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Membuat Stok Zat Pengatur Tumbuh (ZPT)

Esha Flora - Monday, February 08, 2010

Membuat Stok ZPT untuk kebutuhan Kultur Jaringan


Seperti halnya vitamin zat pengatur tumbuh juga dibutuhkan dalam jumlah yang relatif sedikit, umumnya macam
dan jumlah penggunaannya tergantung pada kebutuhan dan orientasi kultur jaringan termasuk pertimbangan bahan
apa
yang
hendak
dikultur
jaringan
.
Umumnya stok zat pengatur tumbuh untuk kultur jaringan dilakukan dengan dasar berat per volume (kepekatan).
Biasanya larutan stok zat pengatur tumbuh dibuat dengan kepekatan antara 1-10 mg/ml. Sehingga pengambilannya
tinggal memperhatikan berapa kebutuhan perliter yang diinginkan dan berapa kepekatan larutan stok yang telah
dibuat.
pH terakhir dari media kultur jaringan setelah penambahan hormon, sebenarnya tidak perlu mengukur dengan tepat
jumlah asam atau basa yang ditambahkan dalam membuat larutan persediaan. Tapi jumlah penambahan asam atau
basa tepat dapat diperkirakan dengan mudah. Cara praktis, dapat dikatakan bahwa 5 ml 0,1 N Natrium hidroksida
(0,4%) tiap 100 mg auksin dan 5 ml 0,1 N asam khlorid (0,86 ml Asam khlorid pekat dalam 100 ml air) tiap 100
mg sitokinin, adalah cukup.
Etil alkohol, dimetil sulfoksida dan pelarut organik yang lain kadang-kadang dipakai sebagai pembantu pelarut
waktu membuat larutan persediaan hormon. Dalam memakai metode ini haruslah diingat bahwa kadar dari
pembantu pelarut dalam media kultur Jaringan tidak boleh tinggi, karena toksik.
Membuat larutan stok zat pengatur tumbuh untuk kultur jaringan sedikit berbeda dengan membuat larutan stok
hara atau vitamin yang mudah larut hanya dengan aquadest. Untuk zat pengatur tumbuh diperlukan asam atau basa
dan kadang pemanasan sebagai cara mempercepat larutnya bahan. Kalau zat pengatur tumbuhnya bersifat asam
maka pelarutannya basa (NaOH 1 N) misalnya: IAA, NAA, IBA, 2,4-D. Sedangkan bila basa, maka pelarutannya
adalah asam (HCI 1 N) misalnya dari golongan sitokinin.
Untuk melarutkan zat pengatur tumbuh selain menggunakan asam dan basa dapat pula melarutkannya dalam
alkohol 40%, atau melalui pemanasan saja. Berdasarkan pengalaman menggunakan tambahan asam atau basa lebih
efektif dan tidak mudah mengalami pengendapan. Kerusakan larutan stok zat pengatur tumbuh umumnya ditandai
dengan munculnya jamur, bakteri, larutan menjadi keruh. Jika demikian lebih baik tidak dipergunakan lagi
Contoh pembuatan larutan stok zat pengatur tumbuh dengan kepekatan 1 mg/ml sebanyak 100 ml, dan prosedurnya
adalah sebagai berikut:
Untuk membuat larutan stok auksin (IAA, NAA, IBA, 2,4-D).

- Menimbang bahan sebanyak 100 mg, kemudian dimasukkan kedalam gelas piala yang diberi aquadest sedikit.
Teteskan sedikit demi sedikit NaOH 1 N kedalam gelas tadi sambil dikocok hingga zat pengatur tumbuh larut
merata.
- Tambahkan aquadest hingga volume mendekati 70 ml, dikocok kembali kemudian tuangkan kedalam labu ukur.
- Bilas gelas piala dengan aquadest sedikit demi sedikit hingga bersih, selanjutnya tambahkan lagi aquadest
kedalam labu ukur hingga volume tepat 100ml.
- Pindahkan larutan tersebut kedalam Erlenmeyer 100ml, ditutup rapat dengan aluminium foil, diberi label dan
kemudian disimpan dilemari es.
- Penggunaanya, misalnya ke dalam 1 liter media akan ditambahkan zat pengatur tumbuh sejumlah 1 mg atau 1
ppm, maka hanya dibutuhkan 1 ml saja dari larutan stok zat pengatur tumbuh. Bila 5 ppm per liter media maka
dibutuhkan 5 ml, demikian seterusnya.
Untuk

larutan

Stok

sitokinin

(Zeitin,

BA,

2-iP,

PBA,

Kinetin)

- Menimbang bahan sebanyak 100 mg, kemudian memasukkan kedalam gelas piala yang diberi aquadest 50 ml.
Teteskan sedikit demi sedikit HCI 1 N kedalam gelas tadi sambil dipanaskan dan dikocok (menggunakan hot plate
stirrer) hingga zat pengatur tumbuh larut merata.
- Tambahkan aquadest hingga volume mendekati 70 ml, dikocok kembali hingga merata kemudian tuangkan
kedalam labu ukur.
- Bilas gelas piala dengan aquadest sedikit demi sedikit hingga bersih, selanjutnya tambahkan aquadest kedalam
labu ukur hingga volume tepat 100 ml.
- Pindahkan larutan tersebut kedalam erlenmeyer 100 ml, tutup rapat dengan aluminium foil, beri label dan
kemudian simpan di lemari es.
- Penggunaanya, misalnya kedalam 1 liter media akan ditambahkan zat pengatur tumbuh sejumlah 2 mg atau 2
ppm, maka hanya dibutuhkan 2 ml saja dari larutan stok zat pengatur tumbuh. Bila diperlukan 8 ppm perliter media
maka dibutuhkan 8 ml, demikian seterusnya.

