Laboratory culture of Chironomus tentans for use in toxicity testing: optimum initial egg-stocking densities

The midge Chironomus tentans Fabricius is a commonly used freshwater invertebrate in sediment toxicity tests. Rigorous laboratory culturing techniques are needed to provide organisms of uniform quality and known age for use in testing and for the continuation of the culture itself. This study was conducted to determine the effect of initial culture stocking density on: (1) post-hatch (larval) dry weight, body length and head-capsule width at 10 and 20 days; (2) time to emergence; (3) number and sex of emergent adults; (4) number of larvae and pupae at test termination (day 42 post hatch); and (5) adult dry weight. Three egg stocking densities were used 690 (1.1 eggs cm^–2), 1043 (1.7 eggs cm^–2) and 1463 (2.4 eggs cm^–2). Mean weight of larvae at 10 days in high density tanks (0.13 mg/organism) was significantly higher (P=0.003) than both the medium and low density tanks (0.10 and 0.09 mg/organism, respectively). No significant differences between the three stocking densities were observed for the body length or head-capsule width at either 10 or 20 days post-hatch. Although not statistically significant, larval dry weight decreased with increased stocking density at day 20. A significantly (P=0.02) greater number of females (173±28) emerged from the low stocking density compared to both the medium and high stocking densities (123±45 and 118±54, respectively). Peak adult emergence for the low and medium stocking densities occurred between days 22 and 25 post-hatch, whereas peak adult emergence occurred between days 30 and 33 for the high stocking density. Survival relative to the initial number of eggs stocked was significantly greater (P=0.007) in the low density treatment compared to that in either the medium or the high density treatments. Mean adult weight exhibited an inverse relationship with initial stocking densities. At test end, there was not a significant difference in the mean number of organisms surviving and emerging in the three density levels. The central tendency for number of organisms surviving for all three treatments was 504 organisms per tank (0.82 organisms cm^–2). The results of this experiment suggest that an optimal egg stocking density of 1.0 egg cm^–2 (600 eggs/tank) be used with the feeding rate identified. This would ensure uniform larvae at the appropriate developmental stage (2nd–3rd instar) needed for toxicological research/testing (e.g. 10 days post-hatch), as well as producing sufficient emergence of males and females for future culture establishment.     

RF radiation-induced changes in the prenatal development of mice

The possible effects of radiofrequency (RF) radiation on prenatal development has been investigated in mice. This study consisted of RF level measurements and in vivo experiments at several places around an “antenna park.” At these locations RF power densities between 168 nW/cm² and 1053 nW/cm² were measured. Twelve pairs of mice, divided in two groups, were placed in locations of different power densities and were repeatedly mated five times. One hundred eighteen newborns were collected. They were measured, weighed, and examined macro- and microscopically. A progressive decrease in the number of newborns per dam was observed, which ended in irreversible infertility. The prenatal development of the newborns, however, evaluated by the crown-rump length, the body weight, and the number of the lumbar, sacral, and coccygeal vertebrae, was improved. Bioelectromagnetics 18:455–461, 1997. © 1997 Wiley-Liss, Inc.     

Electrical and Mechanical Responses in Deep Abdominal Extensor Muscles of Crayfish and Lobster

Electrical and mechanical studies have been made of the deep abdominal extensor muscles, medial (DEAM) and lateral (DEAL), of crayfish and lobster. The medial muscle responds to direct (intracellular) and indirect stimulation with a transient membrane depolarization which exhibits the properties of a propagated non-decremental action potential but does not overshoot the zero level. The amplitude is about 30 mv in crayfish and 50 mv in lobster. It is followed by a fast all-or-none twitch whose duration at 20°C is 30 to 50 msec. and whose developed tension is 500 gm/cm² or about half the tetanic value. Membrane potential is K⁺-dependent and immersion in high K⁺ induces a brief transient tension rise as in other twitch-type muscles. The action potential and twitch are normal even if all external Na⁺ is replaced with sucrose but vary with external Ca⁺⁺, the action potential increasing 8 to 10 mv for a twofold increase in Ca⁺⁺. The lateral muscle (DEAL) is much slower and responds to intracellular stimulation only with an electrotonic or a local response. Mechanical responses and relaxation speeds are slow with minimal duration of contraction of 0.5 to 2 seconds. Immersion in high K solutions induces large maintained tensions. Sarcomere length in the fast DEAM is uniform and about 2 µ at rest, but in the DEAL speed is less and sarcomere length is greater averaging about 4.5 µ but with a mixed population of fibers.     

