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"Cultivo in vitro de clulas y tejidos vegetales. Aplicaciones Biotecnolgicas"
Biotecnologa VegetalPara qu cultivar in vitro clulas, tejidos y rganos vegetales?
micropropagacin mejoramiento de plantas produccin de compuestos de inters in vitro
produccin de metabolitos secundariosproduccin y utilizacin de protenas vegetalesproduccin de polmeros biodegradables
establecimiento de plantas transgnicasplantas resistentes a virusplantas con rutas metablicas modificadasplantas mejoradas en cuanto a composicin de
protenas, aceites, etc. fitorremediacin
remocin de xenobiticosremocin de metales pesados
- Totipotencialidad celular
- Indiferenciacin / Rediferenciacin
Principales conceptos fisiolgicos aplicables a los cultivos in vitro
toda clula vegetal individual es capaz de regenerar una planta entera a partir de un cultivo in vitro sin importar el grado de diferenciacin alcanzado
Concepto de Totipotencia
Haberlandt a principios del siglo veinte
Principales conceptos fisiolgicos aplicables a los cultivos in vitro
Indiferenciacion-rediferenciacin
Tipos de cultivo
-Mejora y seleccin gentica-Conservacin del germoplasma -Sanidad Vegetal
-Produccin de metabolitos primarios, secundarios y de protenas.-Micropropagacin-Estudios metablicos y fisiolgicos
-Bioconversiones y biocatlisis
Aplicaciones
Cultivos organizados: Son aquellos que mantienencaractersticas de organizacin estructural. La progeniepresenta idnticas propiedades que el material original.Por ejemplo el cultivo de rganos.
Cultivos no organizados: Son aquellos en los que lasclulas o tejidos aislados se indiferencian ingresando en unciclo de divisin y crecimiento de duracin indefinida. Laprincipal desventaja de estos el la inestabilidad gentica yaque se genera frecuentemente poliploida. Por ejemplo lassuspensiones celulares.
Cultivos organizados-no organizados: Son aquellos que seoriginan a partir de cultivos indiferenciados (no organizados)que dan origen a callos y agregados celulares, con cierto gradode diferenciacin, o bien a rganos o embriones somticos. Laprincipal desventaja es que la progenie no siempre escompletamente idntica puesto que surgen de cultivospotencialmente poliploides.
Qu tener en cuenta para cultivar in vitro vegetales?
a.- Sala de lavado y esterilizacin
b.- Sala de preparacin de medios
c.- Sala de transferencia de material
d.- Sala de incubacin
Equipos de esterilizacin:EstufasAUTOCLAVES !!!!!
Material de vidrio de uso exclusivo para cultivo
Balanzas, equipos de medicin de pH, agitadores, mantos de calentamiento, microondas, heladeras, freezers
rea de lavado y preparacinDisponibilidad de agua corriente, agua destilada, aguabidestilada y/o desionizada
AREA DE TRANSFERENCIA
consiste en una ZONA ESTRIL con disponibilidad de fuentes de electricidad, vaco, aire comprimido y fuego
Habitacin pequea, libre de polvo,superficies de trabajo lisas,con una lmpara UV sobre la mesada de trabajo, presin positiva, sistema de filtracin de aire
Filtros HEPA (high-efficiency particulate air filter) 0.3-m HEPA de 99.97-99.99% eficiencia
In a LAMINAR FLOW HOOD the air is forced into the unit through a dust filter then passed througha HEPA filter. The air is then either directed downward (VERTICAL FLOW UNIT) or outward(HORIZONTAL FLOW UNIT) over the working surface. The constant flow of microbe-free filtered airprevents non-filtered air and particulate matter in the room from settling on the working surface.
Lo recomendable Cabina de FLUJO LAMINAR
Otra opcin: Caja de Guantes
Sala o cmara de cultivo
LA TEMPERATURAla mayora de las especies desarrollan bien atemperaturas de incubacin que oscilan entre los20 y 28C. Para especies de zonas templadas esideal trabajar a 22 C
El control de la temperatura no es solamente importante porque pueda afectar al crecimiento del cultivo sino tambin porque puede ser un factor que induzca otros procesos fisiolgicos
As, temperaturas bajas (del orden de 4-5C) permiten superar losperiodos de dormicin de algunas leosas y la conservacin prolongadade determinados cultivos in vitro;
mientras que una temperatura constante de 20C induce la formacinde races en la mayora de conferas
Lo importante es el mantenimiento de temperaturas constantes
LA TEMPERATURA
Como se controla?
