Media Recipes
Liquid Media
Sterile Saline
Purpose: Used make dilutions of viable cells for plating or transfer to new media.
This saline concentration of 0.85% w/v (145 mM) is suitable for diluting
E. coli before transferring to new media or plating on agar.
1L |
5L |
Component |
8.5 g |
42.5 g |
NaCl |
Add dH
2O to final volume. Autoclave before use. Prepare large batches in 6 L flask and then aliquot out into bottles. It is not important to measure out exact volumes when dividing a large batch among bottles.
Included topic:
ProtocolsRecipesSaline
LB: Lysogeny Broth / Luria-Bertani Medium (Miller)
Rich media used for routine culture of E. coli and other bacteria at high cell densities.
Add dH
2O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:
1L |
6L |
Component |
10 g |
60 g |
Tryptone |
5 g |
30 g |
Yeast Extract |
10 g |
60 g |
NaCl |
If mixing up large batches and aliquoting, be sure to add exact volumes to final media bottles so that they will be ready for addition of antibiotic to known concentrations. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.
This recipe is from Miller JH. (1992) A Short Course in Bacterial Genetics. CSHL Press. Handbook Unit 25.5.
Note: Be aware that (1) there are several other formulations that may be called LB but vary the amount of NaCl. Using the wrong one can cause large changes in growth. (2) LB was originally supposed to stand for "Lysogeny Broth" and you may also see it called "Luria Broth" (
more about this).
Expected results:
E. coli strains grow to 5×10
9 cells/ml final density in LB.
Variant: 0.1×LB is made with 1 g Tryptone, 0.5 g Yeast Extract, and 10 g NaCl per liter. (It is 0.1× in the nutrients, but
not the salt!)
Included topic:
ProtocolsRecipesLysogenyBroth
DM: Davis-Mingioli
Growth medium used by the long-term
E. coli evolution experiment.
0.5L |
1L |
4.4L |
5L |
Component |
MW |
2.67 g |
5.34 g |
23.32 g |
26.7 g |
Potassium Phosphate (dibasic) K2HPO4 |
MW 174.18 |
1 g |
2 g |
8 g |
10 g |
Potassium Phosphate (monobasic) KH2PO4 |
MW 136.07 |
0.5 g |
1 g |
4.4 g |
5 g |
Ammonium Sulfate (NH4)2 SO4 |
MW 132.08 |
0.25 g |
0.5 g |
2.2 g |
2.5 g |
Sodium Citrate (trisodium, dihydrate) Na3C6H5O7 (H2O)2 |
MW 294.10 |
Add dH
2O to final volume and autoclave.
Note: 4.4 L of DM makes 8 bottles with 550 ml each.
Note: If using Potassium phosphate (dibasic) trihydrate (MW 228.22) use 7g per L
After autoclaving add the following stock solutions:
0.5L |
550mL |
1L |
5L |
Component |
0.5 ml |
550 µl |
1.0 ml |
5 ml |
10% (w/v) Magnesium Sulfate MgSO4 (separately autoclaved stock) |
0.5 ml |
1000 µl |
1.0 ml |
5 ml |
0.2% (w/v) Thiamine (vitamin B1) (filter sterilized) |
And supplement with a carbon source.
If preparing DM-glucose (MW 180.16 g/mol), add this volume of
10% glucose solution (separately autoclaved stock) to get the final concentration desired:
0.5L |
550mL |
1L |
5L |
DMX |
[glucose] (w/v) |
[glucose] (mg/L) |
[glucose] (M) |
125 µl |
137.5 µl |
250 µl |
1.25 ml |
DM25 |
0.0025% |
25 mg/L |
139 µM |
0.5 ml |
0.55 ml |
1 ml |
5 ml |
DM100 |
0.010% |
100 mg/L |
694 µM |
1.25 ml |
1.375 ml |
2.5 ml |
12.5 ml |
DM250 |
0.025% |
250 mg/L |
1.39 µM |
2.5 ml |
2.75 ml |
5 ml |
25 ml |
DM500 |
0.05% |
500 mg/L |
2.78 mM |
5 ml |
5.5 ml |
10 ml |
50 ml |
DM1000 |
0.1% |
1000 mg/L |
5.55 mM |
10 ml |
11 ml |
20 ml |
100 ml |
DM2000 |
0.2% |
2000 mg/L |
11.1 mM |
Remember: DMX = DM + X mg/L glucose. Glucose may no longer limit the final growth density above approximately DM1000.
