children and healthy adults. Comparing the findings of our study with literature data, we hypothesized that, most likely, there is a greater increase in PAD4 expression and more pronounced histone H3 citrullination in children’s neutrophils than in those of adults. Conclusions. Our findings indicate more pronounced ex vivo processes induced by NETosis stimulator in both groups of children (healthy individuals and TB patients) as compared to adults in the respective groups, which should be considered by the researchers interpreting the findings of the ability of neutrophils to form extracellular traps in tuberculosis.

Flipped Well-Plate Hanging-Drop Technique for Growing Three-Dimensional Tumors.

Three-dimensional (3D) tumor culture techniques are gaining popularity as in vitro models of tumoral tissue analogues. Despite the widespread interest, need, and present-day effort, most of the 3D tumor culturing methodologies have not gone beyond the inventors’ laboratories. This, in turn, limits their applicability and standardization. In this study, we introduce a straightforward and user-friendly approach based on standard 96-well plates with basic amenities for growing 3D tumors in a scaffold-free/scaffold-based format. Hanging drop preparation can be easily employed by flipping a universal 96-well plate. The droplets of the medium generated by the well-plate flip (WPF) method can be easily modified to address various mechanisms and processes in cell biology, including cancer. To demonstrate the applicability and practicality of the conceived approach, we utilized human colorectal carcinoma cells (HCT116) to first show the generation of large scaffold-free 3D tumor spheroids over 1.5 mm in diameter in single-well plates. As a proof-of-concept, we also demonstrate matrix-assisted tumor culture techniques in advancing the broader use of 3D culture systems. The conceptualized WPF approach can be adapted for a range of applications in both basic and applied biological/engineering research.

A SEARCH FOR ASSOCIATION BETWEEN BIOFILM STRENGTH AND SITES OF INFECTION CAUSED BY ACINETOBACTER BAUMANNII COMPLEX

INTRODUCTION: In the world of infectious diseases, a group of bacteria known as ESKAPE pathogens, are responsible for most of the life threatening multidrug resistant (MDR) and extensively drug resistant (XDR) infections worldwide. The ESKAPE pathogens comprise of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. AIMS AND OBJECTIVES: The objective of this study is isolate and identify Acinetobactor baumannii complex from clinical samples. Measure the biolm forming capacity of the isolates. Possible association of this biolm strength with the type of clinical sample from which they are isolated. MATERIALS AND METHODS: The samples which were tested in the laboratory were the following: Sputum, End tracheal tube aspirates, Pus, Urine, Blood for culture, Central venous catheter tips & Cerebrospinal uid. Following culture media were taken: MaCconkey agar & Blood agar. The colonies suggestive of Acinetobacter baumannii were selected. On MaCconkey agar, non- pigmented NLF colonies with pink to light lavender hue; and on blood agar, opaque non-hemolytic colonies were obtained. Gram stains from selected colony showed gram negative coccobacilli. Hanging drop preparation showed non-motile bacteria. RESULTS AND ANALYSIS: We found maximum number of strong biolm producers were found among the isolates causing infection of CVC tips (70%). This was closely followed by ETtube isolates (69.5%). The isolates from blood stream, urine, and sputum samples showed all the three patterns, namely, no biolm producer, moderate biolm producer, and strong biolm producer. Our study showed interestingly, no strong biolm producer was found from pus samples, whereas from the CSF isolates no non-producer of biolm emerged. CONCLUSION: This study centers around strength or pattern of biolm formation by this bacteria. It was interesting to nd that there was a statistically signicant association between patterns of biolm produced by Acinetobacter baumannii and the various samples from which they are isolated. This made us to hint at a probable relation between biolm patterns and organotropism as an hypothesis. Only further investigations will tell if it can stand the test of time.

Utility of microbiological profile of symptomatic vaginal discharge in rural women of reproductive age group.

