"Cultivo in vitro de clulas y tejidos vegetales.

Aplicaciones Biotecnolgicas"

Biotecnologa Vegetal
Para qu cultivar in vitro clulas, tejidos y rganos vegetales?
micropropagacin
mejoramiento de plantas
produccin de compuestos de inters in vitro
produccin de metabolitos secundarios
produccin y utilizacin de protenas vegetales
produccin de polmeros biodegradables
establecimiento de plantas transgnicas
plantas resistentes a virus
plantas con rutas metablicas modificadas
plantas mejoradas en cuanto a composicin de
protenas, aceites, etc.
fitorremediacin
remocin de xenobiticos
remocin de metales pesados

Principales conceptos
fisiolgicos aplicables a los
cultivos in vitro

- Totipotencialidad celular

- Indiferenciacin / Rediferenciacin

Concepto de Totipotencia

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

Haberlandt a principios del siglo veinte

Principales conceptos fisiolgicos

aplicables a los cultivos in vitro
Indiferenciacion-rediferenciacin

Tipos de cultivo

Aplicaciones
-Mejora y seleccin gentica
-Conservacin del germoplasma
-Sanidad Vegetal
-Estudios metablicos y fisiolgicos
-Micropropagacin
-Produccin de metabolitos primarios, secundarios y de protenas.
-Bioconversiones y biocatlisis

Cultivos organizados: Son aquellos que mantienen

caractersticas de organizacin estructural. La progenie
presenta idnticas propiedades que el material original.
Por ejemplo el cultivo de rganos.

Cultivos no organizados: Son aquellos en los que las

clulas o tejidos aislados se indiferencian ingresando en un
ciclo de divisin y crecimiento de duracin indefinida. La
principal desventaja de estos el la inestabilidad gentica ya
que se genera frecuentemente poliploida. Por ejemplo las
suspensiones celulares.

Cultivos organizados-no organizados: Son aquellos que se

originan a partir de cultivos indiferenciados (no organizados)
que dan origen a callos y agregados celulares, con cierto grado
de diferenciacin, o bien a rganos o embriones somticos. La
principal desventaja es que la progenie no siempre es
completamente idntica puesto que surgen de cultivos
potencialmente 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

rea de lavado y preparacin

Disponibilidad de agua corriente, agua destilada, agua
bidestilada y/o desionizada
Material de vidrio de uso exclusivo para cultivo
Balanzas, equipos de medicin de pH,
agitadores, mantos de calentamiento,
microondas, heladeras, freezers

Equipos de esterilizacin:
Estufas
AUTOCLAVES !!!!!

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

Lo recomendable Cabina de FLUJO LAMINAR

In a LAMINAR FLOW HOOD the air is forced into the unit through a dust filter then passed through
a 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 air
prevents non-filtered air and particulate matter in the room from settling on the working surface.

Otra opcin: Caja de Guantes

Sala o cmara de cultivo

LA TEMPERATURA
la mayora de las especies desarrollan bien a
temperaturas de incubacin que oscilan entre los
20 y 28C. Para especies de zonas templadas es
ideal 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
Lo importante es el mantenimiento de temperaturas
constantes
As, temperaturas bajas (del orden de 4-5C) permiten superar los
periodos de dormicin de algunas leosas y la conservacin prolongada
de determinados cultivos in vitro;
mientras que una temperatura constante de 20C induce la formacin
de races en la mayora de conferas

LA TEMPERATURA
Como se controla?

Conociendo la temperatura ambiente de la sala donde se site

y el calor generado por las fuentes de luz de que dispone.
El control de la temperatura a la que se desarrolla el cultivo in
vitro se efecta mediante un sistema de refrigeracincalefaccin controlado a travs de un termostato.
El sistema de refrigeracin-calefaccin debe estar
correctamente dimensionado a fin de conseguir que la
temperatura de la zona de cultivo se mantenga dentro de los
lmites 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 que

determinan el desarrollo de los organismos
auttrofos, en ello radica la importancia de
controlar el factor luz en los cultivos in vitro.

LA LUZ
LA 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 LUZ

LA CANTIDAD DE LUZ: LA IRRADIACIN:

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)
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 LUZ
Principales fuentes de luz usadas en las cmaras de cultivo

LAMPARAS
INCANDESCENTES

Producen la luz por fenmenos de incandescencia del filamento calentado por

el paso de la corriente elctrica. Buena parte del espectro se halla en la zona
del 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 arco

voltico. El espectro de la luz producida es rico en la zona del azul, existen
fluorescentes especiales con un espectro rico en la zona azul y roja. Consumen
menos electricidad.

LAMPARAS DE VAPOR DE
MERCURIO Y SODIO

Producen la luz por efecto del paso de la corriente elctrica a travs

de 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 consigue
mediante un programador conectado al circuito de
iluminacin.
El programador del fotoperiodo puede estar
relacionado con el programador de temperaturas,
de forma que se puedan programar diferentes
temperaturas segn sea la fase del fotoperiodo en
la que se halle el cultivo.