Conociendo la temperatura ambiente de la sala donde se sitey el calor generado por las fuentes de luz de que dispone.
El control de la temperatura a la que se desarrolla el cultivo invitro se efecta mediante un sistema de refrigeracin-calefaccin controlado a travs de un termostato.
El sistema de refrigeracin-calefaccin debe estarcorrectamente dimensionado a fin de conseguir que latemperatura de la zona de cultivo se mantenga dentro de loslmites deseados.
LA TEMPERATURA
IMPORTANTE!!!!
LA HOMOGENEIDAD de temperatura: es decir la variacin de la temperatura en diferentes zonas de la cmara. Se puede aumentar la homogeneidad haciendo circular el aire dentro de la cmara mediante un sistema de ventilacin
LA ESTABILIDAD DE LA TEMPERATURA: es decir, una medida de la variacin de la temperatura de la cmara de cultivo a lo largo del tiempo
LA LUZ
La luz es esencial para las plantas debido a que proporciona la energa necesaria para la fotosntesis.
La clorofila y los dems pigmentos fotosintticos captan la energa contenida en diferentes radiaciones para incorporarla a las diversas reacciones qumicas que constituyen el proceso.
Pero la luz tambien puede intervenir en otros procesos fisiolgicos, como el fototropismo, la germinacin, la floracin,etc.
Todos estos fenmenos no son producidos en igual medida por todos los tipos de luz (radiaciones de cualquier longitud de onda) sino que algunas radiaciones concretas tienen un efecto notable mientras que otras tiene poco o ningn efecto. Es por ello que es preciso conocer el espectro de la radiacin que es activo en el proceso fisiolgico estudiado.
LA LUZ
La cmara de cultivo deber reproducir lo mejor posible ese espectro de luz activo, por lo tanto conviene conocer cual es el espectro que emiten nuestras fuentes de luz y en que medida se adapta ste a las necesidades de nuestro cultivo.
La luz es uno de los factores principales quedeterminan el desarrollo de los organismosauttrofos, en ello radica la importancia decontrolar el factor luz en los cultivos in vitro.
LA LUZLA CANTIDAD DE LUZ: LA IRRADIACIN:
la iluminacin de una superficie se puede expresar en lux (en realidad se trata de una unidad definida en trminos de percepcin del ojo humano); o bien midiendo la irradiancia, es decir, la energa radiante que llega a una superficie dada en un intervalo de tiempo. La irradiancia puede ser expresada en funcin de la energa: Wm-2 ; o en funcin de los fotones: mol m-2 s-1
Cuando el sensor del equipo con el que medimos la irradiancia est diseado para detectar solo las longitudes de onda comprendidas entre 400 y 700 nm, obtenemos una medida de la radiacin fotosintticamente activa (PAR)
LA LUZLA CANTIDAD DE LUZ: LA IRRADIACIN:
Se asume que las necesidades de luz de los cultivos in vitro son inferiores a las de la planta in vivo, dado que el medio de cultivo contiene cantidades importantes de sacarosa, los cultivos in vitro se comportan slo parcialmente de forma autotrfica. Adems, una irradiacin excesiva producira un aumento notable de la temperatura dentro del recipiente de cultivo debido al efecto invernadero.
La irradiacin habitual en el campo (a plena insolacin puede llegar a 450 W m-2) resulta nociva en condiciones in vitro
Es habitual usar irradiaciones mucho menores (un 10% del valor de plena insolacin)
LA LUZ
Principales fuentes de luz usadas en las cmaras de cultivo
LAMPARAS INCANDESCENTES
Producen la luz por fenmenos de incandescencia del filamento calentado porel paso de la corriente elctrica. Buena parte del espectro se halla en la zonadel rojo/rojo lejano. Producen gran cantidad de calor y mucho consumo de electricidad.
LAMPARAS FLUORESCENTES
Producen la luz por fenmenos de fluorescencia del gas sometido a un arcovoltico. El espectro de la luz producida es rico en la zona del azul, existenfluorescentes especiales con un espectro rico en la zona azul y roja. Consumenmenos electricidad.