Final composition |
5.1 mM |
Sodium (Na+) |
75.8 mM |
Potassium (K+) |
15.2 mM |
Ammonium (NH4) |
0.83 mM |
Magnesium (Mg2+) |
8.41 mM |
Sulfate (SO42-) |
45.3 mM |
Phosphate (PO43-) |
1.70 mM |
Citrate |
139 µM |
Glucose (in DM25) |
Included topic:
ProtocolsRecipesDavisMingioli
M9 Minimal Medium
1L |
Component |
6 g |
Sodium phosphate dibasic (anhydrous), Na2HPO4 |
3 g |
Potassium phosphate monobasic, KH2PO4 |
0.5 g |
Sodium chloride, NaCl |
1 g |
Ammonium chloride, NH4Cl |
Add dH
2O to 1 liter. Autoclave.
After media is autoclaved add the following sterile ingredients:
1L |
Component |
1 ml |
1M Magnesium sulfate, MgSO4 |
1 ml |
0.1M Calcium chloride CaCl2 |
10 ml |
10% (w/v) glucose (or other carbon source) |
Final composition |
93.0 mM |
Sodium (Na+) |
22.1 mM |
Potassium (K+) |
18.7 mM |
Ammonium (NH4) |
1.0 mM |
Calcium (Ca2+) |
0.1 mM |
Magnesium (Mg2+) |
29.2 mM |
Chloride (Cl- |
0.1 mM |
Sulfate (SO42-) |
42.2 mM |
Phosphate (PO43-) |
Included topic:
ProtocolsRecipesM9Minimal
ABMS: Acinetobacter Minimal Succinate
Courtesy of the Averhoff lab, with modifications for available reagents.
To make standard ABMS, combine the following
pre-sterilized ingredients at room temperature:
1L |
5L |
Component |
880 mL |
4.4 L |
diH2O |
20 mL |
100 mL |
1M Sodium Succinate |
50 mL |
250 mL |
20× Mineral Solution |
50 mL |
250 mL |
20× Phosphate Buffer |
To make ABMS agar:
Per liter of media, add 16 g/L agar and 1 mL/L antifoam to diH
2O, then autoclave in a 2 L flask. When water and agar are cool enough to touch, add component solutions. Optional: warm components at 55°C in a water bath before combining with agar to reduce the temperature difference when combined.
ABMS Component Recipes
SL9 (trace minerals)
1L |
Component |
800 mL |
diH2O |
12.8 g |
Nitrilotriacetic acid |
2 g |
FeSO4•7H2O (note: first dissolve in 2M HCl) |
104 mg |
CoCl2 (anhydrous) |
122 mg |
MnCl2•4H2O |
70 mg |
ZnCl2 |
36 mg |
Na2MoO4•2H2O |
13 mg |
NiCl2 |
Add 1 mL of 10 mL 10× solution:
60 mg |
H3BO3 |
20 mg |
CuCl2•2H2O |
Adjust pH to 6.5 using 1 M NaOH. Bring volume to 1000 mL with diH
2O. Note: this requires a lot of NaOH, so leave room when bringing up to volume.
Mineral Solution (20×)
500 mL |
Component |
10 g |
NH4Cl |
5.8 g |
MgSO4•7H2O |
1 g |
KNO3 |
0.67 g |
CaCl2•2H2O † |
20 mg |
(NH4)6Mo7O24•4H2O |
10 mL |
SL 9 |
† Substitution: You may use 465.8 µL of a 1 M CaCl
2 per 500 ml of stock solution for this component. The final concentration of Ca
2+ in the 20× stock is 9.12 mM.
Bring to 500mL in a graduated cylinder with deionized water.
Phosphate Buffer (20×)
500mL |
Components |
68 g |
KH2PO4 |
132.5 g |
Na2HPO4•H2O |
Bring to 500 mL with diH
2O; pH should be ~6.9-7.0 (you may add 400 mL diH
2O first and bring pH up to 6.8 then complete volume to 500mL)
* Source: Averhoff lab
Included topic:
ProtocolsRecipesABMS
R2A
R2A is a medium that can be used to grow a wide variety of soil microbes.
