In our lab we prepare a 20% stock solution of tween-80 by dissolving it in stirring warm-hot (not boiling) distilled water (heat it in the microwave, think uncomfortably hot bathwater) followed by filter sterilization with a stericup. This stock solution should be protected from light, stored at 4C and discarded after 60 days (trust me, it will go bad). Add this to your cooled 7H9 or Sautons broth as needed before use. As a rule I don't use media with tween that has sat for over a week at room temperature.
I have seen the malachite green concentrations vary by supplier. Its role in the media is antifungal and is more important when growing slow-growing species such as M. tb where mold would have a lot of time to get going. Considering how fast M. smeg. grows I imagine your could could use a lower concentration without much risk. That said, most people use pre-made Middlebrook agar mixes with all of these components already added. I have found the ones from Hardy Diagnostics to work well and to be cost-effective.
It has been my experience that M. smeg. does not grow well (at all) on 7H11, I usually grow it on 7H10. It also grows perfectly well on good old LB agar plates. While M. smeg like all mycobacteria appreciate OADC if you are watching your spend you could probably just supplement your media with glycerol and glucose from sterile stock solutions.
In our lab we prepare a 20% stock solution of tween-80 by dissolving it in stirring warm-hot (not boiling) distilled water (heat it in the microwave, think uncomfortably hot bathwater) followed by filter sterilization with a stericup. This stock solution should be protected from light, stored at 4C and discarded after 60 days (trust me, it will go bad). Add this to your cooled 7H9 or Sautons broth as needed before use. As a rule I don't use media with tween that has sat for over a week at room temperature.
I have seen the malachite green concentrations vary by supplier. Its role in the media is antifungal and is more important when growing slow-growing species such as M. tb where mold would have a lot of time to get going. Considering how fast M. smeg. grows I imagine your could could use a lower concentration without much risk. That said, most people use pre-made Middlebrook agar mixes with all of these components already added. I have found the ones from Hardy Diagnostics to work well and to be cost-effective.
It has been my experience that M. smeg. does not grow well (at all) on 7H11, I usually grow it on 7H10. It also grows perfectly well on good old LB agar plates. While M. smeg like all mycobacteria appreciate OADC if you are watching your spend you could probably just supplement your media with glycerol and glucose from sterile stock solutions.
In our lab we prepare a 20% stock solution of tween-80 by dissolving it in stirring warm-hot (not boiling) distilled water (heat it in the microwave, think uncomfortably hot bathwater) followed by filter sterilization with a stericup. This stock solution should be protected from light, stored at 4C and discarded after 60 days (trust me, it will go bad). Add this to your cooled 7H9 or Sautons broth as needed before use. As a rule I don't use media with tween that has sat for over a week at room temperature.
I have seen the malachite green concentrations vary by supplier. Its role in the media is antifungal and is more important when growing slow-growing species such as M. tb where mold would have a lot of time to get going. Considering how fast M. smeg. grows I imagine your could could use a lower concentration without much risk. That said, most people use pre-made Middlebrook agar mixes with all of these components already added. I have found the ones from Hardy Diagnostics to work well and to be cost-effective.
It has been my experience that M. smeg. does not grow well (at all) on 7H11, I usually grow it on 7H10. It also grows perfectly well on good old LB agar plates. While M. smeg like all mycobacteria appreciate OADC if you are watching your spend you could probably just supplement your media with glycerol and glucose from sterile stock solutions.
In our lab we prepare a 20% stock solution of tween-80 by dissolving it in stirring warm-hot (not boiling) distilled water (heat it in the microwave, think uncomfortably hot bathwater) followed by filter sterilization with a stericup. This stock solution should be protected from light, stored at 4C and discarded after 60 days (trust me, it will go bad). Add this to your cooled 7H9 or Sautons broth as needed before use. As a rule I don't use media with tween that has sat for over a week at room temperature.
I have seen the malachite green concentrations vary by supplier. Its role in the media is antifungal and is more important when growing slow-growing species such as M. tb where mold would have a lot of time to get going. Considering how fast M. smeg. grows I imagine your could could use a lower concentration without much risk. That said, most people use pre-made Middlebrook agar mixes with all of these components already added. I have found the ones from Hardy Diagnostics to work well and to be cost-effective.
It has been my experience that M. smeg. does not grow well (at all) on 7H11, I usually grow it on 7H10. It also grows perfectly well on good old LB agar plates. While M. smeg like all mycobacteria appreciate OADC if you are watching your spend you could probably just supplement your media with glycerol and glucose from sterile stock solutions.
Hi Kieran, unsure about the malachite green, but with regards to the Tween-80, don't autoclave. The best option is to create a working solution with warm distilled water, filter sterilise and store. When required add to your autoclaved media when the temperature reaches around 55C, the same as you would add OADC.
