Effects of nutrient media,different cytokinin types and their concentrations on in vitro multiplication of G × N15 (hybrid of almond × peach) vegetative rootstock

The objective of this research was to assess the effects of different media i.e. Murashige and Skoog (MS) and Quoirin and Lepoivre (QL), cytokinin type i.e. 6-Benzyladenin (BA) and 6-Benzylaminopurine (BAP) and cytokinin concentration on in vitro proliferation of the G × N15 rootstock. To evaluate the effects of different media and cytokinin type, two separate experiments were conducted as factorial based on completely randomized design, and single nodes were used as explants. The results showed that MS nutrient medium was found to be superior to QL nutrient medium. Regarding the interaction between media and growth regulators, the best interaction was found in MS medium supplemented with 1 mg l⁻¹ BAP resulting in 8.5 new micro shoots/explant while 7.75 shoots were observed in MS medium containing 1.25 mg l⁻¹ BA. The longest length of new micro-shoots (2.10 cm) was obtained in hormone-free MS medium. Findings of this study showed that there is a significant correlation between the hormone level and plantlet height and formed callus weight so that an increase in BAP and BA levels in both of MS and QL media resulted significantly in height decrease and callus weight increase. The results also suggest that the best and the worst plantlets in terms of quality were observed in hormone-free QL medium and MS medium supplemented with 1.25 mg l⁻¹, respectively. These results reflect the fact that the presence of high amounts of NH₄NO₃ and cytokinin especially BAP in culture medium triggered inhibitory effect on shoot growth.     

In vitro cultures of Silybum marianum and silymarin accumulation

In this study, a protocol for initiation of callus and shoot cultures from leaves and shoot tips explants of different silybium genotypes collected from different locations in Egypt was established. Callus cultures were initiated from leaves explants and exposed to different concentrations of the precursor (coniferyl alcohol). Shoot cultures were initiated from shoot tips explants. Moreover, the produced plants of the different Silybium shoots as well as intact plants were subjected to protein screening using SDS–PAGE analysis.Results obtained revealed that the optimum medium for growth and maintenance of friable callus was MS medium supplemented with 0.25 mg L⁻¹ 2,4-Dichlorophenoxy acetic acid (2,4-D) + 0.25 mg L⁻¹ Kinetin (Kin). The best medium for proliferation of high number of shoots was MS-medium with 0.25 mg L⁻¹ each of Benzyl Adinine (BA) and Naphthalene Acetic Acid (NAA). Coniferyl alcohol in concentration of 30 μM caused an increase in accumulation of silymarin contents in most callus cultures. SDS–PAGE of different Silybium shoots revealed that the protein profiles of 100% of in vitro produced plantlets similar to their control.     

Direct and residual effects of nitrogen fertilization, foliar application of potassium and plant growth retardant on Egyptian cotton growth, seed yield, seed viability and seedling vigor

In order to obtain high productivity for a cotton crop, one of the major requirements is to establish an adequate plant population. The use of good-quality seed may ultimately be the best approach to attain this goal problem. The objective of this research was to study the effect of N-fertilization (at rates of 95.2 and 142.8 kg of N ha^?1), foliar application of K (at rates of 0, 0.38, 0.77, 1.15 kg of K₂O ha^?1, applied twice during square initiation and boll development stages) and the plant growth retardant (PGR), mepiquat chloride (applied twice, 75 days after planting at 0.0 [control] and 0.048 kg a.i. ha^?1, and 90 days after planting at 0.0 [control] and 0.024 kg a.i. ha^?1), on seed yield, viability, and seedling vigor of Egyptian cotton (Gossypium barbadense cv. Giza 86). A field experiment was conducted at the Agricultural Research Center, Giza, Egypt in two growing seasons. Growth, mineral uptake, seed yield per plant and per ha, seed weight, seed viability, seedling vigor and cool germination test performance were all found to increase significantly due to the addition of the high N-rate, the foliar application of three potassium concentrations, and the PGR mepiquat chloride. The N and K rates as well as application of mepiquat chloride had no significant effect on the germination rate index in both seasons. Under the conditions of this study, applying N at a rate of 142.8 kg ha^?1 combined with spraying cotton plants with K₂O at 1.15 kg ha^?1 and with mepiquat chloride at 0.048 + 0.024 kg ha^?1 were found to improve seed yield as well as seed viability and seedling vigor in the next season.     

