Category: Tissue Culture


Preparation of plant culture Media requires high technicality and also human efforts to achieve the required sterility. Any form of contamination during the stages of preparation will ruin the media particular.

Media

The protocol for preparing a tissue culture Media under aseptic environment is stated as follows

Proceedure

Glass wares to be used for the preparation of the media was thoroughly washed with liquid soap and rinsed with clean water (test tubes, beakers, measuring bowl, and test tube caps)

The washed apparatus was autoclaved.

The components of the media (salts, vitamins, amino acids, growth regulators, sugars, agar, gelrites and water) was weighed at their respective measurements as stated by the protocol.

when using powdered ms ( murashige and skoog basal media)
80% sterile distilled water of the total volume of the medium to be prepared was poured into a beaker.
The beaker was placed on a magnetic stirrer
Except gelrite or agar, all other component of the media was poured into the beaker simultaneously and allowed to stir well.
NB: when using stock prepared ms, the initial volume of sterile water is reduced to about 40% or 50% of water.

pH of the media was adjusted with a pH meter, IM HCl and IM NaOH. pH of 5.8 was used for fluted pupkin, citrus, irvingia and tomatoes.

The volume of the media was completed with sterile distilled water.

The measured amount of gelrite was added into the media and stirred well with the aid of a magnetic stirrer.

The media was boiled with the aid of microwave or oven.
Boiled media was poured into the sterile test tubes (14mls each) or bama bottles (20mls each) and covered well with their caps.

The media was sterilized with the aid of an autoclave
After the sterilization process, the Media is kept in a sterile room for solidification.

Preparation Of Stock Hormones
Hormones used in tissue culture are prepared in stock solutions of concentration mainly in 1mg/ml or 0.1mg/ml. They are used to induce desired growth and development of explants in a media in-vitro. They are kept in a refrigerator after preparation to shield them from degrading.
The procedures for preparing some of these hormones are as follows:

200mls OF 1.05mg/ml 6-Benzylaminopurine (BAP+)
0.21g of 6-benzylaminopurine powder is weighed
About 1ml of1M NaOH is used to dissolve the solute
The solution is bought to volume with sterile distilled water
The solution is poured into a conical flask and stored in a refrigerator. Which will be used in media preparation

50mls OF 1mg/ml 6-Benzylaminopurine (BAP)
0.05g of 6-benzylaminopurine powder is weighed
(ii) About 1ml of1M NaOH is used to dissolve the solute
(iii) The solution is brought to volume with sterile distilled water
The solution is poured into a conical flask and stored in a refrigerator. Which will be used in media preparation

20mls OF 1mg/ml 1-Naphthaleinacetic Acid (NAA)
20mg of 1-naphthaleinacetic acid salt is weighed
About 1ml of1M 70% ethanol is used to dissolve the solute
The solution is brought to volume with sterile distilled water
The solution is poured into a conical flask and stored in a refrigerator. Which will be used in media preparation

20mls OF 1mg/ml  (IAA)

20mg of indole-3-acetic acid salt is weighed
About 1ml of 70% ethanol is used to dissolve the solute
The solution is brought to volume with sterile distilled water
The solution is poured into a conical flask and stored in a refrigerator for future media preparation.

50mls OF 0.1mg/ml Kinetine

5mg of kinetine salt is weighed
About 1ml of1M NaOH was used to dissolve the solute
The solution is brought to volume with sterile distilled water
The solution is poured into a conical flask and stored in a refrigerator for future media preparation

50mls Of 0.1mg/Ml Gibberellic Acid (Ga3)

5mg of gibberellic acid salt is weighed
The solute is dissolved in water
The solution is brought to volume with sterile distilled water
The solution is poured into a conical flask and stored in a refrigerator for media preparation

Preparation Of Ascorbic Acid (Vitamine C)

Ascorbic acid is very essential in tissue culture as it performs a vital role. It functions in the aid to heal up of wounds created by cutting of the explants during culturing. It stops the phenolic compounds that rush out after cutting of explants.
It is prepared as a stock solution and stored in the refrigerator to preserve it from degrading.
The procedures are as follows:

100mls Of Ascorbic Acid400mg of L-Ascorbic acid salt will be weighed

The solute is dissolved in water

The solution is brought to volume with sterile distilled water

The solution is poured into a conical flask and stored in a refrigerator for media preparation.

Precaution

Preparing a media is a primary step towards achieving a successful culture. It is the first line of action by the technician in achieving a great and quality explant culture.

The following precautions can be taken to achieve these high aseptic medium.

  • Always make sure that the glass apparatus are well washed before the process of preparation is being undertaking.
  • Avoid talking, sneezing or other at  acts which can bring contamination into the media being prepared.
  • Ensure that the equipment or machineries used in dispensing or autoclaving the media are in better positions.  Aany deviation in these machines can lead to a large mass contamination of the media. Considering the dispensing machine or the dispenser which cannot be heated during the process, there is need for a high Technical know how or technicality.
  • Always ensure a high aseptic environment during the preparation of a media. An environment filled with dust or free movement of air is  prone to contaminate the media. Hence, there should be a control over the environment during the preparation of Media.

Conclusively,

Media preparation is the chief process in the entire culturing technique. The simple rules has it that:  ensure an aseptic environment and be precise in the measurements of the constituents of the media.

Once these two rules are kept, you are already at the success positions in the preparation of culture media and stock solutions.

Hope these tips are helpful?

Check out equipments used in tissue culture

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