Hi Kieran, unsure about the malachite green, but with regards to the Tween-80, don't autoclave. The best option is to create a working solution with warm distilled water, filter sterilise and store. When required add to your autoclaved media when the temperature reaches around 55C, the same as you would add OADC.
Middle brook 7h10 has a lesser concentration of malachite green when compared to 7h11 and 7h10 is generally used for plating recombinants (electroporation) as too much of malachite green can inhibit their growth. Tween 80 can be autoclaved along with media. It works! If u wanna make stocks of 20% tween 80 then u need to dissolve it in warm water with continuous stirring.. But I suggest u to prepare 20% tween 80 freshly every time u prepare media. It yields better results.
Middle brook 7h10 has a lesser concentration of malachite green when compared to 7h11 and 7h10 is generally used for plating recombinants (electroporation) as too much of malachite green can inhibit their growth. Tween 80 can be autoclaved along with media. It works! If u wanna make stocks of 20% tween 80 then u need to dissolve it in warm water with continuous stirring.. But I suggest u to prepare 20% tween 80 freshly every time u prepare media. It yields better results.
With respect to your question regarding malachite green concentrations, they should be the same regardless of making up 7H10 or 7H11. Although you can alter the concentration of this in your media depending on you perpensity to certain types of mould contamination. Generally I've never used more thant 25mg/L but the highest commercial concentration I've used was 0.25g/L which made for green media but it yielded nice Mycobacterial colonies.
With respect to your second question, you don't NEED Tween-80 unless you're looking for M. bovis in a strict sense or you're looking to do specific strain phenotyping with 7H9. In any case I've never autoclaved Tween-80. Filter sterilise all the way is my view.
With respect to your question regarding malachite green concentrations, they should be the same regardless of making up 7H10 or 7H11. Although you can alter the concentration of this in your media depending on you perpensity to certain types of mould contamination. Generally I've never used more thant 25mg/L but the highest commercial concentration I've used was 0.25g/L which made for green media but it yielded nice Mycobacterial colonies.
With respect to your second question, you don't NEED Tween-80 unless you're looking for M. bovis in a strict sense or you're looking to do specific strain phenotyping with 7H9. In any case I've never autoclaved Tween-80. Filter sterilise all the way is my view.
What should be the concentration of 20% tween 80 in Middlebrook 7H10 agar base media for m. smegmatis ?
What should be the concentration of 20% tween 80 in Middlebrook 7H10 agar base media for m. smegmatis ?
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In our lab we prepare a 20% stock solution of tween-80 by dissolving it in stirring warm-hot (not boiling) distilled water (heat it in the microwave, think uncomfortably hot bathwater) followed by filter sterilization with a stericup. This stock solution should be protected from light, stored at 4C and discarded after 60 days (trust me, it will go bad). Add this to your cooled 7H9 or Sautons broth as needed before use. As a rule I don't use media with tween that has sat for over a week at room temperature.
I have seen the malachite green concentrations vary by supplier. Its role in the media is antifungal and is more important when growing slow-growing species such as M. tb where mold would have a lot of time to get going. Considering how fast M. smeg. grows I imagine your could could use a lower concentration without much risk. That said, most people use pre-made Middlebrook agar mixes with all of these components already added. I have found the ones from Hardy Diagnostics to work well and to be cost-effective.
It has been my experience that M. smeg. does not grow well (at all) on 7H11, I usually grow it on 7H10. It also grows perfectly well on good old LB agar plates. While M. smeg like all mycobacteria appreciate OADC if you are watching your spend you could probably just supplement your media with glycerol and glucose from sterile stock solutions.
In our lab we prepare a 20% stock solution of tween-80 by dissolving it in stirring warm-hot (not boiling) distilled water (heat it in the microwave, think uncomfortably hot bathwater) followed by filter sterilization with a stericup. This stock solution should be protected from light, stored at 4C and discarded after 60 days (trust me, it will go bad). Add this to your cooled 7H9 or Sautons broth as needed before use. As a rule I don't use media with tween that has sat for over a week at room temperature.
I have seen the malachite green concentrations vary by supplier. Its role in the media is antifungal and is more important when growing slow-growing species such as M. tb where mold would have a lot of time to get going. Considering how fast M. smeg. grows I imagine your could could use a lower concentration without much risk. That said, most people use pre-made Middlebrook agar mixes with all of these components already added. I have found the ones from Hardy Diagnostics to work well and to be cost-effective.
It has been my experience that M. smeg. does not grow well (at all) on 7H11, I usually grow it on 7H10. It also grows perfectly well on good old LB agar plates. While M. smeg like all mycobacteria appreciate OADC if you are watching your spend you could probably just supplement your media with glycerol and glucose from sterile stock solutions.