Production of schisantherin A and gomisin G in in vitro cultures of Schisandra chinensis

Methanolic extracts from the biomass of shoot-differentiating and undifferentiating callus cultures of Schisandra chinensis growing respectively on six and two different variants of the Murashige and Skoog (MS) medium, with different concentrations of plant growth regulators, BA (N⁶-benzyladenine) and NAA (α-naphthaleneacetic acid) were analyzed for the accumulation of two lignans–schisantherin A and gomisin G, using the HPLC method. The amounts of the two compounds in the biomass extracts from shoot-differentiating callus cultures were dependent on the concentration of plant growth regulators in the MS medium. The highest amounts of both lignans were obtained on the MS medium supplemented with 3 mg l⁻¹ BA and 1 mg l⁻¹ NAA. The maximum amount of schisantherin A (33.45 mg 100 g⁻¹ DW) was about 1.3 times higher than in the extracts from the leaves and fruits of parent plants. This is the most important result potentially promising from a practical point of view.     

Multiple regeneration pathways in Spathoglottis plicata Blume — A study in vitro

Asymbiotic germination of immature seeds (embryos), and mature seeds and micropropagation of Spathoglottis plicata were described. Effects of three nutrition media namely, Murashige & Skoog (MS); Phytamax (PM); and Phyto-Technology orchid seed sowing medium (P₇₂₃), two carbon sources such as glucose and sucrose at 2–3% (w/v), two plant growth regulators such as 6-benzylaminopurine (BAP; 0.5–3.0 mg l^− 1) and α-naphthalene acetic acid (NAA; 0.5–2.0 mg l^− 1) and peptone (2.0 g l^− 1) were examined on seed germination, early protocorm development and micropropagation. The maximum germination of mature seeds (95%) was recorded in PM medium supplemented with 2% (w/v) sucrose + 2.0 g l^− 1 peptone. For germination of embryos P₇₂₃ medium supplemented with 1.0 mg l^− 1 BAP proved best. Multiple shoot buds or protocorm-like bodies (PLBs) were produced from stem segments of in vitro raised seedlings. Both direct organogenesis and embryogenesis were observed and the morphogenetic response was initiated by different concentrations and combinations of PGRs. The optimum PGR combination for maximal PLB regeneration was 1.0 mg l^− 1 NAA + 2.5 mg l^− 1 BAP, while 1.0 mg l^− 1 NAA + 1.0 mg l^− 1 BAP for shoot bud development. Strong and stout root system was induced in half strength PM medium supplemented with 0.5 mg l^− 1 IAA. The well-rooted plantlets were transferred to pots containing a potting mixture composed of saw dust, coconut coir, humus, and coal pieces at 1:1:1:2 (w/w) with 80% survival in outside environment and flowered after two years of transfer.     

Advantage of menadione-catalyzed chemiluminescent assay for the determination of viable mammalian cell number

A chemiluminescent assay composed of TCPO [bis(2,4,6-trichlorophenyl)oxalate] and harmless rhodamine B is proposed to be superior in the determination of menadione-catalyzed hydrogen peroxide (H₂O₂) production by viable mammalian cells to that composed of TCPO and harmful pyrene [Anal. Biochem. 207 (1992) 255–260]. In tests, the proposed assay showed that the measurable concentration of H₂O₂ and the viable cell number ranged from 10^?9 to 10^?3 M and from 2 × 10² to 2 × 10⁶ cells/100 μl/well in the presence of 10% bovine serum, respectively. The measuring time was approximately 10 min. On the other hand, the measurable cell numbers by the colorimetric WST-1 and MTT assays requiring several hours ranged only from 10³ to 10⁴ cells/100 μl/well and from 10⁴ to 10⁵ cells/100 μl/well, respectively. The cytotoxicity of sodium dodecyl sulfate was also observed at intervals of 1 min by the proposed assay, but not by the above colorimetric assays.     

