Dec 15, 2025
GAM modified supplemented with vitamin K1
This protocol is a draft, published without a DOI.
- geunhui shin1
- 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea
- geunhui
Protocol Citation: geunhui shin 2025. GAM modified supplemented with vitamin K1. protocols.io https://protocols.io/view/gam-modified-supplemented-with-vitamin-k1-hhtub36nx
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 15, 2025
Last Modified: December 15, 2025
Protocol Integer ID: 235092
Keywords: vitamin k1, gam modified
Abstract
How to prepare GAM modified supplemented with vitamin K1
Troubleshooting
Stock solutions preparation
Prepare stock solutions:
HEMIN solution (0.5 g/L ) :
Dissolve 0.25 g Hemin in 500 µL 1 Molarity (M) NaOH ; make up to 50 mL with distilled water and filter sterilize. Store refrigerated.
VITAMIN K1 solution (10 g/L ) :
Dissolve 0.5 g Vitamin K1 of in 50 mL 95 % volume Ethanol and filter sterilize. Store refrigerated in a brown bottle.
Pre-autoclave preparation
20m
For the preparation of 1 L of media (40 petri dishes depending on depth) dilute in 850 mL of distilled water:
35.4 g GAM (Gifu Anaerobic Medium) Broth Modified (MB-G0826).
15 g BD BACTO Agar (214010).
Final pH = 7.1 ± 0.2 at 25 °C
Note
Contents inside GAM (Gifu Anaerobic Medium) Broth Modified (MB-G0826)
10.0 g Peptone
3.0 g Soytone
10.0 g Proteose Peptone
13.5 g Bovine Serum Albumin
5.0 g Yeast Extract
2.2 g Beef Extract
1.2 g Liver Extract
3.0 g Sodium Chloride
0.3 g L-Cysteine
0.3 g Sodium Thioglychollate
3.0 g Dextrose
5.0 g Soluble Starch
2.5 g Monopotassium Phosphate
Pre-autoclave preparation
20m
Stir and heat on a hotplate until completely dissolved (~60 °C is sufficient).
Add distilled water to bring the total volume to 970 mL .
Cover the cap loosely with foil, label, and apply autoclave tape.
Autoclave at 121 °C for 00:20:00 .
20m
Prepare 10% NaHCO₃
In a 500 mL beaker, dissolve 20 g NaHCO₃ in 200 mL dH₂O .
Filter-sterilize using SPL 508252 PES filter.
⚠️ Avoid cellulose nitrate (e.g., Corning 430756) or cellulose acetate (e.g., Sartorius Minisart) filters, as they are less compatible with mildly basic solutions.
Store in the refrigerator (2 °C - 8 °C ).
- It can be stored for 18 months inside the refrigerator.
Post-Autoclave Processing
1h
If the agar has solidified, gently reheat to melt it.
(In a biosafety cabinet) Allow the molten medium to cool to 50 °C .
(In a biosafety cabinet) Add 10 mL of 0.5 mg/mL Hemin solution.
⚠️ At 75 °C, hemin begins to degrade, with a half-life of approximately 2.6 days. When exposed to 95 °C, the degradation accelerates, reducing the half-life to about 0.73 days.
(In a biosafety cabinet) Aseptically add 20 mL of filtered10 % (v/v) NaHCO₃ . Swirl gently to mix.
⚠️ NaHCO₃ starts decomposing when heated over 50 °C .
⚠️ If NaHCO₃ solution is precipitated. Consider swirling it inside 38 °C shaker (220 rpm). Also note that the PS container from SPL filter kit is not heat resistant so it should not be placed on hot plate
(In a biosafety cabinet) Aseptically add 100 µL vitamin K1 solution . Swirl gently to mix.
(In a biosafety cabinet) Pour into sterile Petri dishes.
(In a biosafety cabinet) Let plates dry with lids slightly open for around 01:00:00 .
1h
Store plates in the refrigerator (2 °C - 8 °C ) in dark by wrapping in aluminum foil.
Transfer plates into an anaerobic chamber before use.