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VOTE
In our lab we prepare a 20% stock solution of tween-80 by dissolving it in stirring warm-hot (not boiling) distilled water (heat it in the microwave, think uncomfortably hot bathwater) followed by filter sterilization with a stericup. This stock solution should be protected from light, stored at 4C and discarded after 60 days (trust me, it will go bad). Add this to your cooled 7H9 or Sautons broth as needed before use. As a rule I don't use media with tween that has sat for over a week at room temperature.
I have seen the malachite green concentrations vary by supplier. Its role in the media is antifungal and is more important when growing slow-growing species such as M. tb where mold would have a lot of time to get going. Considering how fast M. smeg. grows I imagine your could could use a lower concentration without much risk. That said, most people use pre-made Middlebrook agar mixes with all of these components already added. I have found the ones from Hardy Diagnostics to work well and to be cost-effective.
It has been my experience that M. smeg. does not grow well (at all) on 7H11, I usually grow it on 7H10. It also grows perfectly well on good old LB agar plates. While M. smeg like all mycobacteria appreciate OADC if you are watching your spend you could probably just supplement your media with glycerol and glucose from sterile stock solutions.
In our lab we prepare a 20% stock solution of tween-80 by dissolving it in stirring warm-hot (not boiling) distilled water (heat it in the microwave, think uncomfortably hot bathwater) followed by filter sterilization with a stericup. This stock solution should be protected from light, stored at 4C and discarded after 60 days (trust me, it will go bad). Add this to your cooled 7H9 or Sautons broth as needed before use. As a rule I don't use media with tween that has sat for over a week at room temperature.
I have seen the malachite green concentrations vary by supplier. Its role in the media is antifungal and is more important when growing slow-growing species such as M. tb where mold would have a lot of time to get going. Considering how fast M. smeg. grows I imagine your could could use a lower concentration without much risk. That said, most people use pre-made Middlebrook agar mixes with all of these components already added. I have found the ones from Hardy Diagnostics to work well and to be cost-effective.
It has been my experience that M. smeg. does not grow well (at all) on 7H11, I usually grow it on 7H10. It also grows perfectly well on good old LB agar plates. While M. smeg like all mycobacteria appreciate OADC if you are watching your spend you could probably just supplement your media with glycerol and glucose from sterile stock solutions.
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Hi Kieran, unsure about the malachite green, but with regards to the Tween-80, don't autoclave. The best option is to create a working solution with warm distilled water, filter sterilise and store. When required add to your autoclaved media when the temperature reaches around 55C, the same as you would add OADC.
Hi Kieran, unsure about the malachite green, but with regards to the Tween-80, don't autoclave. The best option is to create a working solution with warm distilled water, filter sterilise and store. When required add to your autoclaved media when the temperature reaches around 55C, the same as you would add OADC.
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Middle brook 7h10 has a lesser concentration of malachite green when compared to 7h11 and 7h10 is generally used for plating recombinants (electroporation) as too much of malachite green can inhibit their growth. Tween 80 can be autoclaved along with media. It works! If u wanna make stocks of 20% tween 80 then u need to dissolve it in warm water with continuous stirring.. But I suggest u to prepare 20% tween 80 freshly every time u prepare media. It yields better results.
Middle brook 7h10 has a lesser concentration of malachite green when compared to 7h11 and 7h10 is generally used for plating recombinants (electroporation) as too much of malachite green can inhibit their growth. Tween 80 can be autoclaved along with media. It works! If u wanna make stocks of 20% tween 80 then u need to dissolve it in warm water with continuous stirring.. But I suggest u to prepare 20% tween 80 freshly every time u prepare media. It yields better results.
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With respect to your question regarding malachite green concentrations, they should be the same regardless of making up 7H10 or 7H11. Although you can alter the concentration of this in your media depending on you perpensity to certain types of mould contamination. Generally I've never used more thant 25mg/L but the highest commercial concentration I've used was 0.25g/L which made for green media but it yielded nice Mycobacterial colonies.
With respect to your second question, you don't NEED Tween-80 unless you're looking for M. bovis in a strict sense or you're looking to do specific strain phenotyping with 7H9. In any case I've never autoclaved Tween-80. Filter sterilise all the way is my view.
With respect to your question regarding malachite green concentrations, they should be the same regardless of making up 7H10 or 7H11. Although you can alter the concentration of this in your media depending on you perpensity to certain types of mould contamination. Generally I've never used more thant 25mg/L but the highest commercial concentration I've used was 0.25g/L which made for green media but it yielded nice Mycobacterial colonies.
With respect to your second question, you don't NEED Tween-80 unless you're looking for M. bovis in a strict sense or you're looking to do specific strain phenotyping with 7H9. In any case I've never autoclaved Tween-80. Filter sterilise all the way is my view.
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VOTE