For example, the pdf linked to above describes preparing a stock solution of NAA like so:

Code:

NAFTALENACETIC ACID (NAA)


1 Stock solution of NAA 1,000 ppm
Weigh 0.2 g of NAA and dissolve well with some NaOH 1N drops.
2. Add 200 ml of distilled water.
Keep it in a conveniently labeled vial at 0~O.
One ml of stock solution (1,000 ppm) contains 1 mg of NAA.

BAP is also described.


Another site:
Code:
NAA

1 mM stock solution

--> 9.31mg/50ml

disolve in about 2.5 ml 95% etoh or 2.5 ml of 1M KOH or NaOH stir, heat
gently and gradually add water to volume. pH to 5.0
BAP
1 mM stock solution --> 11.25 mg/50ml
Disolve in 2.5 ml of 0.5 M HCl, heat gently and make to volume.
to 5.0

Adjust pH

I was never very good at making solutions, so take what I say with a pinch of salt.

How to convert ppm to percent


1% = 1/100
1ppm = 1/1000000
So
1ppm = 0.0001%
So to convert from ppm to percent, divide the ppm by 10000:
x(%) = x(ppm) / 10000

Example: find how many percent are in 300ppm.


x(%) = 300ppm / 10000 = 0.03%

ppm to percent conversion table


ppm

Percent (%)

0 ppm

0.0000%

1 ppm

0.0001%

2 ppm

0.0002%

3 ppm

0.0003%

4 ppm

0.0004%

5 ppm

0.0005%

6 ppm

0.0006%

7 ppm

0.0007%

8 ppm

0.0008%

9 ppm

0.0009%

10 ppm

0.0010%

20 ppm

0.0020%

30 ppm

0.0030%

40 ppm

0.0040%

50 ppm

0.0050%

60 ppm

0.0060%

70 ppm

0.0070%

80 ppm

0.0080%

90 ppm

0.0090%

100 ppm

0.0100%

200 ppm

0.0200%

300 ppm

0.0300%

400 ppm

0.0400%

500 ppm

0.0500%

600 ppm

0.0600%

700 ppm

0.0700%

800 ppm

0.0800%

900 ppm

0.0900%

1000 ppm

0.1000%

2000 ppm

0.2000%

3000 ppm

0.3000%

4000 ppm

0.4000%

5000 ppm

0.5000%

6000 ppm

0.6000%

7000 ppm

0.7000%

8000 ppm

0.8000%

9000 ppm

0.9000%

10000 ppm

1.0000%

100000 ppm

10.0000%

1000000 ppm

100.0000%

What does "ppm" mean?

Go to "Converting ppm and molarity"


Return to Solutions Menu

The expression "1 ppm" means a given solute exists at a concentration of one part per million parts of the solution.
These are two common ways to think about what the concentration "1 ppm" means:
a) it is one-millionth of a gram per gram of sample solution.

b) it is one gram of solute per million grams of sample solution.


Notice that the more general word 'part' is used above, but 'gram' is used in (a) and (b) just above. This is because
'gram' is used almost exclusively when parts per million is used.
The best way to explain this is by doing some examples:
Problem #1: Sea water contains 3.90 x 106 ppm of dissolved gold. What volume of this sea water would contain
1.00 g of gold?
Solution:
1) 3.90 x 106 ppm means this:
3.90 x 106 g of Au per 1.00 gram of seawater

2) We use a ratio and proportion:


(3.90 x 106 g of Au 1.00 gram of seawater) = (1.00 g of Au x)

3) Cross multiply and divide yields our answer:


2.56 x 105 g of sea water contains 1.00 g of gold

Problem #2: Pollutants in air and water are frequently measured in parts per million (ppm) or parts per billion
(ppb). One part per million would mean that there is one gram of the pollutant in every one million grams of air. At
ordinary temperature and pressure, air has a density of 0.00012 gram per cubic centimeter. What volume of air
would contain one gram of sulfur dioxide, a pollutant that causes acid rain, if the sulfur dioxide concentration is 2
ppm.
Solution:
1) How many grams of air contain one gram of SO2?
2 ppm means 2 grams SO2 per million grams of air

therefore,
one gram of SO2 will be found in 500,000 g of air
2) What volume of air weighs 500,000 g? We use the density (notice I set it up in a ratio and proportion style:

0.00012 g over 1 cm3 = 500,000 g over x

x = 4.17 x 109 cm3

Problem #3: A sample of oil (density = 0.89 g/mL) was found to have dioxin contamination of 2 ppm. How many
mL of the oil would contain 0.01 gram of dioxin?
Solution:
1) Determine grams of oil holding 0.01 g of dioxin. Use a ratio and proportion:
(2 g dioxin over 1,000,000 g solution) = (0.01 g over x)

x = 5000 g
2) Determine the volume of oil:
0.89 g/mL = 5000 g divided by x

x = 5618 mL

Problem #4: Fish need about 5 ppm O2 dissolved in water to survive. Will water with 7 mg O2 per liter sustain
fish?
Solution:
We need to convert 7 mg/L into ppm:
(0.007 g / 1000 g) = (x / 1,000,00 g)

x = 7 ppm
The answer is yes.
Notice the conversion of one liter of water (with 7 mg dissolved oxygen) into 1000 g. I used the density of 1.00
g/mL for the conversion. This is acceptable because solutions in the ppm range are so dilute that the solute (in this
case, the O2, has no effect on the solution density.
Look again at the mg/L value. Another way to explain getting from mg/L to ppm is to multiply both numerator and
denominator by 1000:
(0.007 g / 1000 g) x (1000 / 1000) = 7 g / 1,000,000 g = 7 ppm

You may very well face problems where you get the data in mg/L format and are then asked something about ppm.
Remember!

Problem #5: Show why a mass of 0.145 g of KMnO4 in a 500 mL volumetric flask corresponds to 100 ppm in Mn.
Solution:
1) Using a gravimetric factor, determine grams of Mn in 0.145 g of KMnO4:
0.145 g times (54.938 g/mol over 158.032 g/mol) = 0.0504076 g

2) Calculate ppm of Mn. Use a ratio and proportion:


(0.050 g over 500 g) = (x over 1,000,000)

x = 100 ppm

Problem #6: Symptoms of lead poisoning become apparent after a person has accumulated more than 20 mg in the
body. Express this amount as parts per million for an 80 kg person.
Solution:
1) Convert mg and kg to grams:
20 mg = 20 x 10-3 g
80 kg = 80 x 103 g

2) Determine grams Pb per g of bodyweight:


20 x 10-3 g / 80 x 103 g = 0.25 x 10-6 per g of bodyweight

3) Refer to first description of ppm given at beginning of file:


one ppm "is one-millionth of a gram per gram of sample solution."

In this case, the "solution" is the bodyweight.


answer = 0.25 ppm

Problem #7: If you eat a 6 oz. can (180g) of tuna with 0.20 ppm Hg, how much mercury do you ingest?
Solution:
1) The meaning of 0.20 ppm Hg:
Remember, one ppm "is one-millionth of a gram per gram of sample solution."

Therefore the tuna contains 0.20 x 10-6 g of Hg per gram of tuna.


2) Calculate Hg in 180 g of tuna:

0.20 x 10-6 g of Hg per g of tuna times 180 g of tuna = 3.6 x 10 -5 g of Hg

Problem #8: It is estimated that 3 x 105 tons of sulfur dioxide enters the atmosphere daily owing to the burning of
coal and petroleum products. Assuming an even distribution of the sulfur dioxide throughout the earth's atmosphere
(which is not the case), calculate in parts per million by weight the concentration of SO2 added daily to the
atmosphere. The weight of the atmosphere is 4.5 x 1015 tons.
Solution:
1) Set up a ratio and proportion:
You are adding 3 x 105 parts per 4.5 x 1015 parts.

How many parts per 1 x 106 is this?


2) Solve it:
3 x 105 parts is to 4.5 x 1015 parts as x is to 1 x 106 parts

x = 0.000067 ppm

Problem #9: A solution used to chlorinate a home swimming pool contains 7% chlorine by mass. An ideal chlorine
level for the pool is one part per million chlorine. If you assume densities of 1.10 g/mL for the chlorine solution
and 1.00 g/mL for the swimming pool water, what volume of the chlorine solution in liters, is required to produce a
chlorine level of 1.00 ppm in an 18,000 gallon swimming pool?
Solution:
1) Convert 18,000 gallons to liters:
The conversion factor we will use is 1 gallon = 3.7854 L

18,000 gal x 3.7854 L/gal = 6.81372 x 104 L


2) Determine how many grams of pool water this is:
6.81372 x 107 mL x 1.00 g/mL = 6.81372 x 107 g

Note change from L to mL.


3) At 1 ppm, how much chlorine is required? Use a ratio and proportion:
(1 g chlorine 106 g pool water) = (x 6.81372 x 107 g of pool water)

x = 68.1372 g chlorine required


4) What amount of 7% (by mass) chlorine solution is required to deliver 68.1372 g of chlorine?

(68.1372 g 0.07) = (x/1)

x = 973.3886 g of chlorine solution required


5) What volume (in liters) is this?
973.3886 g 1.10 g/mL = 884.8987 mL

To three sig figs (which seems reasonable to the ChemTeam), the answer is 0.885 L.