Development of an efficient callus proliferation system for Rheum coreanum Nakai,a rare medicinal plant growing in Democratic People’s Republic of Korea

A clonal mass propagation to obtain mountainous sources of Rheum coreanum Nakai, a rare medicinal plant in Democratic People’s Republic of Korea was established by rhizome tissue culture. Whole plants were selected and collected as a vigorous individual free from blights and harmful insects among wild plants of R. coreanum grown on the top of Mt. Langrim (1.540 m above the sea) situated at the northern extremity of Democratic People’s Republic of Korea. Induction of the callus was determined using four organs separated from the whole plant and different plant growth regulators. The callus was successfully induced from rhizome explant on MS medium containing 2.4-D (0.2–0.3 mg/l). In the MS medium supplemented with a combination of BAP (2 mg/l) and NAA (0.2 mg/l), single NAA (0.5 mg/l), or IBA (0.5 mg/l), a higher number of shoot, root and plantlets was achieved. The survival rate on the mountainous region of the plantlets successfully acclimatized (100%) in greenhouse reached 95%, and yields of crude drug and contents of active principles were higher than those obtained by sexual and vegetative propagation. This first report of R. coreanum tissue culture provides an opportunity to control extinction threats and an efficient callus proliferation system for growing resources rapidly on a large scale.     

Anatomical and Biochemical Changes Associated with In Vitro Rhizogenesis in Dendrocalamus giganteus

Successful micropropagation protocol of a difficult-to-root bamboo species, Dendrocalamus giganteus (10–15 years old) along with the analysis of anatomical and biochemical changes during in vitro rhizogenesis was accomplished. Proliferated axillary shoots from nodal segments of 10–15 years old field culms exhibited shoot necrosis during multiple shoot formation phase and was controlled by subculturing in modified MS liquid medium having 825 mg ^l?1 NH₄NO₃, 3800 mg ^l?1 KNO₃, 740 mg ^l?1 MgSO₄ and 9% coconut water, 26.64 μM 6-benzylaminopurine (BA) and 0.46 μM kinetin. These multiple shoots proliferated from field grown culms, failed to root and hence callus was induced on MS solid medium containing 4.44 μM BA, 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.37 μM naphthalene acetic acid (NAA). Organogenesis from the callus was achieved upon transfer to MS medium with 11.10 μM BA and 2.32 pM kinetin. The callus-derived shoots multiplied on modified MS medium were rooted the best (91%) by culturing 3 days on MS medium having glucose (0.5%), sucrose (2.5%) and 98.41 μM indolebutyric acid (IBA) and subsequently to IBA-free MS medium containing 3% sucrose. Studies on peroxidase and IAA oxidase activity and endogenous free- and bound-IAA content showed that IAA oxidase and peroxidase oxidize endogenous IAA resulting in root initials formation. Anatomical studies confirmed the root primordia formation from 3^rd day of IBA treatment and primordia were visible over the surface on 8^th to 10^th day. However, the shoot necrosis symptoms which started on 6^th day of treatment intensified by 10^th day leading to the death of the whole shoot system by 12^th–15^th day. Nevertheless, on the root formation medium with 9.84 μM IBA, new shoot buds were emerged and showed shoot growth in 60% of the rooted cultures, which were successfully acclimatized in shade-house with 100% survival. The present study establishes rooting of callus-derived shoots as the best way for the successful propagation of the difficult-to-root bamboo, D. giganteus when compared to axillary bud proliferated shoots.     