Qu tener en cuenta para cultivar in vitro vegetales?

Tissue Culture Media-Composition

One of the most important factors governing the growth
and morphogenesis of plant tissues in culture is the
composition of the culture medium.
The basic nutrient requirements of cultured plant cells
are very similar to those of whole plants.
Plant tissue and cell culture media are generally made up of some or all of
the following components:
macronutrients, micronutrients, vitamins, amino acids or other nitrogen
supplements, sugar(s), other undefined organic supplements, solidifying
agents 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 tissue
growth

The optimum concentration of each nutrient for achieving

maximum growth rates varies considerably among species

Macronutrients

Culture media should contain at least 25-60 mM of inorganic

nitrogen for adequate plant cell growth

NITRATES RANGE 25-30 mM

Plant cells may grow on nitrates alone, but considerably better results are
obtained when the medium contains both a nitrate and ammonium nitrogen
source

AMMONIUM RANGE 2 - 20 mM
For certain species ammonium concentrations in excess of 8 mM may be
deleterious to cell growth of certain species
Some cells can grow on a culture medium containing ammonium
as the sole nitrogen source if one or more of the TCA cycle acids
(e.g., citrate, succinate, or malate) are also included in the culture
medium at concentrations of approximately 10 mM.
When nitrate and ammonium sources of nitrogen are utilized together in the culture
medium, the ammonium ions will be utilized more rapidly and before the nitrate ions.

Macronutrients

P
Mg
S
Ca

Potassium is required for cell growth of most plant

species. Most media contain K, in the nitrate or chloride
form, at concentrations of 20-30 mM.

The optimum concentrations of P, Mg, S, and Ca

range from 1-3 mM when all other requirements for
cell growth are satisfied.
Higher concentrations of these nutrients may be
required if deficiencies in other nutrients exist.

Micronutrients
The 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

Cu
Co

concentrations of 0.1 M

concentrations 5 M

Mn

concentrations 20-90 M

Mo

Zn

concentrations 1 M

concentrations 5-30 M

concentrations 25-100 M

Componentes (mg/L)

Chu N6

Ammonium nitrate

DKW/Juglan

Gamborg B5

Gamborg B-5
minimal
organics

Hoagland

1416.0

Boric acid
Calcium chloride anhydrous

1650.0

Quoirin
and
Lepoivre

463.0

134.0

134.0

1.6

4.8

3.0

3.0

125.33

112.5

113.24

113.24

1367.0

Cobalt chloride 6H2O

Cupric sulfate 5H2O
37.25

2.86

656.4
0.025

0.025

0.25

0.025

0.025

45.4

37.3

37.25

0.08

Schenk
and
Hildebrandt

300.0

6.2

6.2

72.5

332.2

386.0

6.2

5.0

833.77

200.0

0.025

0.025

0.1

0.25

0.025

0.025

0.2

37.3

37.26

37.3

20.0
2.5

Ferric tartrate 2H2O

5.32

Ferrous sulfate 7H2O

27.85

33.8

27.8

27.85

Magnesium sulfate

90.37

361.49

122.09

122.09

Manganese chloride 4H2O

240.76

27.8

27.8

15.0

180.7

180.7

175.79

195.4

360.0

22.3

16.9

0.76

10.0

5.04

0.25

0.25

0.25

0.1

1.81
3.33

33.5

10.0

10.0

Molybdenum trioxide

0.016

Molybdic acid (sodium salt)

2H2O

0.39

0.25

0.25

Potassium chloride

65.0

Potassium iodide

0.8

0.75

0.75

Potassium nitrate

2830.0

2500.0

2500.0

Potassium phosphate
monobasic

400.0

Potassium sulfate

606.6

265.0

170.0

1559.0

990.0

Sodium phosphate monobasic

130.5

0.83

0.08

1.0

0.75

1900.0

1800.0

2500.0

80.0

170.0

270.0

130.5

16.5

Sodium sulfate

200.0

Zinc nitrate 6H2O

Zinc sulfate 7H2O

1.5

151.0

Ferric sulfate

Manganese sulfate H2O

White

400.0

115.03

Calcium nitrate

Na2-EDTA

Murashige
and
Skoog

400.0

Ammonium phosphate
monobasic
Ammonium sulfate

McCown
woody
plant

17.0
1.5

2.0

2.0

0.22

8.6

8.6

8.6

1.0

2.67

Carbon and Energy Source

Carbohydrates must be supplied to the culture medium because only a few
plant cell lines are fully autotrophic.

The preferred carbohydrate in plant cell culture media is sucrose

Sucrose
concentrations of culture media normally range between 2 and 3 percent

Glucose and fructose are used in some cases

Use of autoclaved fructose can be detrimental to cell growth!!!!