LAMPARAS DE VAPOR DE MERCURIO Y SODIO
Producen la luz por efecto del paso de la corriente elctrica a travsde gases calientes de mercurio (azul y verde) y sodio (naranja). Son altamente eficientes en el consumo de electricidad.
LA LUZ
La alternancia de los ciclos de luz con los de oscuridad: El fotoperiodo: Algunos fenmenos propios del desarrollo de las plantas (germinacin, floracin, tuberizacin, etc.) pueden ser activados por el nmero de horas diarias de luz que recibe la planta.
De forma anloga, el nmero de horas de luz que recibe el explanto cultivado in vitro puede afectar a su desarrollo. En general, el mejor fotoperiodo in vivo ser tambin el mejor fotoperiodo in vitro.
LA LUZ
Control del fotoperiodo
La regulacin del fotoperiodo se consiguemediante un programador conectado al circuito deiluminacin.
El programador del fotoperiodo puede estarrelacionado con el programador de temperaturas,de forma que se puedan programar diferentestemperaturas segn sea la fase del fotoperiodo enla que se halle el cultivo.
Qu tener en cuenta para cultivar in vitro vegetales?
One of the most important factors governing the growthand morphogenesis of plant tissues in culture is thecomposition of the culture medium.
The basic nutrient requirements of cultured plant cellsare very similar to those of whole plants.
Tissue Culture Media-Composition
Plant tissue and cell culture media are generally made up of some or all ofthe following components:
macronutrients, micronutrients, vitamins, amino acids or other nitrogensupplements, sugar(s), other undefined organic supplements, solidifyingagents or support systems, and growth regulators..
Several media formulations are commonly used for the majority of all cell and tissue culture work.
These media formulations include those described by White, Murashige and Skoog, Gamborg et. al., Schenk and Hilderbrandt, Nitsch and Nitsch, and Lloyd and McCown.
Macronutrients
The macronutrients provide the six major elements: nitrogen (N),phosphorus (P),potassium (K),calcium (Ca),magnesium (Mg),and sulfur (S)-required for plant cell or tissuegrowth
The optimum concentration of each nutrient for achievingmaximum growth rates varies considerably among species
Macronutrients
When nitrate and ammonium sources of nitrogen are utilized together in the culturemedium, the ammonium ions will be utilized more rapidly and before the nitrate ions.
Plant cells may grow on nitrates alone, but considerably better results are obtained when the medium contains both a nitrate and ammonium nitrogensource
NITRATES RANGE 25-30 mM
AMMONIUM RANGE 2 - 20 mMFor certain species ammonium concentrations in excess of 8 mM may bedeleterious to cell growth of certain species
Some cells can grow on a culture medium containing ammoniumas the sole nitrogen source if one or more of the TCA cycle acids(e.g., citrate, succinate, or malate) are also included in the culturemedium at concentrations of approximately 10 mM.
Culture media should contain at least 25-60 mM of inorganicnitrogen for adequate plant cell growth
N
Macronutrients
K Potassium is required for cell growth of most plantspecies. Most media contain K, in the nitrate or chlorideform, at concentrations of 20-30 mM.
PMgSCa
The optimum concentrations of P, Mg, S, and Ca range from 1-3 mM when all other requirements forcell growth are satisfied. Higher concentrations of these nutrients may berequired if deficiencies in other nutrients exist.
MicronutrientsThe essential micronutrients for plant cell and tissue growth include:iron (Fe), manganese (Mn), zinc (Zn), boron (B), copper (Cu), and molybdenum (Mo)
Fe Concentration arround 1 M
Iron may be the most critical of all the micronutrients. Iron citrate and tartrate may be used in culture media, but these compounds are difficult to dissolve and frequently precipitate after media are prepared. Murashige and Skoog used an ethylene diaminetetraacetic acid (EDTA)-iron chelate to bypass this problem.