The Effect of Ultraviolet Radiation on the Germination of Nosema algerae Vávra and Undeen (Microsporida: Nosematidae) Spores1

Spores of Nosema algerae Vávra and Undeen were subjected to various dosages of 254 nm ultraviolet radiation (UV). Very high dosages of UV were required to block germination. Germination was normal immediately after UV dosages of 0.2 to 1.0 J/cm², followed by a delayed effect in which both percentage germination and the intrasporal concentration of trehalose decreased with time after UV exposure. Although a few spores were germinated, most of them were inactivated (rendered temporarily unable to germinate) by exposure to UV of 1.1 J/cm². Ultraviolet radiation between 1.1 and 3.4 J/cm² stimulated spores to germinate. However, spores were completely unable to germinate immediately after exposure to dosages above 3.8 J/cm². Ammonia had little effect on stimulation by UV but was inhibitory to germination after stimulation had occurred. These results demonstrate that UV behaves like a germination stimulus and are discussed in terms of the hypothesis that germination is initiated by the breakdown of barriers between trehalose and trehalase.     

Electrical Properties and Acetylcholine Sensitivity of Singly and Multiply Innervated Avian Muscle Fibers

Membrane constants and distribution of acetylcholine (ACh) receptors were determined for multiply innervated fibers of the anterior latissimus dorsi (ALD) and singly innervated fibers of the posterior latissimus dorsi (PLD) muscles of 3–6 month old chickens. The values of the various membrane constants were: length constant, 1.78 mm (mean) in ALD, 0.68 mm in PLD; time constant, 35 msec in ALD, 3.7 msec in PLD; transverse membrane resistance, 4388 Ω cm² in ALD, 561 Ω cm² in PLD; and membrane capacitance, 8.2 µF/cm² in ALD, 7.0 µF/cm² in PLD. Peaks of ACh sensitivity occurred at intervals of ca. 740 µ on ALD fibers with a low sensitivity remaining between peaks. Only one peak of ACh sensitivity was detected on PLD fibers. The maximum ACh sensitivity found was 5 ± 4 mv/ncoul for fibers of the ALD and 77 ± 60 mv/ncoul for fibers of the PLD. The distance over which this sensitivity fell to 0.1 was ca. 225 µ in the ALD and 140 µ in the PLD. The membranes of these two muscle fiber types differ widely regarding some electrical properties and the disposition of ACh-sensitive receptor sites.     

In vitro culture of tobacco protoplasts: Use of feeder techniques to support division of cells plated at low densities

Summary Tobacco protoplasts obtained from leaf mesophyll cells and suspended in agar nutrient medium divided and produced colonies only when plated at high densities, above 10⁴ cells per ml. At such densities coalescence of the expanding colonies occurred at high frequency. Nondividing X-irradiated protoplasts used as feeder cells supported division of viable protoplasts plated at densities as low as 10² cells per ml. The feeder cell technique should thus facilitate the application of screening procedures for the isolation of colonies originating from single mutated cells occurring in a suspended population.     

Protein-free medium for C-1300 mouse neuroblastoma cells

Summary An optimized medium, designated MCDB 411, has been developed for mouse neuroblastoma cells. At cell densities of 1×10⁴ cells/cm² or greater, several different clones of C1300 mouse neuroblastoma cells can be cultured serially in Medium MCDB 411 with no serum or other undefined supplementation and with a doubling time of about 24 h. At clonal densities it is necessary to supplement the medium with 1.0 μg/ml insulin. Alternately, good clonal growth can be obtained without direct supplementation by coating the culture dishes with insulin and rinsing off all that is not tightly bound. Primary cultures of cells from serially transplanted C1300 tumors that have never been cultured previously in vitro can be established directly in unsupplemented Medium MCBD 411 with rapid initiation of multiplication and no apparent crisis or selection for minority cell types. The availability of a synthetic medium that supports growth of neuroblastoma cells without supplementation should facilitate the use of these cells as model systems for the study of neuronal function and differentiation. This work was supported by Grant CA-15305 from the National Cancer Institute.     