Symptomatic vaginal discharge is the most frequent symptom in women of reproductive age group. Owing to social stigma majority of affected women hesitate to seek medical consultation. Therefore the actual incidence of vaginal discharge is much more than what is reported. The aim of the study is to determine the microbiological profile of symptomatic vaginal discharge in rural area and its utility in the management of genital tract infection. This was a descriptive type of observational study, conducted in sexually active women of reproductive age group (18-45 years) attending the OPD/IPD of Obstetrics and Gynaecology Department of National Institute of Medical Sciences, Shobhanagar, Jaipur (Rajasthan), over a period of 18 months from June 2012 to December 2013. Hundred sexually active non pregnant women of reproductive age group (18-45 years) were included in the study. After taking consent general physical examination along with pelvic examination was performed. Two high vaginal swabs and blood sample were collected for various tests. Hanging drop preparation was immediately made. This was followed by gram staining and culture. Chlamydia trachomatis IgM antibody was detected by ELISA method. Out of 100 women with symptomatic vaginal discharge, specific diagnosis was obtained in 89% of cases whereas no specific aetiology was found in 11% cases. Mean age was 32.60 years. Fifty-three percent patient had Bacterial vaginosis, candidiasis was found in 14% cases, 16% had Chlamydia trachomatis infection while Trichomonas vaginalis infection was detected in 6% cases. Homogenous discharge was most prevalent (52%), followed by mucopurulant discharge in 23% of women. Patient with symptomatic vaginal discharge need to be actively managed with appropriate antimicrobial agents. Judicious management may be helpful in prevention of HIV, HPV, CIN and post infection sequelae.

Novel co-enrichment method for isolation of magnetotactic bacteria.

A novel co-enrichment technique was designed for enrichment of magnetotactic bacteria from soil, water, and sediments. Delayed addition of iron uptake inducer and the iron source proved amenable to induce magnetosome synthesis by MTB followed by their separation from consortium using magnetic flux. We successfully enriched and isolated both North seeking as well as South seeking magnetotactic bacteria from Lonar Lake (Buldhana), Moti Lake (Jalna), Ghanewadi Lake (Jalna), Ganesh Lake (Miraj), Rankala Lake (Kolhapur), and industrial metal-contaminated glaying soils (Jalna) and a soil (Karad), (MS, India) exposed to high-voltage electric current. The hanging drop preparations and growth under magnetic stress on low-agar media allowed conformation of magnetotactic behavior of the isolates. Both Gram positive and Gram negative MTB were isolated with diverse morphologies. South seeking population was more predominant. The soil inhabitants showed little dwelling property which was more prominent in case of aquatic inhabitants. The use of in situ pH and salt concentrations during enrichment and isolation found suited. The simultaneous growth of whole consortium in the system ensured the in situ simulation of microenvironment needful for proper growth of fastidious MTB.

English

Bacillus anthracis is the causative agent of Anthrax. The aim was to detect the presence of Bacillus anthracis in a case of suspected Cutaneous Anthrax in a 30 year old male who had history of handling a sick cow and noticed a painless ulcer on his palm 4 days later. Microbiological investigations revealed the presence of Bacillus anthracis. A diagnosis of Cutaneous Anthrax was made and the concerned authority was immediately notified. INTRODUCTION: The genus Bacillus is comprised of a large number of spore forming, Gram positive rods, few of which are pathogenic and a few useful for man. For e.g., Bacillus species are used in manufacture of antibiotics (B. subtilis), as indicators of sterilization procedure (G. stearothermophilus) etc. The most famous member is Bacillus anthracis, which is an organism of historical importance; being the first pathogenic bacterium to be observed under microscope, the first bacillus to be isolated in pure culture (Koch 1876) and the first bacterium used for the preparation of attenuated vaccine. It however became a household name after its use as a weapon of bio-terrorism. CASE REPORT: A 30 yrs old Muslim male from Laxmikantapur, Karimpur (a district town in West Bengal), gave history of handling a sick cow which eventually died. He consumed its meat on 18.04.2013. He noticed a painless ulcer developing on his right palm 4 days later. The lesion started discharging pus, gradually hardened and turned black (Eschar). No other symptom was reported by the patient and he had no significant medical or surgical history, including Diabetes and Hypertension. The swabs and smears from the lesion were sent to us by the CMOH of District hospital of Nadia, WB for the possible presence of Bacillus anthracis in them. As a precautionary measure all the procedures were performed in Bio safety Cabinet type 2 B with proper personal protection1. The direct smears were stained with Gram stain, Polychrome Methylene blue stain, India Ink and Ziehl-Neelsen stain. The swabs were inoculated in 5% Sheep Blood Agar, Nutrient Agar and Peptone water and incubated at 370C in O2 for 24 hrs. Colonies obtained were stained and hanging drop preparation for motility was done. Relevant biochemical tests and antibiotic sensitivity was also done. The results obtained were as per table 1.