Seed storage and germination in Kumara plicatilis,a tree aloe endemic to mountain fynbos in the Boland,south-western Cape,South Africa

Seed storage under appropriate conditions is a relatively inexpensive means of safeguarding plant genetic material for ex situ conservation. Post-storage germination trials are used to determine the viability of stored seeds, and hence the efficacy of the particular storage treatment. Kumara plicatilis (= Aloe plicatilis) is a tree aloe endemic to mountain fynbos in the Boland, south-western Cape. The viability and germination behaviour of K. plicatilis seeds were assessed for seeds stored for four and nine months at − 80 °C, 4 °C, 25 °C and under ambient conditions in a laboratory. Seeds were germinated under controlled conditions and germination rates and percentages determined. Ungerminated seeds were tested for viability using tetrazolium salt. Seed viability was not significantly reduced during storage. Seeds stored at − 80 °C for four and nine months exhibited the fastest germination rate overall (both 5.9 ± 0.3 weeks, mean ± S.E.), and slowest was for seeds stored under ambient conditions for four and nine months (both 7.8 ± 0.4 weeks). All seed lots showed similar percentage germination after four months of storage (78.0–90.4%). The highest percentage germination overall was for seeds stored at − 80 °C for four months (90.4%) and the lowest was for seeds kept at 4 °C and − 80 °C for nine months (39.2 and 39.6%, respectively). Respective percentage viability for ungerminated seeds in these two treatments was 82% and 87%, respectively, indicating the induction of secondary dormancy. Induced dormancy triggered by protracted cold temperatures may be an adaptation that enables seeds to survive prolonged extreme conditions that are unfavourable for germination. Further research on the long-term storage of aloe seeds would be beneficial for developing long-term seed storage and germination testing protocols for ex situ conservation.     

Uprising the antioxidant power of Argania spinosa L. callus through abiotic elicitation

This study was carried out in order to investigate the ability of tissues of Argania spinosa (L.) to undergo unlimited cell divisions by triggering their proliferative potential via callogenesis. Axenic cultures were efficiently established using axillary buds cultured on half-strength Murashige and Skoog (MS) medium after 20 min of surface sterilization with sodium hypochlorite 6% (v/v). The highest callus rate was achieved with 1.0 mg L⁻¹ of naphthaleneacetic acid (NAA) and 1.0 mg L⁻¹ of 2,4-dichlorophenoxyacetic acid (2,4D) or similarly with 0.01 mg L⁻¹ of 6-benzylaminopurine (BAP) and 1.0 mg L⁻¹ of 2,4D at pH of 5.8, under dark conditions. The results of this study show also a significant increase in the callus's antioxidant power under abiotic pressure induced by NaCl. Catalase (CAT), peroxidase (PO), and superoxide dismutase (SOD) activities were significantly triggered, which protected the cells from the stimulated oxidative stress, under hydrogen peroxide (H₂O₂) significant release. This reaction favors subsequently the tissue recover process linked to the low abundance of polyphenol oxidase (PPO) activity and malondialdehyde (MDA) content. This work proves the efficiency of salt stress in boosting the argan cell's antioxidant status, which could be commercially applied in the field of cells regenerative therapy.     

Effects of simulated nitrogen deposition on growth and photosynthesis of 1-year-old Chinese fir (Cunninghamia lanceolata) seedlings

To study the impact of nitrogen deposition on 1-year-old Chinese fir (Cunninghamia lanceolata) seedlings in pots, the dissolved NH₄NO₃ was sprayed on the seedlings every 3 days for 1 year. The simulated elevated N depositions were equivalent to N0(0), N1(6 gN/(m² a)), N2(12 gN/(m² a)), N3(24 gN/(m² a)) and N4(48 gN/(m² a)). The results indicated that medium N treatments (N2, N3) enhanced growth significantly. The height, stem base diameter and per-seedling biomass of Chinese fir seedlings increased with N loads and decreased in the high N treatments. Compared to N0, the height and per-seedling biomass were highest in N2 treatment and increased by 10.77% and 12.35%, respectively. The stem base diameter was highest in N3 treatment and increased by 8.81% compared to N0. The net photosynthetic rate (Pn) in treatments N1, N2, N3, N4 increased by 1.20%, 9.28%, 24.23% and 4.30%, and the highest photosynthetic rate by 67.09%, 125.32%, 148.10% and 51.90%, respectively. The N1–N3 treatments, especially N2, stimulated light compensation point (LCP) of the seedlings significantly, but N4 exhibited inhibitive effect. Compared with LCP, light saturation point (LSP) showed weaker response to N loads, positive to N2, but negative to all other N treatments. Low-to-medium N treatments (N1, N2) enhanced Chl (a + b) by 2.19% and 37.15%, while medium-to-high N treatments (N3, N4) reduced Chl (a + b) by 7.95% and 15.56%, respectively. Water use efficiency (WUE) and stomatal conductance (C) decreased slightly with N loads.     