1L |
5L |
Component |
0.5 g |
2.5 g |
Yeast Extract |
0.5 g |
2.5 g |
Proteose Peptone No. 3 |
0.5 g |
2.5 g |
Casamino Acids |
0.5 g |
2.5 g |
Dextrose |
0.5 g |
2.5 g |
Soluble Starch |
0.3 g |
1.5 g |
Sodium Pyruvate |
0.3 g |
1.5 g |
Dipotassium Phosphate |
0.05 g |
0.25 g |
Magnesium Sulfate |
Add dH
2O to final volume and autoclave.
If making R2A solid media also add 15 g of agar and 1 ml of 5% antifoam per liter.
Included topic:
ProtocolsRecipesR2A
RCV Medium
RCV is a complex media consisting of 3 different sub-components that must be prepared ahead of time if they are not already made.
Main recipe
1L |
Component |
10 mL |
10% (NH4)2 SO4 |
40 mL |
10% DL-malate, pH 6.8 |
50 mL |
Super salts (see below) |
15 mL |
0.64 KPO4, pH 6.8 (see below) |
add 3/4 H
2O before adding the KPO
4
adjust pH to 6.8 if needed
if making plates add 16 g Agar per L
0.64M KPO4, pH 6.8
500mL |
Component |
20 g |
KH2PO4 |
30 g |
K2HPO4 |
Super salts
1L |
Component |
40 mL |
1.0% EDTA |
20 mL |
20% MgSO4 5H2O |
20 mL |
7.5% CaCl2 2H2O |
20 mL |
Trace elements (see below) |
48 mL |
0.5% FeSO4 7H2O |
20 mL |
0.1% thiamine-HCl |
Trace Elements
250mL |
Component |
0.3975 g |
MnSO4H2O |
0.7 g |
H3BO3 (Boric Acid) |
0.01 g |
Cu(NO3)2 3H2O |
0.06 g |
ZnSO4 7H2O |
0.1875 g |
NaMoO4 2H2O (Sodium molybdate VI dihydrate) |
Included topic:
ProtocolsRecipesRCVMedium
SOB/SOC: Super Optimal Broth
For SOB:
200mL |
250mL |
1L |
Final [ ] |
Component |
1 g |
1.25 g |
5 g |
0.5% |
yeast extract |
4 g |
5 g |
20 g |
2.0% |
tryptone |
0.12 g |
0.125 g |
0.5 g |
10 mM |
NaCl |
0.037 g* |
0.042 g* |
0.186 g |
2 mM |
KCl |
0.48 g |
0.6 g |
2.4 g |
20 mM |
MgSO4 (anhydrous) |
*Don't measure; it's just a pinch.
Adjust to pH 7.5 with 1M NaOH.
Note: 200 ml of SOC makes 25 vials of 8 ml each.
For 1 L: Add 800 ml of dH
2O and dissolve components. Adjust to pH 7.5 prior to use with 1 M NaOH (approximately 25 ml). Add dH
2O to a final volume 1 L.
Autoclave.
For SOC (=SOB+glucose)
Bulk scale: Make SOB, except add dH
2O to a final volume of 960 ml instead of 1 L. Autoclave. When solution has cooled to 50°C (cool enough to touch with bare hands for a few seconds), add 40 ml of sterile 10% (w/v) glucose. (That's a final concentration of 0.4% glucose or about 20 mM.)
Smaller scale: To achieve a 20 mM concentration of glucose for a known final volume
n of SOC, you can calculate the amount of 10% w/v glucose to be added as 0.036
n. The amount of SOB to add is then 0.964
n. Keep units constant, so if
n is in µL, the final amounts will also be in µL and so on.
- Note: If you have a known volume of SOB x and want to know how much 10% w/v glucose to add to make SOC containing 20 mM glucose, simply divide x/0.964 to get your final volume n and subtract x from n to get the volume of 10% w/v glucose to add.
Generally when making stocks of SOC it is advisable to make smaller volume aliquots rather than larger ones. SOC media is extremely rich and can contaminate easily particularly after glucose is added. Additionally, smaller volumes tend to be pulled from it repeatedly with pipettes, which increases the chances of contamination over time.
Note: Some recipes for SOB use 10 mM MgSO
4 and 10 mM MgCl.
Included topic:
ProtocolsRecipesSOB
TGY Medium
1L |
5L |
Component |
5 g |
25 g |
Pancreatic digest of casein |
5 g |
25 g |
Yeast Extract |
1g |
5 g |
Glucose |
1 g |
5 g |
K2HPO4 |
Add dH
2O to final volume and autoclave.