English

INTRODUCTION- Sub clinical ascending infections through the lower female genital tract are predominant worldwide. Important morbidities related to poor perinatal outcome both for the mother and for the fetus and new born comprise preterm birth, prelabor rupture of membranes, post partum sepsis and maternal anaemia. In the fetus, sepsis and intrauterine growth retardation are suspected to be the consequences of ascending maternal infection. Both the direct effect of the infection and the maternal immune response contribute to these eventualities. This study was done to identify antenatal women with various genital infections and to know the outcome of pregnancy in presence of these infections. OBJECTIVES- Diagnosis treatment and preventive measures in genital infections in Antenatal period to improve the pregnancy out come . MATERIAL AND METHODS- One thousand women were studied for lower genital tract infection by ELISA KITS for chlamydia trachomatis, hanging drop preparation of vaginal discharge for trichomoniasis and mycelia of candida albicans can be seen by wet mount of vaginal dischage in 10% KOH. Gardnerella vaginal infection was diagnosed by Amsel's criteria . RESULT-out of thousand women 53% were positive for single or multiple infections (gp-1), 47% women had no infection (gp-11) . Labour outcome was studied in 415 antenatal women of gp -1 &395 antenatal women of gp -11 . In gp-1 51.80% had no effect ,7.71% had abortions and 40.48% had pre term labour and/or PROM .Which was significantly higher than in non infected group . IN INFECTED GROUP-41.92% neonates had no effect others had some effect in terms of LBW, birth asphyxia, neonatal death & IUD. Which was significantly higher than in non infected group. P=0.00 CONCLUSION- The study showed significantly higher incidence of obstetrical morbidities in women with lower genital tract infection,since genital infections are the root cause of these obstetrical morbidities so early detection and treatment of these infections is very important to

Infectivity of Bufo melanostictus (Amphibia: Bufonidae) to Two New Species of Haematozoan Parasites from Rohilkhand, India

Micro-haematotechniques (hanging drop preparations, microhaematocrit, permanent smears) on blood of Bufo melanostictus revealed 2.10 % prevalence of Trypanosoma bisalpurensis n. sp. (intensity 1.0–2.0/100 RBCs) and Hepatozoon gangwarii n. sp. (intensity 1.0–3.0/100 RBCs). The permanent preparations of the parasites were cyto-morpho-taxonomically compared with the previously described forms. The characteristic ‘S’-shaped body, the unusual close proximity of the nucleus and kinetoplast, the slender and elongated body and a shortened free flagellum of the Trypanosoma species and the differential size, nuclear position and dimensions, nuclear displacement ratio, absence of capsule and gametocyte characteristics of Hepatozoon are regarded as novel identity of the parasites and are being reported as new species with specific characters as provided in this communication. The displacement of the host nucleus is consequential due to Hepatozoon infection and is also described herein.

Cardio-myogenic differentiation of human amniotic fluid colony derived stem cells in the form of embryonic body-like structure

To investigate cardio-myogenic differentiation potential of human amniotic fluid colony derived stem cells (HAFCDSC) in the form of embryonic body (EB)-like structure in vitro. HAFCDSC were isolated from second trimester amniotic fluid which was backup of amniocentesis specimens. The forth passage of HAFCDSC were cultured by hanging-drop preparation in complete medium for 5 days to form EB-like structures followed by inducing medium in regular tissue culture dishes for 2 weeks (experiment group). The EB-like structures cultured in complete medium were served as control group. Cardiomyocyte phenotypes were detected by RT-PCR and immunofluorescence staining. HAFCDSC could form EB-like structures 24 h after hanging drop culture, and the diameter of EB-like structures gradually increased with culture duration and the mean diameter of EB-like structures reached 237.3 ± 26.7 µm on 5(th) day. The formation rate of EB-like structures was 93.5%. RT-PCR analysis showed EB-like structures expressed stem cell related mRNA. Immunofluorescence staining demonstrated that some cardiomyocyte phenotypes such as Desmin, α-Actinin and Tn I were expressed by HAFCDSCs in EB-like structures after cardio-myogenic induction. Cardiomyocytes specific mRNA including Desmin, α-Actin, Tn I, Tn T, T-box and NK2.5 were also transcripted as detected by RT-PCR. No positive results was found in control group. HAFCDSC can form uniform EB-like structures after hanging drop culture for 5 days. HAFCDSC differentiate into cardiomyocyte-like cells through the form of EB-like structures after induction by 5-Aza.