Improved survival of mesenchymal stromal cell after hypoxia preconditioning: role of oxidative stress

AimsTo investigate the mechanisms underlying the beneficial effect of hypoxia preconditioning (HPC) on mesenchymal stromal cells (MSCs) and optimize novel non-invasive methods to assess the effect of biological interventions aimed to increased cell survival.Main methodsMSCs from rat femur, with or without HPC, were exposed to hypoxic conditions in cell culture (1% O₂ for 24 h) and cell survival (by the LDH release assay and Annexin-V staining) was measured. Oxidant status (conversion of dichloro-fluorescein-DCF- and dihydro-ethidium-DHE-, protein expression of oxidant enzymes) was characterized, together with the mobility pattern of cells under stress. Furthermore, cell survival was assessed non-invasively using state-of-the-art molecular imaging.Key findingsCompared to controls, Hypoxia resulted in increased expression of the oxidative stress enzyme NAD(P)H oxidase (subunit 67^phox: 0.05 ± 0.01 AU and 0.48 ± 0.02 AU, respectively, p < 0.05) and in the amount of ROS (DCF: 13 ± 1 and 42 ± 3 RFU/μg protein, respectively, p < 0.05) which led to a decrease in stem cell viability. Hypoxia preconditioning preserved cell biology, as evidenced by preservation of oxidant status (16 ± 1 RFU/μg protein, p < 0.05 vs. hypoxia), and cell viability. Most importantly, the beneficial effect of HPC can be assessed non-invasively using molecular imaging.SignificanceHPC preserves cell viability and function, in part through preservation of oxidant status, and its effects can be assessed using state-of-the-art molecular imaging. Understanding of the mechanisms underlying the fate of stem cells will be critical for the advancement of the field of stem cell therapy.     

Specific activity and viability of Nitrosomonas europaea during discontinuous and continuous fermentation

The energy conservation and number of viable cells of Nitrosomonas europaea fluctuate dramatically during cultivation. In discontinuous culture the specific activity (SA) reaches its maximum after 9 h with about 2700 nmol O₂ (mg protein)^?1 min^?1, where the highest number of viable N. europaea cells is detectable after 21 h with 2 × 10⁸ cell ml^?1. Afterwards, both SA and viable cell number immediately start to decrease. Accordingly, the exponential growth turns into a linear growth, whereby the number of viable cells permanently decreases. The exponential growth phase can be extended from about 21 to 38 h by increasing the concentration of CO₂ or trace elements. In continuous fermentation of N. europaea, SA of about 2500 nmol O₂ (mg protein)^?1 min^?1 and viable cell number of 2.5 × 10⁸ cell ml^?1 is detectable at dilution rates between 1 and 1.8 day^?1. At dilution rates below 1 day^?1, SA and number of viable cells are reduced. The minimal doubling time is 13 and 15 h during continuous and discontinuous fermentation, respectively. Consequently, cell production of N. europaea should be performed in continuous fermentation. When bacteria are grown in discontinuous systems, they should be harvested in the early exponential growth phase.     