Included topic:
ProtocolsRecipesTGY
DR: Defined minimal media for D. radiodurans
250 ml |
500 ml |
Component |
50 ml |
100 ml |
5x M9 salts |
250 ul |
500 ul |
5mM MnCl2 |
250 ul |
500 ul |
0.8M MgCl2 |
250 ul |
500 ul |
0.18 M CaCl2 |
2.5 ml |
5 ml |
Syringe filtered Vitamins Mix |
500 ul |
1 ml |
Syringe filtered Amino Acid mix (see below) |
Add dH
2O to final volume.
Note: All components are either pre-sterilized or syringe filtered so the only things that need to be autoclaved are the receiving bottle and the added water.
To prepare the amino acids:
Amount |
Amino Acid |
1 g |
L-Cys |
0.5 g |
L-His |
0.5 g |
L-Met |
Dissolve these amino acids in 40 ml of dH
2O
Included topic:
ProtocolsRecipesDR
YPS Medium
1L |
1.5L |
Component |
3.0 g |
4.5 g |
Yeast Extract |
3.0 g |
4.5 g |
Peptone |
2.0 mL |
3.0 mL |
1 M Magnesium sulfate |
2.0 mL |
3.0 mL |
1 M Calcium chloride |
add water to final volume, adjust pH to 7.0
autoclave
Included topic:
ProtocolsRecipesYPSMedium
1416: 4-hydroxybenzoic acid medium (for JJ-1b, Bacillus sp.)
1L |
4L |
Component |
4.25 g |
17.0 g |
Potassium Phosphate (dibasic) K2HPO4 trihydrate |
1.00 g |
4.00 g |
Sodium Phosphate (monobasic) NaH2PO4 monohydrate |
2.00 g |
8.00 g |
Ammonium chloride NH4Cl |
0.20 g |
0.40 g |
Magnesium Sulfate MgSO4 heptahydrate |
12.0 mg |
48.0 mg |
Ferrous Sulfate FeSO4 heptahydrate |
3.00 mg |
12.0 mg |
Manganese Sulfate MnSO4 monohydrate |
3.00 mg |
12.0 mg |
Zinc Sulfate ZnSO4 heptahydrate |
1.00 mg |
4.00 mg |
Cobalt (II) Sulfate CoSO4 |
0.10 g |
0.40 g |
Nitrilotriacetic acid * |
1.00 g |
4.00 g |
4-Hydroxybenzoic acid * |
* Dissolve these components in water made alkaline with concentrated NaOH before adding other components.
Adjust medium for final pH 7.2, +/- 0.2. Autoclave for at least 15 minutes.
If plates needed, add 15.0 g of agar per liter to the above recipe.
Included topic:
ProtocolsRecipes1416,4-hydroxybenzoicacidmedium
YPD Medium
General media used for culturing Yeast. Adapted from
Cold spring harbor
In 1 L bottle:
1L |
Component |
20g |
Peptone |
10 g |
Yeast Extract |
- Dissolve above in ~900 mL water.
- Bring to 950 mL (final volume will be 1L after autoclaving)
In separate 500mL bottle, prepare 40% Glucose w/v
Autoclave separately.
WARNING If glucose added before autoclaving, media will caramelize and be unusable.
- Add 50 mL of 40% glucose solution to YPD media.
Included topic:
ProtocolsRecipesYPDmedium
Czapek Broth (CB)
Combine in a flask:
500 mL |
1 L |
Component |
1.5 g |
3 g |
sodium nitrate |
0.5 g |
1 g |
potassium phosphate dibasic |
0.25 g |
0.5 g |
potassium chloride |
9 mg |
18 mg |
ferrous sulfate heptahydrate |
2.075 mL |
4.15 mL |
1 M sterile magnesium sulfate |
- In a flask, combine sodium nitrate, potassium phosphate dibasic, potassium chloride, and ferrous sulfate heptahydrate in diH2O
- If making 500 mL of media, use 995.85 mL diH2O
- If making 1 L of media, use 497.925 mL diH2O
- Once reagents are combined and dissolved, measure pH and adjust until it reaches 7.5
- Once pH of 7.5 is achieved, autoclave the solution and allow to cool
- Once cooled, add 1 M magnesium sulfate
Source:
Throne-Holst et. al 2007
Included topic:
ProtocolsRecipesCzapekBroth
Solid Media
LB: Lysogeny Broth / Luria-Bertani (Miller) Agar
Add dH
2O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:
1L |
6L |
Component |
10 g |
60 g |
Tryptone |
5 g |
30 g |
Yeast Extract |
10 g |
60 g |
NaCl |
1 ml |
6 ml |
5% Antifoam* |
16 g |
48 g |
Agar* |
ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.