Contraction of protoplasm. I. Cinematographic analysis of the anodally stimulated contraction of Spirostomum ambiguum

Electrically stimulated contraction of Spirostomum ambiguum was investigated by high speed cinematography (up to 6,000 pps). Contraction is completed in about 4 msec following a latent period of up to 30 msec. Reduction in length during contraction followed a sigmoid curve, and final length was about 50% of the original length. Contraction always started at the end of the animal directed towards the anode. When the length of each half was measured separately, it was found that the cathodal end lagged about 1 msec in all cases observed. Rate of contraction was increased when the external calcium contraction was increased, and was decreased in Ca-free and K-free solutions, but was unchanged in K-rich solutions. These results are interpreted in terms of contraction being associated with a relative increase of calcium bound to the contractile protein. The differential migration of potassium and calcium ions in an electric current would result in a temporary lowering of K⁺ at the anodal end of the animal, hence a relative rise would take place in the Ca⁺⁺ available for binding. The results of experiments using changed calcium and potassium concentrations can be explained by this hypothesis which is in general agreement with modern work on muscle contraction and relaxation.     

Cloning and characterization of endosymbiotic algae isolated fromParamecium bursaria

Summary The green parameciumParamecium bursaria has many endosymbiotic algae in its cytoplasm. Here, we cloned and characterized endosymbiotic algae fromP. bursaria and examined in detail the interaction between the cloned algae and algae-free paramecia. Homogenates ofP. bursaria were cultured on agar plates containing various kinds of media to establish clones of the endosymbiotic algae. Many algal colonies were obtained from poorly nutritious medium (CA medium) after one month in culture. Algae were picked up from these colonies and inoculations were repeated 9 times on agar plates containing CA medium. On enriched media including bacto-peptone, glucose, proteose-peptone and/or yeast extract, however, bacteria and mold grew rapidly and no algal colonies were formed. When the cloned algae were cultured in liquid CA medium, they grew faster than on agar plates and the numbers stayed constant at 1 × 10⁷ algae/ml after 7 days in culture. They revealed high infectivity to algae-free paramecia, and an incubation period of 24 h and at least 1 × 10³ algae/paramecium were required to achieve successful infection (80–90%). The growth and infection rate did not change through 74 repeated inoculations of algae in liquid CA medium. Optical microscopic observations revealed marked morphological similarity between endosymbiotic algae and free-livingChlorella, but the latter showed no infectivity to algae-free paramecia. The cloned endosymbiotic algae presented here will provide an excellent opportunity to examine the mechanism of symbiont-host interaction.     

In vitro study of microwave effects on calcium efflux in rat brain tissue

In this study we investigated the prospect of microwave-induced alteration of ⁴⁵Ca²⁺ efflux from rat neural tissue at low pulse repetition frequencies and low power densities under in vitro conditions. Rat cerebral tissue, preloaded with ⁴⁵Ca²⁺, was exposed to pulsed-microwave radiation (1-GHz carrier frequency) according to one of several PRF-power density exposure schemes: 16 Hz at 0.5, 1.0, 2.0, or 15 mW/cm², or 32 Hz at 1.0 or 2.0 mW/cm² average power density. Measurements of radioactivity in the efflux medium and in the tissue sample were used to calculate an efflux value for each sample. The results indicate that the radiation conditions used did not alter calcium efflux in rat brain tissue.     

Differential Modulation of Corticospinal Excitability by Different Current Densities of Anodal Transcranial Direct Current Stimulation

Background

Novel non-invasive brain stimulation techniques such as transcranial direct current stimulation (tDCS) have been developed in recent years. TDCS-induced corticospinal excitability changes depend on two important factors current intensity and stimulation duration. Despite clinical success with existing tDCS parameters, optimal protocols are still not entirely set.

Objective/hypothesis

The current study aimed to investigate the effects of four different anodal tDCS (a-tDCS) current densities on corticospinal excitability.

Methods

Four current intensities of 0.3, 0.7, 1.4 and 2 mA resulting in current densities (CDs) of 0.013, 0.029, 0.058 and 0.083 mA/cm² were applied on twelve right-handed (mean age 34.5±10.32 yrs) healthy individuals in different sessions at least 48 hours apart. a-tDCS was applied continuously for 10 minute, with constant active and reference electrode sizes of 24 and 35 cm² respectively. The corticospinal excitability of the extensor carpi radialis muscle (ECR) was measured before and immediately after the intervention and at 10, 20 and 30 minutes thereafter.

