Stock solutions

1. UNIVERSITY OF AGRICULTURE
SCIENCE, DHARWAD
College of agriculture, Hanumanamatti
BTH - 311
Topic - Stock solutions and Do’s and don‘ts of tissue culture
Laboratory
Submitted by,
Parvati D U
UGS20AGR9918

2. Definition:
A Stock solution is a
concentrated solution
which is used to prepare
dilute solution of some
lower concentration for
actual use.

3. Prepation of stock solutions -
A stock solution can be prepared by weighing out an
accurate amount of pure solid or by measuring out an
accurate volume of pure liquid and diluting to a
known concentartion

4. Stock solution can be prepared by three different
concentration terms like:
1.Molar stock solution
2.Normal stock solution
3.Percent stock solution

5. Stock solution dilution formula

6. M1×V1 = M2×V2
0.150 M × 1.25 L = M2 × 2.50L
M2 = 0.07 M
• 0.125M is a molarity of diluted solution.

7. Preparing stock solution
• To prepare a stock solution, weigh out the required
amount of the compound and place it in a clean flask.
• It is common practice to make a stock solution 10x or
100x, depending upon the solubility of the compound.
• Once the chemical is in the flask, dissolved it in a small
amount of water, ethyl alcohol, 1 N NaOH, or 1 N HCL.

8. • Next, slowly add double-distilled water to the flask,
while agitating.
• Continue this until the proper volume is reached. Label
the flask with the name of the solution, preparation
and expiration dates, and the name of the person who
prepared the solution.
• Certain items, e.g., IAA, must be prepared and stored in
amber bottles to prevent photodecomposition.

9. Macronutrients : Stock solutions of macronutrients can
be prepared at 10 times the concentration of the final
medium. 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°-4°C.
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.
Iron stock solutions should be prepared and stored
separately from other micronutrients in an amber storage
bottle.

10. Vitamins: Vitamins are prepared as 100X or 1000X stock
solutions and stored in a freezer (-20°C) 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 4°C for
several months
Auxin stock solutions are generally prepared at 100-
1000 times the final desired concentrations. Solution of NAA
and 2,4-D can be stored for several months in a refrigerator or
indefinitely at -20°C.

11. Advantages:
• Since stock solutions are highly concentrated,
their storage requires less space.
• It is also convenient to transport stock solutions
due to their small volume.
• The concentration of various components of a
working solution is more accurate when it is made
from the stock solution rather than individual
components from its original form.

12. • Stock solutions of biologically active
substances (e.g., enzymes, inhibitors, DNA,
RNA, etc.) are generally more stable in highly
concentrated form.
• They are especially helpful in situations
where you need a very minute amount of a
component to prepare a complex solution.

13. Disadvantages
• Since stock solutions are highly concentrated, often
dissolving them requires more time and effort.
Sometimes, heating of the solution is required to
dissolve the substances completely.
• Long-term storage of stock solutions (especially salt
solution, at low temperature) may lead to precipitation
of solute.

14. Storage of stock solution:
Storage conditions for most stock solutions
have already been pointed out; however, some
additional points can be made. 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.

15. We recommend that stock solutions, once
prepared, are stored as aliquots in tightly sealed
vials at -20°C. Generally, these will be useable for
up to one month.
[An aliquot is a piece or a sub-sample of a larger
whole, particularly one extracted for chemical
analysis or other treatment.]

16. Of tissue culture laboratory
DO’S AND DON’TS

17. Do’s
• Always wear personal protective equipment (PPE): To save
your cultures from getting contaminated and yourself
from harmful chemicals always wear a mask, gloves,
disposable caps to cover hair, and a clean lab coat. While
working with liquid nitrogen, wear thermally isolated
gloves, eye protection, and a splash-proof apron.
• Try to keep two PPE kits for the washing and media
preparation area and the tissue culture area of the lab. You
can make it easier by keeping two different colored coats
in both labs.
• Keeping the culture area, media preparation area, and
transfer area clutter-free helps to reduce mistakes while

18. • The most common mistake done while working in
the lab is preparing multiple reagents or solutions
simultaneously and not labeling them. This creates
confusion and may lead to the disposal of solutions.
So, always label the solutions or reagents with
content and dates of preparation as soon as you
prepare. This also helps to avoid the use of a solution
after its expiration date.
• Work with one kind of tissue or perform one
experiment at a time. Don’t try to multitask but be
arranged as much as possible. This will also reduce
the chances of cross-contamination by mislabeling
and the spread of microbes from numerous opened
media bottles due to the generation of aerosols.

19. • Always use 70% isopropanol or ethanol to clean
the work area or surface. You can use a clean
paper towel to wipe the area. This should be done
before you initiate the experiment or in between
operations.
• If you are working with multiple cell lines then it’s
advisable to maintain separate bottles of media
for each cell line in culture/cultivation.
• Regularly examine your cultures and commercially purchased
media for any signs of gross bacterial and fungal contamination.
This also prevents the spreading of microbial contamination (if
any) invaded your cultures.

20. • After you buy media or reagents always check its
quality control information and ideally examine
its performance before involving it in your
experiments. Checking the information will tell
you what conditions are required to store your
chemicals to maintain their performance.
• Keep minimum cardboard packaging in all the
cell culture areas.
• Always ensure at regular intervals that all the
machines installed in the media preparation area,
transfer area, and culture area are working

21. • Mycoplasmas are endophytic bacteria that
live inside cells. So, always test
explants/starting material whenever you
are going to work with any for the
presence of this bacteria.

22. Don’ts
• The continuous use of antibiotics should be strictly
avoided as this can lead to the generation of
resistant strains in the culture and may mask the
underlying contamination. The use of antibiotics is
also avoided in plant tissue culture because it slows
down the growth of cultures.
• Avoid the accumulation of any kind of waste in
microbiological safety cabinets or incubators.
• Do not make the culture area crowded at one time.
It is advisable to have only one person at a time
while working especially in the culture or transfer

23. • Don’t bring cells from unauthenticated sources
in the main culture area without completing the
quality control checks. You can also use a cell
supplier that authenticates cells.
• Don’t keep cell lines continually in cultures
without returning frozen stock.
• Avoid cultures to run out of media. So, always
subculture them after 70% of the media is
utilized or after a fixed interval of days suitable
for a particular culture.

24. • Do not allow the media to cross the
expiration date. The shelf life is only 4-6
weeks at +4°C after adding glutamine and
serum.
• Don’t let the water baths get dirty. So,
clean them regularly.
• Don’t allow the essential equipment used
in the labs to become out of calibration.
This will reduce analytical mistakes.

25. Thank you