AGAR: Measure 8 grams of agar per 500 ml bottle and
add to each individual bottle before aliquoting the LB. Autoclave to homogenize.
Each liter of LB Agar makes 2 bottles of 500 ml.
Notes and variations:
- Common synonyms are Luria-Broth and Luria-Bertani medium.
- This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
- If you are performing sacB counter-selection, you may need to use even lower salt concentrations.
- To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.
Included topic:
ProtocolsRecipesLysogenyBrothAgar
TA: Tetrazolium Sugar (TA, TM, TL, ...)
Combine in a 2 L flask:
1.5L |
Component |
15 g |
Tryptone |
1.5 g |
Yeast Extract |
7.5 g |
NaCl |
24 g |
Agar |
1.5 ml |
5% Antifoam |
Add dH
2O to 1.3 liter.
Combine in a 500 ml flask:
1.5L |
Component |
15 g |
Sugar (arabinose = TA, maltose = TM, lactose = TL) |
Add dH
2O to 0.2 liter.
Autoclave sugar and media separately.
Total combined volume is 1.5 liters.
Combine and add:
1.5L |
Component |
1.5 ml |
5% (w/v) Triphenyl Tetrazolium chloride (TTC), filter sterilized, stored at 4°C |
Source:
Lenski Lab Protocol
Included topic:
ProtocolsRecipesTetrazoliumSugar
MG: Minimal Glucose agar, aka DM: Davis Mingioli agar
Note: When making these plates it is necessary to prepare and autoclave the 3 main parts (salts, agar, and sugar) separately. Compounds that inhibit growth are produced when agar and phosphate or phosphate and glucose are autoclaved together.
1L |
1.5L |
Component |
MW |
5.3 g |
8 g |
Potassium Phosphate (dibasic) K2HPO4 |
MW 174.18 |
2 g |
3 g |
Potassium Phosphate (monobasic) KH2PO4 |
MW 136.07 |
1 g |
1.5 g |
Ammonium Sulfate (NH4)2 SO4 |
MW 132.08 |
0.5 g |
0.75 g |
Sodium Citrate (trisodium, dihydrate) Na3C6H5O7 (H2O)2 |
MW 294.10 |
333 ml |
500 ml |
dH2O |
|
Autoclave.
Next, prepare the agar:
1L |
1.5L |
Component |
16 g |
24 g |
Agar |
1 ml |
1.5 ml |
Antifoam (5%) |
333 ml |
500 ml |
dH2O |
Autoclave.
Next, prepare the sugar. The final concentration is 0.4% w/v:
1L |
1.5L |
Component |
4 g |
6 g |
Glucose (or other sugar) |
333 ml |
500 ml |
dH2O |
Autoclave.
After the 3 parts have been autoclaved, combine the contents of the three flasks together while they are still warm add the following stock solutions:
1L |
1.5L |
Component |
1.0 ml |
1.5 ml |
10% Magnesium Sulfate MgSO4 (separately autoclaved stock) |
1.0 ml |
1.5 ml |
0.2% Thiamin (vitamin B1) (filter sterilized stock) |
Source:
Lenski Lab Protocol
Included topic:
ProtocolsRecipesMinimalGlucose
MC: Minimal Citrate
Prepared the same as
MG: Minimal Glucose with the following changes:
- No glucose
- 4.5 g/L Sodium Citrate (trisodium, dihydrate)
Included topic:
ProtocolsRecipesMinimalCitrate
M9 Minimal Media Plates
As with DM and MG media, make sure to autoclave the agar and phosphate separately.
For 1 liter of media:
1L |
Component |
6 g |
Sodium phosphate, Na2HPO4 (anhydrous) |
3 g |
Potassium phosphate, KH2PO4 |
0.5 g |
Sodium chloride, NaCl |
1 g |
Ammonium chloride, NH4Cl |
Add dH
2O to
500 mL
1L |
Component |
16 g |
Agar |
1 ml |
Antifoam |
Add dH
2O to
500 mL.
Autoclave media. Once the bottles are cooled to 55°C, mix the agar and M9 components.
Next, add the following sterile ingredients:
1L |
Component |
1 ml |
1M Magnesium sulfate, MgSO4 |
1 ml |
0.1M Calcium chloride CaCl2 |
10 ml |
10% (w/v) glucose (or other carbon source) |
Included topic:
ProtocolsRecipesM9
PA: Lac Papillation Agar
Make 10× Minimal A Salts.