Results

Post hoc comparisons showed significant differences in corticospinal excitability changes for CDs of 0.013 mA/cm² and 0.029 mA/cm² (P = 0.003). There were no significant differences between excitability changes for the 0.013 mA/cm² and 0.058 mA/cm² (P = 0.080) or 0.013 mA/cm² and 0.083 mA/cm² (P = 0.484) conditions.

Conclusion

This study found that a-tDCS with a current density of 0.013 mA/cm² induces significantly larger corticospinal excitability changes than CDs of 0.029 mA/cm². The implication is that might help to avoid applying unwanted amount of current to the cortical areas.     

Efficiency in thin-film liquid system for <Emphasis Type="Italic">Hosta</Emphasis> micropropagation

Three varieties of Hosta (Striptease, Minuteman and Stiletto) at four densities (40, 80, 120 and 200 explants per litre) were micropropagated on semi-solid agar and a thin-film liquid system with intermittent wetting of plant tissue. The mechanics of wetting by a small wave front required a larger rectangular vessel (11 × 27=297 cm ²) compared to the common cylindrical baby food jar (18 cm ²). Plants multiplied more rapidly in the agitated thin-film system than on agar. Lower plant densities increased rates of multiplication in liquid, but had little or no effect on multiplication rate on agar. Increasing plant density lowered the overall multiplication rate, but yielded greater numbers of plants per vessel. Yield, tabulated for utilization of shelf-space in growth room, was greater at all densities in rectangular vessels of liquid than conventional jars of agar media. Increased plant density lowered the sugar residual in media following the culture cycle and liquid media had less residual sugar than agar media. A liquid medium with 50 g l^–1 sucrose was concentrated enough so that sugar depletion did not limit growth, even at the highest densities. The liquid system allows the technician to skip the step of manually spacing and orienting the freshly cut bud tissue at the transfer station. Harvesting 75–100 plants per vessel from the large rectangular vessels resulted in most efficient use of technician time. Plants from liquid and agar acclimatized to greenhouse. Increased multiplication, space utilization, sugar availability and worker efficiency was demonstrated to be greater in thin-film liquid than more conventional agar-based system.     

The meiobenthos of five mangrove vegetation types in Gazi Bay,Kenya

The vertical distribution of meiofauna in the sediments ofAvicennia marina,Bruguiera gymnorrhiza,Ceriops tagal,Rhizophora mucronata andSonneratia alba at Gazi Bay (Kenya), is described. Seventeen taxa were observed, with highest densities in the sediments ofBruguiera (6707 ind. 10 cm^–2), followed byRhizophora (3998 ind. 10 cm^–2),Avicennia (3442 ind. 10 cm^–2),Sonneratia (2889 ind. 10 cm^–2) andCeriops (1976 ind. 10 cm^–2). Nematodes accounted for up to 95% of total densities; other common taxa were copepods, turbellarians, oligochaetes, polychaetes, ostracods and rotifers. High densities occurred to about 20 cm depth in the sediment. EspeciallyCeriops sediments show still high densities of nematodes (342 ind. 10 cm^–2) and copepods (11 ind. 10 cm^–2) in the deepest layer (15–22 cm). Particle size and oxygen conditions were major factors influencing meiobenthic distribution;Uca burrows had a major impact on distribution and abundance of meiofauna.     

Effects of Microwave Exposure at Various Power Densities on Mitochondrial Marker Enzymes in Mouse Brains

Four groups of C₅₇BL mice were irradiated with 3 GHz pulse (PW) microwaves for 3 hours at incident power densities of 0.1, 0.5, 1 and 5 mW/cm² respectively. The amount of mitochondria1 marker enzymes succinate dehydrogenase (SDH) and monoamine oxidase (MAO) in the hypothalamus and hippocampus were determined by microspectrophotometry. SDH and MA0 in the irradiated groups (except 0.1 mW/cm²) were significantly lower compared to the control group (p < 0.01). The lowest level occurred in the 5 mW/cm² group. The threshold level was 0.5 mW/cm². To compare the effects of PW with continuous wave (CW) exposure, two experimental groups were exosed to 2.45 GHz, using CW; the enzymes were decreased only in the 5 mW/cm² group. The results show that PW radiation is more effective then CW radiation in decreasing SDH and MA0 levels.     