Culture medium improvement for Isaria fumosorosea submerged conidia production

Submerged conidia and blastospores of the entomopathogenic fungus Isaria fumosorosea are produced in several liquid culture media. However, yields and the ecological fitness of these propagules vary according to culture media composition. In most culture media, hyphae, blastospores and submerged conidia are white but we found that in some media they develop a brown pigmentation. A dark pigment was extracted from brown-pigmented propagules and analyzed by IR spectroscopy. Adsorption bands coincided to those characteristics of melanins.Hadamard's matrices were employed in order to increase submerged conidia yields and brown pigmentation of fungal propagules. Media containing 20–30 mg/l of FeSO₄·7H₂O and 6–12 mg/l of CuSO₄·5H₂O allowed reaching the highest pigmentation (9 in a hedonic scale). A maximal concentration of submerged conidia of 1.0 (±1.2) × 10¹² cell/l was achieved after 120 h of liquid culture in a improved culture medium, containing 25 ml/l of Polyethylene glycol (MW 200), substance which enhanced submerged conidia production, reducing free mycelia or mycelial pellets formation. In the improved medium, it was estimated that more than 60% of produced biomass corresponded to submerged conidia and blastospores, while in other media, mycelia were the main product (80–97%).     

Protocorm culture of Dendrobium candidum in balloon type bubble bioreactors

Protocorm cultures of Dendrobium candidum were established in balloon type bubble bioreactors using Murashige and Skoog (MS) medium with 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), 2.5% (w/v) sucrose, 5:25 mM NH₄:NO₃ and 1% (v/v) banana homogenate for the production of biomass and bioactive compounds. In 3 l bioreactor containing 2 l medium, a maximum protocorm biomass (21.0 g l⁻¹ dry biomass) and also optimum quantities of total polysaccharides (389.3 mg g⁻¹ DW), coumarins (18.0 mg g⁻¹ DW), polyphenolics (11.9 mg g⁻¹ DW), and flavonoids (4.5 mg g⁻¹ DW) were achieved after 7 weeks of culture. Based on these studies, 5 and 10 l bioreactor cultures were established to harvest 80 g and 160 g dry biomass. In 10 l bioreactors, the protocorms grown were accumulated with optimal levels of polysaccharides (424.1 mg g⁻¹ DW), coumarins (15.8 mg g⁻¹ DW), polyphenols (9.03 mg g⁻¹ DW) and flavonoids (4.7 mg g⁻¹ DW). The bioreactor technology developed here will be useful for the production of important bioactive compounds from D. candidum.     

Production of rhoifolin and tiliroside from callus cultures of Chorisia chodatii and Chorisia speciosa

The current work aims to stimulate the production of rhoifolin and tiliroside as two valuable phytochemicals from Chorisia chodatii Hassl. and Chorisia speciosa A. St.-Hil. callus cultures. A comparison between three explants from the in vitro germinated seedlings of both species for callus induction and accumulation of both flavonoids was carried out. Highly efficient calluses were induced from the leaves, stems and roots of C. chodatii seedlings on Gamborg’s B5 (B5) and Murashige and Skoog (MS) media containing 2.0 mg/l β-naphthalene acetic acid (NAA) and 0.5 mg/l 6-benzyladenin (BA) or kinetin (Kn), while those of C. speciosa seedlings efficiently produced calluses on both media supplemented with 0.5 or 1.0 mg/l NAA and 0.5 mg/l BA. Besides, the highest contents of rhoifolin (1.927 mg/g DW) and tiliroside (1.776 mg/g DW) from C. speciosa cultures were obtained from the calluses of seedlings’ roots and stems maintained on B5 medium containing 1.0 mg/l NAA and 0.5 mg/l BA, respectively. On the other hand, the maximum rhoifolin content (0.555 mg/g DW) from C. chodatii cultures was obtained from the calluses of seedlings’ stems grown on B5 medium supplemented with 2.0 mg/l NAA and 0.5 mg/l BA, whereas the highest tiliroside content (0.547 mg/g DW) was provided by the root explants on B5 medium containing 2.0 mg/l NAA and 0.5 mg/l Kn. Both flavonoids were bioaccumulated in greater amounts than the wild and cultivated intact plants, which provides a promising tool for their future commercial production under a controlled environment, independent of climate and soil conditions.     