10× Minimal A Salts |
1L |
Component |
MW |
80 g |
Potassium Phosphate (dibasic) K2HPO4 |
MW 174.18 |
45 g |
Potassium Phosphate (monobasic) KH2PO4 |
MW 136.07 |
10 g |
Ammonium Sulfate (NH4)2 SO4 |
MW 132.08 |
5 g |
Sodium Citrate (trisodium, dihydrate) Na3C6H5O7 (H2O)2 |
MW 294.10 |
to 1L |
dH2O |
|
Autoclave. Salt solution can be stored at room temperature.
Agar Solution |
1L |
Component |
15 g |
Agar |
to 490 ml |
dH2O |
Autoclave.
Sugar Solution |
1L |
Component |
2 g |
Glucose |
to 400 ml |
dH2O |
Autoclave.
After autoclaving, combine agar and sugar and add:
1L |
Component |
100 ml |
10× Minimal A Salts |
6 ml |
10% w/v (0.083M Mg2+) Magnesium Sulfate, anhydrous MgSO4 (autoclaved) MW 120.37 |
2.5 ml |
0.2% w/v Thiamin (vitamin B1) (filter sterilized) |
1 ml |
500 mg/ml P-Gal (phenyl β-D-galactoside) (filter sterilized) |
1 ml |
40 mg/ml X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) (filter sterilized) |
Note: The amounts added of some standard components (including agar) are slightly different for this recipe. Volumes have also been altered from the original Miller recipe to use the same stock solutions we have available for making DM plates. The final concentrations are 0.2% glucose, 0.05% P-Gal, and 0.004% X-Gal (w/v).
Sources:
- Nghiem, Y., Cabrera, M., Cupples, C.G., Miller, J.H. (1988) The mutY gene : A mutator locus in Escherichia coli that generates G•C→T•A transversions. Proc. Natl. Acad. Sci. U.S.A. 85:2709-2713.
- Miller J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Included topic:
ProtocolsPapillationAgar
TGY Medium
1.5L |
Component |
7.5 g |
Pancreatic digest of casein |
7.5 g |
Yeast Extract |
1.5g |
Glucose |
1.5 g |
K2HPO4 |
24g |
Agar |
1.5mL |
Antifoam |
Add dH
2O to final volume and autoclave.
Included topic:
ProtocolsTGYPlates
MOB: Mobility Media
This media is useful for enhancing the mobility of ADP1, which moves across the plate by "twitching".
1L |
Final [ ] |
Component |
5 g |
0.5% |
agarose |
5 g |
2.0% |
tryptone |
2.5 g |
10 mM |
NaCl |
After pouring your gel, let it set overnight at room temperature. The next day, spot with 1µl of your overnight-grown culture, leave the plate to dry for 15 minutes at room temperature, wrap in Parafilm, invert, and incubate at 30°C. You should see spreading over the next 24 hours.
Included topic:
ProtocolsMobilityMedia
Blood - Heart Infusion Agar
Heart Infusion Agar supplemented with sterile Sheep's Blood. Used for the cultivation of fastidious organisms (Bee gut microbiome species
et al)
1L |
Component |
44 g |
Heart Infusion Agar (HIA) (Difco brand) |
50mL |
Sterile Sheep's Blood |
Autoclave water and media (without blood) on a 20min liquid cycle. Transfer autoclaved media to hot water bath at 55 deg Celsius. Allow to equilibrate for 30mins to 1hr. Add 100mL blood (using PipetAid) and stir to mix (a stir bar isn't necessary, but do not shake). Smooth, small circles while the bottle is on a flat surface is sufficient to mix the blood and warm agar. Add necessary antibiotics and supplements (remember the volume is now 1100 mL, not 1 L) and pour into plates.
As with other agar, after autoclaving the HIA you can let it solidify at room temperature. After microwaving to dissolve, it still needs to be cooled before adding blood.
Included topic:
ProtocolsRecipesBloodHeartInfusion
Stab Agar
Making agar stabs for storage and transport of bacterial strains.
1L |
Component |
10 g |
Tryptone |
5 g |
Yeast extract |
10 g |
Sodium chloride |
6 g |
Agar |
Autoclave. Transfer to cryovials before media cools. Can store the vials at 4 C for a few months.
Included topic:
ProtocolsRecipesStabAgar