Morphological transformation of C3H/10T1/2 cells subcultured at low cell densities

Morphological transformation in $\text{C3H/10T}\text{1}\text{2}$ cells treated with varied concentrations of benzo (α) pyrene (BP) was measured following subculture at low cell densities. Subconfluent cultures exposed to BP were allowed to grow to confluence, trypsinized, and reseeded at cell densities ranging from 5 to 2,300 surviving cells/cm². These secondary cultures were incubated for 8 to 9 weeks, stained, and examined for evidence of morphological transformation. BP-treated cells reseeded in virtual isolation in microwells (approx. 5 surviving cells/cm²) transformed at frequencies up to 14.5%. At these low initial cell densities, transformation frequency did not demonstrate a significant dependence on BP concentration. However, BP-treated cells reseeded at higher densities (11 to 2,300 surviving cells/cm²) showed both density-dependent transformation frequencies and BP-concentration dependence of transformation. As reported previously (Haber et al., Cancer Res. 37 1644, 1977), the subculturing of treated cells did not affect the BP-concentration dependence of focus formation in the $\text{C3H/10T}\text{1}\text{2}$ transformation assay. Cell density-dependent suppression of morphological transformation has now been observed over a wide range of BP concentration. We suggest that this phenomenon is associated with colony interactions and consider various possible mechanisms of BP involvement.     

The in vivo effects of 2.45 GHz microwave radiation on rabbit serum components and sleeping times

Summary An investigation was conducted to determine the effects of relatively low power density microwave exposures on various serum components of the Dutch rabbit. Both continuous wave and pulsed mode exposures at 2.45 GHz were used at power densities of 25, 10 and 5 mW/cm². Studies of 10 serum components were performed. Additional studies were conducted on changes in sleeping times of pentobarbital-sedated rabbits at various power densities. Gross and histopathological examinations were performed on representative samples of animals.Changes in the blood chemistry of irradiated animals were consistent with a dose-dependent response to a non-specific thermal stress at all power densities used. Observed physiological response, as well as rectal temperature measurements, indicated that the thermoregulatory capability of the rabbits was sufficient to compensate for the thermal burden at 5 and 10 mW/cm², but could be overridden by a 2 h exposure at 25 mW/cm². Pathology findings included a mild, repairable nephrosis in animals exposed at a power density of 25 mW/cm².A further investigation of analeptic effects at power densities varying from 5 mW/cm² to 50 mW/cm² resulted in a statistically significant decrease in sleeping times, apparently proportional to power density below 15 mW/cm².This research was partially supported by the US Army Medical Research and Development Command, Contract No. DADA17-72-C-2144. (The views expressed are those of the authors and do not necessarily reflect those of the Department of the Army)     

Response to cadmium of Daucus carota hairy roots dual cultures with Glomus intraradices or Gigaspora margarita

Ri T-DNA-transformed carrot roots were cultivated in two experiments either non-inoculated or inoculated with the arbuscular mycorrhizal (AM) fungi Glomus intraradices or Gigaspora margarita. The influence of two concentrations of cadmium (Cd) in the medium (2 mg l^–1, 4 mg l^–1) on both root and mycelium growth was tested. Both parameters were estimated at 10-day intervals for 70 or 100 days for G. intraradices and Gi. margarita, respectively. In the first experiment, G. intraradices showed a rapid spread of extraradical mycelium (ERM) and reached average densities per treatment of about 90 cm cm^–2 agar medium after 70 days. At the higher Cd level, the growth of ERM was delayed in comparison to the treatment without Cd addition. Root growth was inhibited by both Cd levels; the inhibition was, however, significantly lower in the treatments inoculated with G. intraradices compared to the non-inoculated control. In the second experiment, the ERM of Gi. margarita started to grow after a period of 50 days and reached average densities per treatment of only up to 27 cm cm^–2 by the end of the cultivation. The growth of Gi. margarita mycelium was not inhibited by Cd. No differences in root growth were observed between the Gi. margarita inoculated and non-inoculated treatments. The inhibitory effect of Cd on root growth differed between the non-inoculated treatments in both experiments. The study has shown that the AM fungus Glomus intraradices can alleviate Cd-induced growth inhibition to carrot hairy roots. The potential and limits of the monoxenic system in studying the interaction between AM fungi and heavy metals are discussed.     