Effect of cyanobacterial extracellular products on high-frequency in vitro induction and elongation of Gossypium hirsutum L organs through shoot apex explants

The challenging task of bringing high efficiency transformed plants attracts lot of attention in recent times. In search for this, there have been many attempts made using, different techniques like tissue culture and plant breeding methods. Here we report a suitable alternative facile route, where cyanobacterial extracellular products are utilized as growth regulators and its performance validated on Gossypium hirsutum L. MS medium is tested with cyanobacterial extracellular products of Nostoc ellipsosporum, Dolichospermum flos-aquae and Oscillatoria acuminata .Our best results show that the addition of O. acuminata extracellular product with plant growth hormones gives the excellent induction and elongation in cotton. In addition to this, the multiple shoot was obtained on MS medium fortified with 1.0 mg L^?1 BA with 8% O. acuminata and 1.5 mg L^?1 TDZ with 12% O. acuminata. High frequency of shoot elongation supplemented with MS medium, iP 2.5 mg L^?1 and 16% O. acuminata and root production MS medium fortified with 12% O. acuminata best responsible for regeneration in cotton plants. The rooted plants were hardened and transferred to soil with 90% survival rate.     

Heat activation and germination–promotion of Bacillus subtilis spores by infrared irradiation

The influence of infrared (IR) radiation on the viability and heat-activation of Bacillus subtilis spores, suspended in phosphate-buffered saline, was investigated. Two types of IR heaters with different spectral distributions were used. Near-infrared (NIR) and far-infrared (FIR) heaters with main wavelengths of approximately 1 μm and 3–6 μm, respectively, were utilized. Although both irradiation treatments decreased the number of B. subtilis colonies at a bulk temperature of approximately 75 °C, the mode of action was clearly different. In the case of the NIR heater, the number of colony-counts decreased gradually. In contrast, use of the FIR heater resulted in heat activation of the spores during the early stage of irradiation at a low bulk temperature (40–60 °C) over several minutes, followed by a decrease in the number of colonies. Consequently, FIR irradiation inactivated 90% of B. subtilis spores more effectively as compared to NIR irradiation for 20 min with a suspension volume of 20 ml and irradiation energy of 7.57 kW m^?2. Spore exposure to FIR irradiation accelerated their germination rate in nutrient broth; however, this was not true for treatment with the NIR heater. The absorption IR spectrum of B. subtilis spores indicated that FIR radiation was absorbed easily by the spore cell components and might activate the bioactive substances involved in germination. Even at the same irradiation energy, the influence of infrared radiation on spore germination was dependent on the IR spectral distribution. Bacterial spores undergoing germination lose their resistance to stressors, such as heat, chemicals and ultraviolet rays. FIR heating promotes heat activation and germination, thereby producing vegetative cells that are more susceptible to other killing methods, enabling the killing of bacterial spores at lower stress without product damage.     

Validation of cell density and viability assays using Cedex automated cell counter

A method using Cedex automatic cell counter (Innovatis) to determine the cell density and viability of a whole cell-based immunotherapy product has been developed and validated for the assay performance characteristics including specificity, accuracy, precision, linearity, range, and robustness. Instrument-to-instrument variation due to intrinsic differences in handmade flow cells was also evaluated. For cell density, Cedex demonstrated acceptable specificity, accuracy and precision for cell densities ranging from 3.13 × 10⁵ to approximately 1.0 × 10⁷ cells/mL, with intermediate precision of about 5% relative standard deviation (RSD). However, a marked difference was observed between the two instruments studied and they therefore could not be used interchangeably without additional calibration procedures that went beyond the manufacturer's recommendation. For viability, mixing known numbers of non-viable cells with highly viable cells allowed evaluation of the specificity, accuracy and linearity of the viability determination. Acceptable levels of accuracy (95.3–106.4% recovery) and precision (RSD < 5%) were demonstrated for the viability range from 50 to 100%. The instrument-to-instrument difference was less than 4.6%. The assays for both cell density and viability were sufficiently robust for assay parameters. However, the effect of certain parameters was cell line-dependent, suggesting that Cedex performance should be verified for each cell line of interest.