Micronutrients
CuCo
concentrations of 0.1 M Mo concentrations 1 M
I concentrations 5 M Zn concentrations 5-30 M
Mn concentrations 20-90 M B concentrations 25-100 M
Componentes (mg/L) Chu N6 DKW/Juglan Gamborg B5Gamborg B-5 minimal organics
HoaglandMcCown woody plant
Murashige and Skoog
Quoirin andLepoivre
Schenk andHildebrandt
White
Ammonium nitrate 1416.0 400.0 1650.0 400.0
Ammonium phosphate monobasic
115.03 300.0
Ammonium sulfate 463.0 134.0 134.0
Boric acid 1.6 4.8 3.0 3.0 2.86 6.2 6.2 6.2 5.0 1.5
Calcium chloride anhydrous 125.33 112.5 113.24 113.24 72.5 332.2 151.0
Calcium nitrate 1367.0 656.4 386.0 833.77 200.0
Cobalt chloride 6H2O 0.025 0.025 0.025 0.025 0.1
Cupric sulfate 5H2O 0.25 0.025 0.025 0.08 0.25 0.025 0.025 0.2
Na2-EDTA 37.25 45.4 37.3 37.25 37.3 37.26 37.3 20.0
Ferric sulfate 2.5
Ferric tartrate 2H2O 5.32
Ferrous sulfate 7H2O 27.85 33.8 27.8 27.85 27.8 27.8 15.0
Magnesium sulfate 90.37 361.49 122.09 122.09 240.76 180.7 180.7 175.79 195.4 360.0
Manganese chloride 4H2O 1.81
Manganese sulfate H2O 3.33 33.5 10.0 10.0 22.3 16.9 0.76 10.0 5.04
Molybdenum trioxide 0.016
Molybdic acid (sodium salt) 2H2O
0.39 0.25 0.25 0.25 0.25 0.25 0.1
Potassium chloride 65.0
Potassium iodide 0.8 0.75 0.75 0.83 0.08 1.0 0.75
Potassium nitrate 2830.0 2500.0 2500.0 606.6 1900.0 1800.0 2500.0 80.0
Potassium phosphate monobasic
400.0 265.0 170.0 170.0 270.0
Potassium sulfate 1559.0 990.0
Sodium phosphate monobasic 130.5 130.5 16.5
Sodium sulfate 200.0
Zinc nitrate 6H2O 17.0
Zinc sulfate 7H2O 1.5 2.0 2.0 0.22 8.6 8.6 8.6 1.0 2.67
Carbon and Energy Source
Glucose and fructose are used in some cases
The preferred carbohydrate in plant cell culture media is sucrose
Carbohydrates must be supplied to the culture medium because only a fewplant cell lines are fully autotrophic.
Other carbohydrates that have been tested include lactose, galactose, rafinose, maltose, and starch.
Sucroseconcentrations of culture media normally range between 2 and 3 percent
Use of autoclaved fructose can be detrimental to cell growth!!!!
Vitamins
Normal plants synthesize the vitamins required for their growthand development.Vitamins are required by plants as catalysts in various metabolicprocesses
When plant cells and tissues are grown in vitro, some vitamins may become limiting factors forcell growth
The vitamins most frequently used in cell and tissue culturemedia include thiamin (B1), nicotinic acid, pyridoxine (B6), and myo-inositol
Vitamins
It is basically required by all cells for growth. Concentrations range from 0.1 to 10.0 mg/l.
ThiaminB1
concentrations of 0.1-5.0 mg/lNicotinic acid
used at 0.1-10.0 mg/lPyridoxine B2
Nicotinic acid and pyridoxine are often added to culture media but are not essential for cell growth in many species.
Vitamins
Other vitamins such as biotin, folic acid, ascorbic acid, pantothenic acid, vitamin E (tocopherol), riboflavin, and p-aminobenzoic acid have been included in some cellculture media.
The requirement for these vitamins by plant cell cultures is generallynegligible, and they are not considered growth-limiting factors. These vitamins are generally added to the culture medium onlywhen the concentration of thiamin is below the desired level or whenit is desirable to grow cells at very low population densities.
it has been shown to stimulate growth in certain cell cultures.