Analysis of ciliary beat frequency and ovum transport ability in the mouse oviduct

The oviduct is important in reproduction where fertilization occurs, and the fertilized eggs are conveyed to the uterus. Multi-ciliated cells of the oviductal epithelium and muscle contractions are believed to generate this unidirectional flow. Although there are many studies in human oviducts, there are few reports on mouse oviductal ciliary movements where we can dissect underlying genetic programs. To study ciliary movements in the mouse oviduct, we exposed the ovary-side of the oviduct (infundibulum) longitudinally and recorded the ciliary beatings in a hanging drop preparation. We calculated the ciliary beat frequency (CBF) by automated image analysis and found that the average CBF was 10.9 ± 3.3 and 8.5 ± 2.5 Hz (±standard deviation) during the diestrus and estrus stages, respectively. Mapping of the CBF to multiple locations in the epithelium showed that the cilia beat regularly at a local level, but have a range of frequencies within the entire plane. We also observed ova with cumulus cells were transported to the uterus side by the opened oviduct at the diestrus and estrus stages. These results suggest that the ciliated cells of the infundibulum can generate unidirectional flows and are able to deliver ova by their ciliary activities despite their discordance in beating periodicity.

A Hemogram of Clarias batrachus Parasitized by Two Species of Hemoflagellates (Trypanosoma batrachi Qadri, 1962 and Trypanoplasma haematalis n.sp.) as an Indicator of Infectivity

The blood of a freshwater catfish, Clarias batrachus Linn. was scanned for hemoflagellate infectivity by examination of fresh blood (hanging drop preparations and hematocrit) and stained smears. Trypanosoma batrachiQadri, 1962 occurring at a prevalence of 29.5% (intensity: 4 to 20 forms/20 min scan) is reported as a new locality record (Bareilly, Uttar Pradesh, India). A new species of monomorphic biflagellate was also found in 8.1% of the infected fish concurrently with Trypanosoma. Based on its morpho-cytostatistical features, flagellar length, and AF:PF ratio, the new species is named Trypanoplasma haematalis n.sp. A few selected hematological parameters (Hb%, ESR, PCV, and MCH) of infected fish were estimated to ascertain the validity as an indicator of flagellate infection under natural and experimentally-induced conditions, by inoculation of 3 ml of infected blood in laboratory-reared, healthy uninfected fish. In either case, the blood parameters recorded significant changes up to a maximum of 55.79% (Hb%, Group B3), 151.04% (ESR, Group B3), 65.45% (PCV, Group B3), and 106.26% (MCH, Group B3) suggesting that hemograms of fishes may be utilized as indicators of some infestation, which offers scope to corroborate it with flagellate infectivity.

Prevalence of cholera in pediatric patients with acute dehydrating diarrhea

To estimate the prevalence of culture-confirmed cholera in patients with acute dehydrating diarrhea, at a tertiary care center in north India, during a 6-month period from March to August, 2006. We studied 145 children, who presented to the pediatric emergency services of a tertiary care teaching hospital in north India with acute dehydrating diarrhea. Each patient had his/her stool sample collected for Vibrio cholerae culture and hanging drop preparation for darting motility. The stool specimen for hanging drop analysis was immediately transported to the emergency laboratory, where a trained technician prepared the slides and examined them for darting motility characteristic of Vibrio cholerae. V. cholerae was isolated in 36 (24.8%) patients. Forty-nine (33.7%) patients had a positive hanging drop examination. Hanging drop examination had a sensitivity and specificity of 85.8% and 81.7%, respectively. Severe dehydration (OR 4.3; P<0.01) and hanging drop positivity (OR 12.42; P<0.001) were associated with higher odds of cholera after adjustment for other risk factors. Cholera is an important cause of acute watery diarrhea in pediatric patients in urban north India and should be ruled out in all children presenting with acute dehydrating diarrhea, particularly those with severe dehydration. Hanging drop test is useful for diagnosis in the emergency setting.

