Cryopreservation of the microalgae Chaetoceros calcitrans (Paulsen): analysis of the effect of DMSO temperature and light regime during different equilibrium periods

Abstract

ABSTRACT.We evaluated the effect of three variables (cryoprotectant temperature, light regime, and time of exposure to the cryoprotectant) throughout the equilibrium period during cryopreservation on the viability of the microalga Chaetoceros calcitrans (Paulsen). For this, the cryoprotectant dimethyl sulfoxide (DMSO) at 5% (v/v) was added at three different temperatures (4, 10, and 25ºC) before placing the microalgae in cryobiological straws for freezing. Once inside the cryobiological straws, the microalgal-cryoprotectant suspensions were subjected to the following light regimes for 15 or 45 min: complete light, complete darkness, light/darkness, and darkness/light. Suspensions were then frozen under controlled conditions and stored in liquid nitrogen. The viability index proposed by Cañavate & Lubian (1995b) was used to measure microalgal viability after cryopreservation. Results indicated that it is necessary to use a cryoprotectant to ensure the viable cryopreservation of C. calcitrans. Statistical analyses showed that the temperature of the DMSO influenced the viability of cryopreserved microalgae and that there was no synergistic effect between the other variables studied. The viabilities obtained with DMSO at 25ºC, 10ºC, and 4ºC were 34.9%, 27.8%, and 20.6%, respectively. Keywords: cryopreservation, diatoms, DMSO, temperature, light, equilibrium period.Criopreservación de las microalgas Chaetoceros calcitrans (Paulsen): análisis del efecto de la temperatura de DMSO y régimen de luz durante diferentes períodos de equilibrio RESUMEN. Se evaluó el efecto de tres variables − la temperatura del crioprotector, el régimen de luz y el tiempo de exposición al crioprotector durante el período de equilibrio en el proceso de criopreservación − sobre la viabilidad de la microalga Chaetoceros calcitrans (Paulsen). Para ello se utilizó, el crioprotector dimetilsulfóxido (DMSO) al 5% (v/v), el cual fue adicionado a tres temperaturas diferentes (4, 10 y 25ºC) antes de que las microalgas fueran introducidas dentro de pajuelas criobiológicas para su congelación. Una vez dentro de las pajuelas, las suspensiones de microalgas con crioprotector se sometieron durante 15 o 45 min a luz completa, oscuridad completa, luz/oscuridad y oscuridad/luz. Luego se congelaron de manera controlada y se almacenaron en nitrógeno líquido. La viabilidad de las microalgas posterior a la criopreservación fue medida a través del índice de viabilidad propuesto por Cañavate & Lubian (1995b). Los resultados indicaron que es necesario el uso de criprotector para la criopreservación viable de C. calcitrans. El análisis estadístico demostró que la temperatura del DMSO influye sobre la viabilidad de la microalga criopreservada y que no existe efecto sinérgico entre las demás variables de estudio. Las viabilidades alcanzadas usando DMSO a 25ºC, 10ºC y 4ºC fueron de 34,9%, 27,8% y 20,6% respectivamente.

Oikopleura dioicaculturing made easy: A Low‐Cost facility for an emerging animal model inEvoDevo

“…With the exception of C. calcitrans (Salas‐Leiva, ), we did not found specific protocols designed for the cryopreservation of the microalgae that we use for O. dioica feeding, and therefore, we modified the conditions of some general protocols that used dimethyl sulfoxide (DMSO) as cryoprotectant (Day, ). To cryopreserve our microalgae, 1 mL of densely growth culture (i.e., OD > 0.25 for Isochrysis sp .…”

Optimization of Cryopreservation Process Using Response Surface Methodology for Chlorella saccharophila and Chlorella zofingiensis

“…The most important factors, which effects on algal viability, are the type, concentration and timing of cryoprotective agents, pretreatment with cryoprotectant and duration of cryopreservation [11]. Salas-Leiva and Dupré [12] reported that concentration, temperature and time of exposure are related to the use of cryoprotectant in additionally there are various methods of handling them emphasized in the literature. The cryoprotectant agent as DMSO is used for the treatment of microalgae because it easily permeates cell membranes and its low hydrophilicity [13].…”

Proximate composition analysis posterior to the cryopreservation of Chaetoceros calcitrans

“…Batch cultures of C. calcitrans using micro-filtered sterile sea water were grown. Culture conditions, cryopreservation protocol and viability estimations are broadly described in Salas-Leiva and Dupré (2). A brief description of the cryopreservartion protocol is presented here: dimethylsulfoxide (Me2SO, 10%) (Sigma, St. Louis, MO) was added to the cells at 4 and 25°C in complete darkness (1:1 proportion in mL).…”

“…Sin embargo, cuando la viabilidad posterior a la criopreservación es baja, como lo es en el caso de Chaetoceros calcitrans (viabilidad <40%) (2), es necesario indagar si los microorganismos sobrevivientes presentan una composición proximal similar a la observada en la cepa antes de ser criopreservada, es decir, si la cepa conserva las características nutricionales iniciales. Resultados obtenidos por Sánchez-Saavedra y Nuñez-Zarco (3) indican que el almacenamiento a baja temperatura de refrigeración (4°C) (durante 1 a 6 semanas) influye en la densidad celular y en la composición proximal de los cultivos de las diatomeas Navicula sp, N. incerta, N. thermalis y Nitzschia laevis.…”