Spatial variation in density, mean size and physiological condition of the holarctic amphipod Diporeia spp. in Lake Michigan

• 1 We examined spatial patterns in population characteristics (density, biomass, mean body length) and physiological condition (lipid content, length‐weight) of the amphipod Diporeia spp. in Lake Michigan by collecting samples at up to 85 sites in late summer 1994 and 1995. Variables were examined relative to water depth and three lake regions: south, central and north. Most major river systems are found in the south, and this region is more nutrient‐enriched compared to the north.
• 2 Over all sites, mean density was 5240^‐2, biomass was 4.1g dry wt m^‐2, and mean body length was 5 mm. While maximum densities were related to depth, with a peak at 30–70 m, greatest densities occurred on the west side of the lake, and low densities were found in the south‐east, north‐east and lower Green Bay. High densities in the west probably resulted from upwelling, and reduced densities in the south‐east may reflect food competition with Dreissena polymorpha (zebra mussel).
• 3 Lipid content, weight per unit length, and mean length declined with increased water depth, but depth‐related trends were most evident in the south. Overall, mean lipid content and weight per unit length were significantly lower in the south (16.6% dry wt, 0.59 mg at 5 mm body length) compared to the north (23.7% dry wt, 0.78 mg at 5 mm body length). These regional differences may have resulted from greater diatom availability in the north and competition from D. polymorpha in the south. Triacylglycerols and phospholipids were the dominant lipid classes in all three regions. Although the mean proportion of triacylglycerols, the energy‐storage lipid, was lower in the south than in the north, regional differences in proportions of lipid classes were not significant.
• 4 Mean lipid content and weight per unit length of Diporeia in the south were lower than values found in the late 1980s prior to the establishment of Dreissena. Mean lipid content of mature individuals is now at levels considered a minimum for successful reproduction.

     

Microwave radiation (2450 MHz) alters the endotoxin-induced hypothermic response of rats

The parenteral administration of bacterial endotoxin to rats causes a hypothermia that is maximal after approximately 90 minutes. When endotoxin-injected rats were held in a controlled environment at 22°C and 50% relative humidity and exposed for 90 minutes to microwaves (2450 MHz, CW) at 1 mW/cm², significant increases were observed in body temperature compared with endotoxintreated, sham-irradiated rats. The magnitude of the response was related to power density (10 mW/cm² > 5 mW/cm² > 1 mW/cm²). Saline-injected rats exposed for 90 minutes at 5 mW/cm² (specific absorption rate approximately 1.0 mW/g) showed no significant increase in body temperature compared with saline-injected, sham-irradiated rats. The hypothermia induced by endotoxin in rats was also found to be affected by ambient temperature alone. Increases in ambient temperature above 22°C in the absence of microwaves caused a concomitant increase in body temperature. This study reveals that subtle microwave heating is detectable in endotoxin-treated rats that have an impaired thermoregulatory capability. These results indicate that the interpretation of microwave-induced biological effects observed in animals at comparable rates and levels of energy absorption should include a consideration of the thermogenic potential of microwaves.     

Feeder cell density—A key parameter in human embryonic stem cell culture

Summary A key issue in human embryonic stem (ES) cell culture that has largely been ignored is the high degree of variability in the murine embryonic fibroblast (MEF) feeder cell density, which has been reported by different studies and protocols. Presumably, too low a feeder cell density would result in insufficient levels of secreted factors, extracellular matrix, and cellular contacts provided by the feeder cells for the maintenance of human ES cells in the undifferentiated state. Too high a feeder cell density, on the other hand, may result in a more rapid depletion of nutrients and oxygen within the in vitro culture milieu, as well as physically hinder the attachment and growth of ES colonies during serial passaging. Preliminary investigations by our group revealed that an elevated MEF cell density of 32,000 cells/cm², above the recommended value of 20,000 cells/cm², appeared to be highly detrimental to the attachment and growth of serially passaged ES colonies of the H9 line (WiCell Research Institute Inc., Wilmington, MA, USA). At the edge of ES colonies that have attached to the higher density feeder layer (32,000 cells/cm²), the ES cells appear to stack up to form a “bulge.” This was not observed under the recommended feeder cell density of 20,000 cells/cm². By contrast, other established ES cell lines are routinely propagated at much higher feeder densities of 60,000 to 70,000 cells/cm². This report briefly discusses the issue of MEF feeder cell density in relation to our preliminary observations, and the results of other studies.