Budding and direct division of adult human erythrocytes in serum-free cultures

This paper documents the budding and division process of human erythrocytes in vitro using time-lapse light microscopy of hanging-drop preparations. The erythrocytes were prepared from normal adult human peripheral blood. Red blood cells showed cytoplasmic budding, segmentation, and direct division with delayed addition of erythropoietin in serum-free Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham. These observations are thought to be useful for developing model of definitive erythropoiesis and simple expansion of human erythrocytes.

Learamoeba waccamawensis, N. G., N. Sp. (Heterolobosea: Vahlkampfiidae), a New Temperature‐Tolerant Cyst‐Forming Soil Amoeba

Soil from a roadside ditch at Lake Waccamaw, North Carolina, was tested for cyst‐forming free‐living amoebae, and water from the same ditch was tested for fecal coliform bacteria. Soil samples incubated at room temperature (21‐23° C) yielded Acanthamoeba polyphaga, Amoeba sp., Hyperamaeba sp., Mayorella penardi. Naegleria gruberi, Naegleria minor, and unidentified ciliates, testaceans and slime molds. Incubation at 37‐39° C yielded Acanthamoeba jacobsi, Platyamoeba schaefferi, and a new amoeba to be described herein. Fecal contamination of the ditch was confirmed by a direct membrane filtration technique that yielded a mean of 1,410 + 134 bacteria/ ml. The new amoeba is assigned to the class Heterolobosea Page &amp; Blanton, 1985 on the basis of promitotic nuclear division, and a flagellated stage with a cytostome as seen in the genus Tetramitus. Amoebae studied in hanging drop preparations were flattened and irregular as described for the genus Stachyamoeba in the family Gruberellidae but changed to a lobose cylindrical form as described for species of Vahlkampfia in the family Vahlkampfiidae. A new genus and species, Learamoeba waccamawensis, is proposed for the amoeba described herein.

The role of cell fusion in erythropoiesis — Part 1

The present haemopoietic pathways have been deduced from fixed material, and can be criticised on the grounds that they are an interpretation of a highly complex cytological picture and open to error. Furthermore, the pathways have not helped in the search for the haemopoietic stem cell, the initiating and most essential element of the pathways. Hence a different approach to the study of haemopoiesis may be useful, and in this regard Sabin's work is valuable. Sabin is one of the few investigators who has carried out a study of erythropoiesis on the living organism. She studied hanging drop preparations of explanted blastoderms of the chicken, and found that special embryonic cells called angioblasts fused to form well demarcated aggregates which were transformed into erythroblastic islands. In this paper, cell fusion leading to erythropoiesis has been further explored. Leishman stained smears of the blastoderm of the chicken, the splanchnic mesenchyme of the tadpole, the bone marrow of the juvenile chicken and the bone marrow of the juvenile frog were studied. Aggregates consisting of a set of nuclei enclosed by a common cytoplasm were found in all four tissues, and the origin and fate of the aggregates could be deciphered. The aggregates arose by a fusion of embryonic cells, and after the aggregate had attained a certain size, the cytoplasm underwent dissolution, denuding the nuclei. The bare nuclei, by manufacturing a haemoglobinised cytoplasm, were transformed into erythroblasts. The findings not only confirmed Sabin's observations on erythropoiesis in the chicken blastoderm but also showed that cell fusion was an integral part of erythropoiesis on a wider scale.

Eubacterium plautii (Seguin 1928) comb. nov.

The type strain (ATCC 29863 = VPI 0310) of Fusobacterium plautii has a multilayered cell envelope structure consisting of a barely visible cell membrane, a dense intermediate layer, and a single-layered outer membrane. The cells do not have flagella and are not motile in hanging drop preparations. The results of this and earlier investigations indicate that despite having a negative Gram reaction, this strain is a gram-positive bacterium with a gram-variable cell wall structure. Since the type strain of F. plautii fits the generic description of Eubacterium, the species is transferred to this genus as Eubacterium plautii (Seguin) comb. nov.

Effect of cyclic AMP and cytochalasin B on tissue cultured melanophores of Xenopus laevis.

AbstractThe effects of cytochalasin B or low concentrations of adenosine 3′,5′‐monophosphate (cyclic AMP) were tested on melanophores in hanging drop preparations of neural fold explants from Xenopus laevis embryos in Barths' solution. After one week in culture, the melanophores were punctate in this medium. Cyclic AMP at 5 mM consistently caused reversible morphological transformation of these cells to the stellate state, whether they were situated within an epithelial outgrowth or isolated on the surface of the coverglass. Only the isolated melanophores consistently responded to 1 mM cyclic AMP. Cytochalasin B at 1–10 μg/ml caused aggregation of melanin granules in stellate cells, but left long, narrow cell branches containing some melanosomes. Its effect was at least partially reversible and appeared to be dose dependent. At 1% concentration, dimethyl sulfoxide caused melanin dispersion.

