A manual of rice seed health testing - IRRI books - International Rice ...

Appendix 2. Media, stains, buffer and their
preparations.
I. Media preparation
Unless otherwise specified, all media are
made up to 1 liter with distilled water and
sterilized at 121 °C for 15 min (15 psi).
Filter-sterilized cycloheximide can be added
to the molten media to give a final concentration
of 0.01%. [Dissolve 1 g
cycloheximide in 10 ml distilled water and
filter sterilize (10% solution). Add 1 ml to 1
liter molten medium.]
Aqueous Lugol's iodine
Iodine powdered crystals (I 2
)
Potassium iodide (KI)
Distilled water
5.0 g
10.0 g
100.0 ml
Method of preparation:
1) Stock solution. Dissolve KI in distilled
water. Slowly add l 2
crystals and shake until
dissolved. Keep the solution in a tightly
closed brown bottle.
2) Working solution. Make up a 1:5 dilution
of the stock solution with distilled water
each time before using.
Arginine medium ( Thornley's Medium 2A )
Phenol red
0.01 g
Dipotassium phosphate 0.3 g
(K 2 HPO 4 )
Peptone
1.0 g
Agar 3.0 g
Sodium chloride (NaCI) 5.0 g
Arginine HCI
10.0 g
Distilled water
1.0 liter
In case it has an orange color, adjust pH to
6.8 with 1N HCI until the medium turns yellow.
Ayers et al mineral salts medium
Potassium chloride (KCI) 0.2 g
Magnesium sulfate
0.2 g
(MgSO 4 ·7H 2
O)
Ammonium phosphate
1.0 g
(NH 4
H 2
PO 4
)
Agar
12.0 g
Distilled water
1.0 liter
Sugars to be tested: to the autoclaved basal
medium (at 45 °C), add filter-sterilized
solutions of the test carbohydrates for a
0.5% final concentration.
To demonstrate acid production:
—Incorporate 1 ml bromthymol blue (1.6%
alcohol solution) in the basal medium.
—Adjust to pH 7.0 prior to autoclaving.
—Add filter-sterilized solutions of the test
carbohydrates for a 1% final concentration.
—Dispense the medium in test tubes instead
of in petri dishes.
—Record presence or absence of a yellow
color indicating acid production.
Gluconate peptone broth
Dipotassium phosphate
(K 2 HPO 4 )
Yeast extract
Peptone
Potassium gluconate
Distilled water
1.0 g
1.0 g
1.5 g
40.0 g
1.0 liter
Benedict's reagent
Copper sulfate (CuSO 4 5H 2 O) 1.73 g
Sodium carbonate (Na 2
CO 3
) 10.0 g
Sodium citrate 17.3g
[C 3
H 4
OH(COO) 3
Na 3
]
Distilled water
0.1 liter
Preparation:
1) Solution 1. Dissolve sodium carbonate
and citrate in 60.0 ml of distilled water.
2) Solution 2. Dissolve copper sulfate in
20.0 mI of distilled water.
3) Add solution 2 to solution 1, while stirring
constantly.
4) Adjust to a total volume of 100 ml with
distilled water.
Hugh and Leifson's oxidation-fermentation
medium (0-F)
Bromthymol blue
0.08 g
K 2
HPO 4
0.3 g
Peptone
2.0 g
Agar
3.0 g
NaCl
5.0 g
Distilled water 1.0 liter
Add 10 ml of 10% aqueous D-glucose solution
(filter-sterilized) to the sterile molten
medium to obtain a final concentration of
1% glucose. Dispense in 6-ml volumes in
sterile, narrow tubes. Seal one set of tubes
with mineral oil.
King's medium B (KMB)
Glycerol
K 2
HPO 4
MgSO 4 ·7H 2
O
Agar
Peptone
Distilled water
Kligler's iron agar, pH 7.4
Ingredients, BBL
Phenol red
Ferric ammonium citrate
(50:50 mixture)
a. Ferric citrate (FeC 6
H 5
O 7
)
b. Ammonium citrate
[(NH 4
) 3
C 6
H 5
O 7
]
Sodium thiosulfate (Na 2
S 2
O 3
)
Dextrose (glucose)
Sodium chloride (NaCI)
Lactose
Agar
Polypeptone
Distilled water
Modified Wakimoto 's medium
Ferrous sulfate
Calcium nitrate
Sodium phosphate
Bacto peptone
Agar
Sucrose
Distilled water
15.0 ml
1.5 g
1.5 g
17.0 g
20.0 g
1.0 liter
0.025 g
0.5 g
0.5 g
1.0 g
5.0 g
10.0 g
15.0 g
20.0 g
1.0 liter
0.05 g
0.50 g
0.82 g
5.00 g
17.00 g
20.00 g
1.00 liter
Nutrient agar (NA)
Beef extract
Peptone
Agar
Distilled water
3.0 g
5.0 g
17.0 g
1.0 liter
Nutrient broth (NB) is nutrient agar without
the agar.
Peptone sucrose agar (PSA)
Sodium glutamate
Peptone
Sucrose
Agar
Distilled water
1.00 g
10.00 g
10.00 g
17.00 g
1.00 liter
Peptone sucrose broth (PSB) is PSA without
the agar.
Potassium nitrate agar
Potassium nitrate (KNO 3
)
Beef extract
Peptone
Agar
Distilled water
1.0 g
3.0 g
5.0 g
7.0 g
1.0 liter
Selective medium for P. fuscovaginae
(Miyajima 1989)
Penicillin G
750,000 units
Novobiocin
45 mg
Cycloheximide
75 mg
75% ethanol 3 ml
Distilled water
50 ml
King's medium B
940 ml
Selective medium for P. glumae PPGA +
0.1% CaCl 2
(Matsuda et al 1988)
KH 2
PO 4
0.5 g
CaCl 2
1.0 g
NaCl
3.0 g
Na 2
HPO 4 · 12H 2
O
3.0 g
Glucose
5.0 g
Peptone
5.0 g
Agar
20.0 g
Decoction of 200 g
1.0 liter
potato
Selective medium for P. glumae S-PG
(Tsushima et al 1986)
Cetrimide 0.01 g
EDTA-Fe
0.01 g
Phenol red
0.02 g
Na 2
MoO 4 · 2H 2
O
0.024 g
MgSO 4 · 7H 2
O
0.25 g
Na 2 HPO 4 1.2 g
KH 2
PO 4
1.3 g
(NH 4
) 2
SO 4
5.0 g
D-sorbitol 10.0 g
Agar 20.0 g
L-cystine 10.0 g
(0.1 ml of a 0.01%
aqueous solutiondissolve
0.01 g in
100 tnl distilled H 2
O
by heating)
Methyl violet
1.0 mg
(1 ml of a 0.01%
aqueous solution
(0.01 g dissolved in
Distilled water
100 ml distilled H 2
O)
1.0 liter
Appendix 109