1494 . . OMIZ-PAT Medium (modified)

Equipment needed: Centrifuge Filter

Final pH: 6.9

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	Compound	Amount	Unit
	Distilled water	100	ml
	Solution A	10	ml
	Solution B	10	ml
	Solution C	10	ml
	Solution D	10	ml
	Solution E	10	ml
	Solution F	10	ml
	Solution G	10	ml
	Solution H	200	µl
	Solution I	8	µl
	Solution J	2	µl
	Solution K	40	µl
	Solution L	2	ml
	Trace elements I (pre-dilute 1:1000; use)	2	ml
	Trace elements II (pre-dilute 1:1000; use)	2	ml
	Trace elements III (pre-dilute 1:1000; use)	200	µl
	Distilled water (add up to 200 ml)	200	ml
	Supplement 1	200	µl
	Supplement 2	2	ml
	Supplement 3	2	ml
	Supplement 4	200	µl
	Supplement 5	2	ml
1 Add 10 ml rabbit serum, flush the medium with N₂CO₂, adjust the pH to 6.9 and filter-sterilize the completed medium into tubes.
	Rabbit serum	10	ml
2 This recipe describes the preparation of 200 ml medium, which can be used for about 4 weeks when kept at 4°C.

Solution A #

100

	Compound	Amount	Unit
	L-Alanine	90	mg
	L-Arginine	350	mg
	L-Asparagine	300	mg
	L-Aspartic acid	236	mg
	L-Cysteine HCl	700	mg
	L-Glutamine	1360	mg
	L-Glutamic acid	600	mg
	Glycine	150	mg
	L-Histidine	1240	mg
	L-Isoleucine	262	mg
	L-Leucine	262	mg
	L-Lysine HCl	364	mg
	L-Methionine	300	mg
	L-Ornithine HCl	340	mg
	L-Phenylalanine	330	mg
	L-Proline	230	mg
	L-Serine	1050	mg
	L-Threonine	240	mg
	L-Tryptophan	204	mg
	L-Tyrosine	180	mg
	L-Valine	234	mg
	Distilled water	100	ml
1 Prepare Solutions A, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C

Solution B #

100

	Compound	Amount	Unit
	KCl	1936	mg
	MgSO₄ x 7 H₂O	500	mg
	NaH₂PO₄ x H₂O	280	mg
	NH₄Cl	3200	mg
	Citric acid , trisodium salt	400	mg
	Distilled water	100	ml
1 Prepare Solutions B, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C

Solution C #

100

	Compound	Amount	Unit
	Calcium D-(+)-pantothenate	10	mg
	Choline chloride	100	mg
	Myo-inositol	100	mg
	Thiamine HCl	10	mg
	Thiamin pyrophosphate	10	mg
	Coenzym A , sodium salt	2	mg
	Flavin adenine dinucleotide (FAD)	2	mg
	ß-nicotinamide adenine dinucleotide (NAD)	2	mg
	2-Mercaptoethanesulfonic acid , sodium salt (MES)	20	mg
	N-acetylmuramic acid	50	mg
	N-Acetylglucosamine	400	mg
	D-glucuronic acid , sodium salt	1600	mg
	D-galacturonic acid	1600	mg
	Pyruvic acid , sodium salt	1100	mg
	Fumaric acid , disodium salt	1000	mg
	Formic acid , sodium salt	600	mg
	Distilled water	100	ml
1 Prepare Solutions C, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C

Solution D #

100

	Compound	Amount	Unit
	Pyridoxal hydrochloride	10.0	mg
	Pyridoxal phosphate	10.0	mg
	D , L-lactic acid, sodium salt	1120.0	mg
	Hypoxanthine	2.8	mg
	Uracil	2.2	mg
	Thymidine	4.8	mg
	N-(2-acetamido)-2-aminoethanesulfonic acid	3.6	mg
	DL-carnitine	400.0	mg
	Putrescine 2 HCl	10.0	mg
	Phenol red , sodium salt	20.0	mg
	Distilled water	100.0	ml
1 Prepare Solutions D, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C

Solution E #

100

	Compound	Amount	Unit
	D-Glucose	4.0	g
	D-Fructose	1.6	g
	D-Maltose	1.6	g
	D-Mannitol	1.6	g
	D-Mannose	4.0	g
	D-Arabinose	4.0	g
	L-Fucose	4.0	g
	D-Trehalose	4.0	g
	D-Sucrose	4.0	g
	L-Rhamnose	4.0	g
	Distilled water	100.0	ml
1 Prepare Solutions E, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C