Myo-inositol is commonly includedin many vitamin stock solutions
Although it is a polialcohol derived from carbohydrates not a vitamin
Myo-inositol is generally used in plant cell and tissue culture media at
concentrations of 50-5000 mg/l
HOHO
OH
HO
OH
OH
Vitamin complex
MS Vitamins contains: 2.0 mg Glycine, 0.5 mg Nicotinic Acid, 100 mg myo-Inositol, 0.5 mg Pyridoxine HCl, 0.1 mg Thiamine HCl
LS Vitamins contains: 100 mg myo-Inositol0.1 mg Thiamine HCl
GB Vitamins contains: 1.0 mg Nicotinic Acid, 100 mg myo-Inositol, 1.0 mg Pyridoxine HCl, 10.0 mg Thiamine HCl
AMINO ACIDS OR OTHER NITROGEN SUPPLEMENTS
Although cultured cells are normally capable of synthesizing all of the required amino acids, the addition of certain amino acids oramino acid mixtures may be used to further stimulate cell growth
The use of amino acids is particularly important for establishingcell cultures and protoplast cultures
Amino acids provide plant cells with an immediately available source of nitrogen, which generally can be taken up by the cells more rapidly thaninorganic nitrogen
When amino acids are added alone, care must be taken, as they can be inhibitory to cell growth
Tyrosine has been used to stimulate morphogenesis in cellcultures but should only be used in an agar medium.
Supplementation of the culture medium with adenine sulfate can stimulate cell growth and greatly enhance shoot formation.
Casein hydrolysate concentrations between 0.05 and 0.1 percent
Examples of amino acids included in culture media to enhancecell growth:
glycine at 2 mg/liter, glutamine up to 8 mM, asparagine at 100 mg/liter,L-arginine and cysteine at 10 mg/liter, L-tyrosine at 100 mg/liter.
AMINO ACIDS OR OTHER NITROGEN SUPPLEMENTS
The most common sources of organic nitrogen used in culturemedia are amino acid mixtures (e.g., casein hydrolysate), L-glutamine, L-asparagine, and adenine
coconut milk is commonly used at 5-20% (v/v).
Undefined Organic SupplementsAddition of a wide variety of organic extracts to culture media often results in favorable tissue responses
Supplements that have been tested include protein hydrolysates, coconut milk, yeast extracts, malt extracts, ground banana, orange juice, and tomato juice
They should only be used as a last resort, and only coconut milkand protein hydrolysates are used to any extent today
The inhibition of growth in the presence of AC is generally attributed to the absorption of phytoregulators, such as 1-Napthaleneacetic acid (NAA), kinetin, 6-benzylaminopurine (BA), indole-3-acetic acid (IAA), and 6---dimethylallylaminopurine (2iP)
Undefined Organic Supplements
The addition of activated charcoal (AC) to culture media may have a beneficial effect
AC may cause:absorption of inhibitory compoundsabsorption of growth regulators from the culture mediumand darkening of the medium
The stimulation of cell growth by AC is generally attributed to its ability to bind to toxic phenolic compounds produced during culture
AC is generally acid-washed prior to addition to the culture mediumat a concentration of 0.5-3.0 percent
Solidifying Agents or Support SystemsAgar is the most commonly used gelling agent for preparing semisolid and solid plant tissue culture media
Agar advantages over other gelling agents
Mixed with water, it forms a gel that melts at approximately 60-100 C and solidifies at approximately 45C; thus, agar gels are stable at all feasible incubation temperatures
Agar gels do not react with media constituents and are not digested by plant enzymes
The firmness of an agar gel is controlled by the concentration and brand of agar used in the culture medium and the pH of the medium
The agar concentrations commonly used in plant cell culture media range between 0.5 and 1.0%; these concentrations give a firm gel at the pHs typical of plant cell culture media
Solidifying Agents or Support Systems
Carrageenans or carrageenins are a family of linear sulphated polysaccharides extracted from red seaweeds
The name is derived from a type of seaweed that is abundant along the Irishcoastline near the village of Carragheen. Gelatinous extracts of carrageenseaweed (also known as Irish moss) have been used as food additives forhundreds of years
Solidifying Agents or Support Systems
Alginic acid (algine, alginate) is a viscous gum that is abundant in the cell wallsof brown algae.
Chemically, it is a linear copolymer with homopolymeric blocks of (1-4)-linked -D-mannuronate (M) and its C-5 epimer -L-guluronate (G) residues, respectively,covalently linked together in different sequences or blocks.
The monomers can appear in homopolymeric blocks of consecutive G-residues(G-blocks), consecutive M-residues (M-blocks), alternating M and G-residues(MG-blocks) or randomly organized blocks.