Other carbohydrates that have been tested include lactose, galactose, rafinose,
maltose, and starch.

Vitamins
When plant cells and tissues are grown in vitro,
some vitamins may become limiting factors for
cell growth
Normal plants synthesize the vitamins required for their growth
and development.
Vitamins are required by plants as catalysts in various metabolic
processes

The vitamins most frequently used in cell and tissue culture

media include thiamin (B1), nicotinic acid, pyridoxine (B6),
and myo-inositol

Vitamins

Thiamin
B1

It is basically required by all cells for growth.

Concentrations range from 0.1 to 10.0 mg/l.

Nicotinic acid

concentrations of 0.1-5.0 mg/l

Pyridoxine B2

used at 0.1-10.0 mg/l

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 cell
culture media.

The requirement for these vitamins by plant cell cultures is generally

negligible, and they are not considered growth-limiting factors.
These vitamins are generally added to the culture medium only
when the concentration of thiamin is below the desired level or when
it is desirable to grow cells at very low population densities.

Myo-inositol is commonly included

in many vitamin stock solutions

OH
HO
HO

Although it is a polialcohol derived

from carbohydrates not a vitamin

OH

HO
OH

it has been shown to stimulate growth in

certain cell cultures.

Myo-inositol is generally used in plant cell

and tissue culture media at

concentrations of 50-5000 mg/l

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

GB Vitamins contains:
1.0 mg Nicotinic Acid,
100 mg myo-Inositol,
1.0 mg Pyridoxine HCl,
10.0 mg Thiamine HCl

LS Vitamins contains:
100 mg myo-Inositol
0.1 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 or
amino acid mixtures may be used to further stimulate cell growth
The use of amino acids is particularly important for establishing
cell 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 than
inorganic 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 cell
cultures 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.

AMINO ACIDS OR OTHER NITROGEN SUPPLEMENTS

The most common sources of organic nitrogen used in culture

media are amino acid mixtures (e.g., casein hydrolysate), Lglutamine, L-asparagine, and adenine

Casein hydrolysate concentrations between 0.05 and 0.1 percent

Examples of amino acids included in culture media to enhance

cell 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.

Undefined Organic Supplements

Addition 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 milk
and protein hydrolysates are used to any extent today

coconut milk is commonly used at 5-20% (v/v).

Undefined Organic Supplements

The addition of activated charcoal (AC) to culture media may have
a beneficial effect
AC may cause:
absorption of inhibitory compounds
absorption of growth regulators from the culture medium
and 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
The inhibition of growth in the presence of AC is generally
attributed to the absorption of phytoregulators, such as 1Napthaleneacetic acid (NAA), kinetin, 6-benzylaminopurine
(BA), indole-3-acetic acid (IAA), and 6--dimethylallylaminopurine (2iP)
AC is generally acid-washed prior to addition to the culture medium
at a concentration of 0.5-3.0 percent

Solidifying Agents or Support Systems

Agar 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 Irish
coastline near the village of Carragheen. Gelatinous extracts of carrageen
seaweed (also known as Irish moss) have been used as food additives for
hundreds of years

Solidifying Agents or Support Systems

Alginic acid (algine, alginate) is a viscous gum that is abundant in the cell walls
of brown algae.
Chemically, it is a linear copolymer with homopolymeric blocks of (1-4)-linked -Dmannuronate (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. Its
empirical chemical formula is NaC6H7O6. Its form as a gum, when extracted
from the cell walls of brown algae, is used by the foods industry to increase
viscosity and as an emulsifier. It is also used in indigestion tablets and the
preparation 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.

pH
El 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

pH
En 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.

Preparing stock solutions

The use of stock solutions reduces the number of
repetitive operations involved in media preparation and,
hence, the chance of human or experimental error

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 to
prevent precipitation

Stock solution of macronutrients can be stored safely

for several weeks in a refrigerator at 2- 4C

Micronutrients
Micronutrient stock solutions are generally made up at
100 times their final strength
It is recommended that micronutrient stocks be
stored in either a refrigerator or freezer until
needed
Micronutrient stock solutions could be stored in a refrigerator
for up to 1 year without appreciable deterioration

Iron stock solutions should be prepared and stored

separately from other micronutrients in an amber
storage 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 several
months in a refrigerator or indefinitely at -20C
Auxin stock solutions are generally prepared at 1001000 times the final desired concentrations
Generally IAA and 2,4-D are dissolved in a small volume
of 95% ethyl alcohol or KOH and then brought to volume
with double-distilled water
NAA can be dissolved in a small amount of 1 N NaOH or
KOH, 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 from
1 to 10 liter of medium; these aliquots are stored in small
vials or plastic bags in a freezer
This procedure removes the inconvenience of having to
thaw a large volume of frozen stock each time medium
is prepared.

Some have found that heating in a microwave oven is

a satisfactory and quick method of thawing
concentrated 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