Solution F #

100

	Compound	Amount	Unit
	CaCl₂ x H₂O	300	mg
	Distilled water	100	ml
1 Prepare Solution F, flush with nitrogen to make it anoxic, filter-sterilize and store at 4°C

Solution G #

	Compound	Amount	Unit
	Asialofetuin	1	mg/ml
1 Prepare Solutions G, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C

Solution H #

100

	Compound	Amount	Unit
	Vitamine B₁₂	5	mg
	D-(+)-biotin	5	mg
	Folic acid	5	mg
	Folinic acid , calcium salt	10	mg
	Nicotinamide	5	mg
	Nicotinic acid	10	mg
	Riboflavine	1	mg
	Distilled water	100	ml
1 Prepare Solutions H, flush each with nitrogen to make them anoxic, filter-sterilize and store at -20°C

Solution I #

	Compound	Amount	Unit
	Isobutyric acid	10	µl
	2-Methylbutyric acid	10	µl
	Valeric acid	10	µl
	Isovaleric acid	10	µl
1 Prepare Solution I and store at -20°C

Solution J #

	Compound	Amount	Unit
	DL-alpha-lipoic acid	1.0	mg/ml
	2-Mercaptoethanol	0.1	ml
	Ethanol	0.9	ml
1 D,L-alpha-lipoic acid (1 mg/ml); in 2-mercaptoethanol-ethanol (1:10); store at -20°C

Solution K #

	Compound	Amount	Unit
	Hemin	0.2	mM
	NaOH	10.0	mM
1 Hemin (0.2 mM) in NaOH (10 mM)

Solution L #

140

	Compound	Amount	Unit
	Yeast extract (fresh)	42	g
	Distilled water	140	ml
1 Homogenize 42 g fresh baker yeast in 140 ml distilled water, incubate for 24h at 56°C and centrifuge to harvest the supernatant. Adjust pH to 7 with NaOH, make anoxic, filter-sterilize and store at -80°C

Trace elements I #

100

	Compound	Amount	Unit
	CuSO₄	7.89	mg
	MnSO₄ x H₂O	169.00	mg
	ZnSO₄ x 7 H₂O	2.87	g
	HCl (10 mM)	100.00	ml
1 Prepare Trace elements I, flush with nitrogen to make them anoxic, filter-sterilize and store at 4°C

Trace elements II #

100

	Compound	Amount	Unit
	Na₂SeO₃	173	mg
	HCl (10mM)	100	ml
1 Prepare Trace elements II, flush with nitrogen to make them anoxic, filter-sterilize and store at 4°C

Trace elements III #

100

	Compound	Amount	Unit
	NiSO₄ x 6 H₂O	13.1	mg
	SnCl₂ x 2 H₂O	11.8	mg
	NaVO₃	61.0	mg
	(NH₄)₆Mo₇O₂₄ x 4 H₂O	1240.0	mg
	HCl (10 mM)	100.0	ml
1 Prepare Trace elements III, flush with nitrogen to make them anoxic, filter-sterilize and store at 4°C

Supplement 1 #

	Compound	Amount	Unit
	Fe SO₄ x 7 H₂O	10	mM
	HCl	10	mM
1 FeSO₄ x 7H2O (10 mM) in HCl (10 mM)
2 Prepare Supplements 1, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C

Supplement 2 #

	Compound	Amount	Unit
	Ascorbic acid	10	%(w/v)
	Distilled water	2	ml
1 Ascorbic acid (10%; w/v); in distilled water, neutralize with NaOH
2 Prepare Supplements 2, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C

Supplement 3 #

	Compound	Amount	Unit
	Glutathione	0.5	M
	Distilled water	2.0	ml
1 Glutathion (0.5 M); in distilled water, neutralize with NaOH
2 Prepare Supplements 3, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C

Supplement 4 #

	Compound	Amount	Unit
	Cholesterol	1	mg/ml
	Ethanol (100 %)	1	ml
1 Cholesterol (1 mg/ml); in Ethanol (100%); store at -20°C

Supplement 5 #

	Compound	Amount	Unit
	NaHCO₃	10	%(w/v)
	Distilled water	2	ml
1 NaHCO₃ (10%; w/v) in distilled water, store at 4°C

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