# C89: SBBM


## Mail sol. C89

970	ml	**Mail sol. C14**	
30	ml	**SE2 (Soil Extract 2)**	

1. Used for some filamentous green algae e.g. Trentepohlia, can give better growth
than 3N-BBM+V (Mail sol. C 14) or JM:SE (Mail sol. C 41). Make both media and sterilise, then mix aseptically in
the ratio of 97% 3N-BBM+V and 3% SE2.


## Mail sol. C14

10	ml	NaNO3 (75 g/l stock solution)	
10	ml	CaCl2 x 2 H2O (2.5 g/l stock solution)	
10	ml	MgSO4 x 7 H2O (7.5 g/l stock solution)	
10	ml	K2HPO4 x 3 H2O (7.5 g/l stock solution)	
10	ml	KH2PO4 (17.5 g/l stock solution)	
10	ml	NaCl (2.5 g/l stock solution)	
6	ml	**Trace Elements (PIV)**	

1. Once elements are dissolved autoclave at 15 psi for 15 minutes.

1	ml	**Vitamin B1 solution**	
1	ml	**Vitamin B12 solution**	
15	g	Agar, for solid medium	

2. Make up to 1 litre with distilled water. For agar add 15 g per litre Bacterialogical Agar.
Autoclave at 15 psi for 15 minutes.


## Vitamin B1 solution

0.12	g	Thiamine hydrochloride (Vitamin B1)	

1. Filter sterile


## Trace Elements (PIV)

1. Ensure elements are added in the following sequence:

0.75	g	Na2-EDTA	
0.097	g	FeCl3 x 6 H2O	
0.041	g	MnCl2 x 4 H2O	
0.005	g	ZnCl2	
0.002	g	CoCl2 x 6 H2O	
0.004	g	Na2 MoO4 x 2 H2O	

2. Once elements are dissolved autoclave at 15 psi for 15 minutes.


## Vitamin B12 solution

0.1	g	Cyanocobalamine (Vitamin B12)	

1. Take 1 ml of this solution and add 99 ml Deionised water. Filter sterile.


## SE2 (Soil Extract 2)

105	g	Soil (air­dried, sieved)	
660	ml	Deionized water	

1. Site selection for a good soil is very important and for most purposes a soil from 
undisturbed deciduous woodland is best.  Sites to avoid are those showing obvious 
signs of man's activity and particular care should be taken to avoid areas where 
fertilizers, crop sprays or other toxic chemicals may have been used.
2. A rich loam with good crumb structure should be sought.  Stones, roots and larger 
invertebrates should be removed during an initial sieving through a 1 cm mesh.  The sieved soil should be spread to air dry and hand picked for smaller 
invertebrates and roots.  It should be turned periodically and picked over again.  When dry it may be sieved through a finer mesh (2­4 mm) or stored as it is prior to
use.
3. Soil is prepared as above.  105 g of air­dried sieved soil and 660 ml of deionized 
water are placed in a 1 litre bottle and autoclaved once at 15 psi for 15 minutes, 
then again after 24 hours.  The contents of the bottle are left to settle (usually for 
at least a week) and then the supernatant is decanted and filtered.  The final pH
should be 7.0 - 8.0.


## Mail sol. C41

0.7	ml	Ca(NO3)2 x 4 H2O (4.0 g/200 ml stock solution)	
0.7	ml	KH2PO4 (2.48 g/200 ml stock solution)	
0.7	ml	MgSO4 x 7 H2O (10.0 g/200 ml stock solution)	
0.7	ml	NaHCO3 (3.18 g/200 ml stock solution)	
0.7	ml	**Solution 1**	
0.7	ml	**Solution 2**	
0.7	ml	**Solution 3**	
0.7	ml	NaNO3 (16.0 g/200 ml stock solution)	
0.7	ml	Na2HPO4 x 12 H2O (7.2 g/200 ml stock solution)	
300	ml	**SE2 (Soil Extract 2)**	
1000	ml	Deionized water	
15	g	Agar, for solid medium	

1. This recipe is a 7:3 mixture of JM and SE2. Make up to 1 litre with deionized water. For agar,
add 15.0 g per litre of Bacteriological Agar (Thermo Scientific™ Oxoid™ Agar No.1). Autoclave at 15 psi for 15 minutes.


## Solution 1

0.45	g	FeNa-EDTA	
0.45	g	Na2-EDTA	


## Solution 2

0.496	g	H3BO3	
0.278	g	MnCl2 x 4 H2O	
0.2	g	(NH4)6Mo7O24 x 4 H2O	


## Solution 3

0.008	g	Cyanocobalamine	
0.008	g	Thiamine HCl	
0.008	g	Biotine	


## SE2 (Soil Extract 2)

x		Soil (air-dried)	
x		Distilled water	

1. Site selection for a good soil is very important and for most purposes a soil from undisturbed
deciduous woodland is best. Sites to avoid are those showing obvious signs of man's activity
and particular care should be taken to avoid areas where fertilizers, crop sprays or other toxic
chemicals may have been used.
2. A rich loam with good crumb structure should be sought. Stones, roots and larger
invertebrates should be removed during an initial sieving through a 1 cm mesh. The sieved
soil should be spread to air dry and handpicked for smaller invertebrates and roots. It should
be turned periodically and picked over again. When dry sieve through a finer mesh (2-4 mm)
and store in an airtight container away from light and heat.
3. Soil is prepared as above. Air-dried soil and twice its volume of supernatant distilled water
are autoclaved together at 15 psi for 2 hours and left to cool. The supernatant is then
decanted and then distributed to containers in volumes suitable for making up batches of
media. The aliquots and their containers are autoclaved for an appropriate length of time
(e.g. 1 litre or less for 15 minutes) and are then kept in a cool place (e.g. a refrigerator) until
required.