Genetic and developmental characteristics of a new color variant, axanthic, in the Mexican axolotl, Ambystoma mexicanum shaw

A new genetic trait, axanthic, is described in the Mexican axolotl with a phenotype completely lacking visible xanthophores and iridophores. Animals received from the colony of Dr. L. E. DeLanney at Ithaca, New York, have been mated among themselves for a total of seven spawnings, in which the axanthic trait was inherited as a simple Mendelian recessive. The symbol for this newly found gene is ax. Homozygous recessive animals appear normal except for their lack of visible xanthophores and iridophores. The results of the spawnings also indicate that the axanthic gene is not linked to the melanoid gene ( m). Its linkage with other known mutants in the axolotl is being tested. Hanging-drop preparations in Niu-Twitty's solution of neural fold explants were made using axanthic and normal embryos. Xanthogenesis was never visible in cultures from axanthic animals, whereas all those made from yellow animals had fully differentiated xanthophores within 9 days after the explants were made. Counts of cells in cultures from axanthic animals showed a complement of axanthic xanthoblasts, that is, otherwise normal xanthophores without pigment. Epidermal vesicles were made by combining flank somatopleure from early tailbud embryos with neural fold from neurulae. Axanthic somatopleure was combined with axanthic and xanthic neural fold (in separate vesicles) and xanthic somatopleure combined with folds from axanthic embryos. Only the vesicles with neural fold from xanthic donors had visible xanthophores. Homotopic reciprocal neural crest transplants were made between axanthic and yellow larvae at midtailbud stages. The grafted regions were obvious after pigment synthesis had begun. Xanthophores were visible in these regions whenever the donor material was derived from a yellow larva. The graft region was devoid of visible xanthophores whenever the donor material was axanthic neural crest, even when the crest was transplanted onto xanthic animals. Chromatographic analyses of the pteridine contents in axanthic as compared to normal animals showed that the axanthic animals were lacking five fluorescent spots found in normal larvae. Two of these spots from normal animals had yellow fluorescence; three had blue fluorescence. The three blue fluorescent compounds were tentatively identified as Ranachrome-3, Hynobius-blue, and 2-amino-4-hydroxypteridine-6-car☐ylic acid. One yellow spot appeared to be sepiapterin. The other could not be identified. The chromatographic results indicate that the axanthic mutant is deficient in these pteridine compounds, which are evidently necessary for the pigmentation of xanthophores. The results from the experimental work offer support to the hypothesis that the axanthic trait, inherited as a simple Mendelian recessive, acts at the level of the xanthoblasts to prevent the accumulation of yellow pigments in these cells. As there are no visible iridophores in these mutants either, it may also prevent the accumulation of pigmentary purines in iridoblasts.

The effect of nerve growth factor on dispersed neuronal-Hela heterokaryons

Dorsal root ganglia from 7-day chick embryos were minced and slowly trypsinized to produce a suspension of cells. This suspension was centrifuged in an albumin density gradient and the fraction enriched with neuronal perikarya collected. This fraction was combined with an equivalent number of HeLa cells. The two cell types are easily identifiable with interference microscopy. The two cell types were fused by the addition of β-propiolactone-inactivated Sendai virus. The resultant heterokaryons were cultured in modified hanging drop preparations in the presence of nerve growth factor and supplemented medium. Nerve growth factor stimulated the growth of long processes from the neuronal-HeLa heterokaryons when added to both long- and short-term cultures thereby demonstrating retention by the heterokaryon of a differentiated property of the neuron. We conclude that differentiated properties can be retained in somatic cell hybrids.

Ultrastructural organization of spheroplasts induced in Mycobacterium sp. smegmatis by lysozyme or glycine.

The conversion of Mycobacterium sp. smegmatis cells into spheroplasts was achieved at a high rate by their treatment with either lysozyme (100 μg/ml) or glycine (2%) in a liquid Dubos medium without albumine, stabilized by sucrose (up to 0.34 M). The dynamic of conversion was followed in hanging-drop preparations.