36 replies to this topic

[SunsetSatine]

I know that you have to be careful with sugars as they can carmalize, but what about starch? I prepared media with starch and after I autoclaved it, I noticed a gelatinous substance at the bottom. I'm assuming that is the starch?

[Trof]

Ever baked a bread or cooked a sauce?

[El Crazy Xabi]

Soluble starch?

Did you add agar and did you boil it well before? I don't do it and it tends to form a viscous layer on the bottom.

It shouldn't be a problem, I consulted a media handbook and all the starch-containing media are prepared and autoclaved all together, except for some anaerobic media where it is autoclaved separatedly

[hobglobin]

I guess what you observed is starch retrogradation.
If it's no problem as in the previous post mentioned then it's okay, otherwise you might can try modified starch or soluble starch (if it's acceptable for your bacteria and not too expensive)

[SunsetSatine]

Hi guys!

I used soluble starch. I am preparing media for anerobes, but the protocol didn't mention anything about how to handle the starch, so I autoclaved it together with the other components and did notice that viscous layer at the bottom after I pulled it from the autoclave.

This was for an enrichment broth, so I didn't have agar, but I need to prepare plates now and am worried about this happening again, because I can't pour plates with that viscous layer at the bottom. If I autoclave first with the agar, add the soluble starch and then autoclave again, is that going to affect my other components? The manuals don't have anything about what happens if you autoclave media twice.

[El Crazy Xabi]

What's the name of the medium?

Is the starch autoclaved with the reducing agents?

Edited by El Crazy Xabi, 25 November 2012 - 09:41 PM.

[SunsetSatine]

It's a modified basal medium and contains the following:

Tryptone
Yeast Extract
Starch
NaHCO3
Resazurin
Mineral Solutions
Rumen Fluid
L-Cysteine HCl (my reducing agent)

The mineral solutions have K2HPO4, KH2PO4, NaCl, and (NH4)2SO4

I'll be preparing both plates and broth tubes

[SunsetSatine]

The recipe doesn't have instructions on how to prepare the media, so I've been combining all the ingrediants and autoclaving them together.

[El Crazy Xabi]

You didn't even sparge with CO2/N2?

Well, based in the info I could get from some handbooks

• Carbohydrates and other carbon sources, almost always autoclaved separately
  
• NaHCO3, always prepared separately and filter sterilise. Likely to decompose if autoclaved
  
• L-Cysteine HCl also prepared separately many times used together with Na2S and prepared in the same solution, though some recipes autoclave the cysteine with the general initial mix

This is an example from a handbook of media, I think it is easier to follow with an example:

Quote

Clostridium cellobioparum Medium
Composition per 1010.0mL:
NaCl .............................................................................................. 1.0g
K2HPO4 ........................................................................................ 0.5g
KH2PO4 ........................................................................................ 0.5g
(NH4)2SO4 .................................................................................... 0.5g
CaCl2·2H2O .................................................................................. 0.1g
MgSO4·7H2O............................................................................... 0.1g
Resazurin ................................................................................... 1.0mg
Rumen fluid, clarified ............................................................300.0mL
Cellobiose solution ..................................................................50.0mL
NaHCO3 solution .....................................................................30.0mL
Na2S·9H2O solution.................................................................20.0mL
L-Cysteine·HCl solution ..........................................................10.0mL
pH 6.8 ± 0.2 at 25°C
Cellobiose Solution:
Composition per 50.0mL:
D-Cellobiose.................................................................................. 5.0g
Preparation of Cellobiose Solution: Add cellobiose to distilled/
deionized water and bring volume to 50.0mL. Mix thoroughly. Sparge
under 100% N2 gas for 3 min. Filter sterilize. Store under N2 gas.
NaHCO3 Solution:
Composition per 30.0mL:
NaHCO3...................................................................................... 10.0g
Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized
water and bring volume to 30.0mL. Mix thoroughly. Sparge
with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.
Na2S·9H2O Solution:
Composition per 20.0mL:
Na2S·9H2O.................................................................................. 0.25g
Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/
deionized water and bring volume to 20.0mL. Mix thoroughly.
Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.
L-Cysteine·HCl Solution:
Composition per 10.0mL:
L-Cysteine·HCl ........................................................................... 0.25g
Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to
distilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.
Preparation of Medium: Add components, except cellobiose solution,
NaHCO3 solution, Na2S·9H2O solution, and L-cysteine·HCl solution,
to distilled/deionized water and bring volume to 900.0mL. Mix
thoroughly. Sparge with 100% CO2. Autoclave for 15 min at 15 psi
pressure–121°C. Aseptically and anaerobically add 50.0mL of sterile
cellobiose solution, 30.0mL of sterile NaHCO3 solution, 20.0mL of
sterile Na2S·9H2O solution, and 10.0mL of sterile L-cysteine·HCl solution.
Mix thoroughly. Aseptically and anaerobically distribute into
sterile screw-capped bottles.
Use: For the cultivation and maintenance of Clostridium cellobioparum
and Clostridium polysaccharolyticum.

If moderators consider necessary to remove it (as it is extracted from a book) just send me a message.

[SunsetSatine]

Hi,

I did sparge with the CO2/N2 gas mix after taking it out of the autoclave. But then, what do you do with agar? Add everything, but the carbohydrate, and then aseptically
add, before pouring the plates?

Thank you for the example. That really helps. However, for the recipe that I have, everything is in grams.....For example, it says that I have to add 0.4g NaHCO3 per 100 ml. So, in that case, should I prepare 0.4g NaHCO3 in 100 ml of DI water? But, then how can I calculate how much of that solution I need to add to my media after it comes out of the autoclave? Because, I would need to decrease the amount of DI water I use in my media correct?

The amount of starch that I'm using is 0.6g per 100 ml of media....so I would do the same thing and prepare 0.6g of soluble starch in 100 ml DI water? Sparging and then autoclaving of course.

[El Crazy Xabi]

You have to take into account the solubility of the extra solutions you have to make.

e.g. To make 100 mL, if the final concentration of NaHCO3 is 0.4 g/100mL. Make 0.4 g in 10mL. I'm pretty sure you won't have solubility problems with bicarbonate but you have to check it for the starch.
Off course you have to subtract the volume of each separated solution to the main solution (the one with all the other things you don't do separately). In the example the recipe says to bring that solution to 900 mL before

[SunsetSatine]

Hmmm, okay. How do I determine how much of the 0.4g in 10ml, I need to add to my final media?

[El Crazy Xabi]

if you need 0.4 g in your final volume, all

[SunsetSatine]

So, when I make plates with this recipe, I'll prepare everything, except the NaHCO3, Starch, and L-Cysteine, autoclave. Then, while the media is cooling, I will aseptically, add those three components, which have been filter-sterilized/autoclaved seperately?

[El Crazy Xabi]

Aseptically under N2/CO2 atmosphere, yes