The chemical compound sodium alginate is the sodium salt of alginic acid. Itsempirical chemical formula is NaC6H7O6. Its form as a gum, when extractedfrom the cell walls of brown algae, is used by the foods industry to increaseviscosity and as an emulsifier. It is also used in indigestion tablets and thepreparation of dental impressions.
Solidifying Agents or Support Systems
Another gelling agent commonly used for commercial as well as research purposes is Gelrite
Gelrite is synthetic and should be used at 1.25-2.5 g/l, resulting in a clear gel which aids in detecting contamination
Alternative methods of support have included use of perforated cellophane, filter paper bridges, filter paper wicks, polyurethane foam, and polyester fleece.
pHEl pH final del medio de cultivo es un factor importante por diversas razones:
Valores bajos, inferiores a 3.5 impiden la solidificacin de los agentes gelificantes aadidos a los medios slidos Si la evolucin del pH del medio lo hace bajar por debajo de 3.5 se puede producir su licuacin
El valor del pH puede afectar a la solubilidad de algunos componentes del medio de cultivo
El valor del pH puede afectar a la absorcin de determinados nutrientes por parte del explanto (p.e. la absorcin de iones NO3- aumenta con la acidez del medio)
El valor del pH del medio puede afectar al pH del citoplasma y como consecuencia a la actividad de muchos enzimas
pHEn general, se trabaja a pH entre 5.2 y 5.8.
Una vez ajustado el pH del medio, este sufrir una ligera acidificacin durante el proceso de esterilizacin en autoclave, para, despus, evolucionar nuevamente durante el curso del cultivo, de forma que habitualmente se ir acidificando progresivamente como resultado de la absorcin diferencial de algunos componentes del medio de cultivo, as como de la excrecin de exudados por parte del explanto.
El control del pH inicial del medio y de su dinmica durante el cultivo tiene una gran importancia en el desarrollo de cualquier proyecto de cultivo in vitro
A pesar de su importancia, en muchos experimentos este control se limita a fijar su valor inicial sin reparar en los posibles efectos de su dinmica.
Macronutrients
Stock solutions of macronutrients can be prepared at 10 times the concentration of the final medium
Sometimes a separate stock solution for calcium salts may be required toprevent precipitation
Stock solution of macronutrients can be stored safelyfor several weeks in a refrigerator at 2- 4C
The use of stock solutions reduces the number of repetitive operations involved in media preparation and, hence, the chance of human or experimental error
Preparing stock solutions
Micronutrients
Micronutrient stock solutions are generally made up at 100 times their final strength
It is recommended that micronutrient stocks bestored in either a refrigerator or freezer untilneeded
Micronutrient stock solutions could be stored in a refrigeratorfor up to 1 year without appreciable deterioration
Iron stock solutions should be prepared and storedseparately from other micronutrients in an amberstorage bottle
Vitamins
Vitamins are prepared as 100 X or 1000 X stock solutions and stored in a freezer (-20C) until used
Vitamin stock solutions can be stored safely in a refrigerator for 2-3 months but should be discarded after that time.
Growth Regulators The auxins NAA and 2,4-D are considered to be stable and can be stored at 4C for several months
IAA should be stored at -20C
Solution of NAA and 2,4-D can be stored for severalmonths in a refrigerator or indefinitely at -20C
Auxin stock solutions are generally prepared at 100-1000 times the final desired concentrations
Generally IAA and 2,4-D are dissolved in a small volumeof 95% ethyl alcohol or KOH and then brought to volumewith double-distilled water
NAA can be dissolved in a small amount of 1 N NaOH orKOH, which also can be used to dissolve 2,4-D and IAA
Storage of Stock Solutions
For convenience, many labs prepare stock solutions and then divide them into aliquots sufficient to prepare from1 to 10 liter of medium; these aliquots are stored in smallvials or plastic bags in a freezer
This procedure removes the inconvenience of having tothaw a large volume of frozen stock each time mediumis prepared.
Some have found that heating in a microwave oven isa satisfactory and quick method of thawingconcentrated medium.
Conviene tener en cuenta que algunos de los componentes del medio de cultivo pueden ser termolbiles (algunas vitaminas, algunos reguladores). En este caso, la esterilizacin de la solucin que contienen la sustancia termolbil debe hacerse por filtracin y aadirse una vez esterilizada al medio de cultivo previamente esterilizado y parcialmente (en medios slidos) o totalmente (en medios lquidos) enfriado