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Growth Medium FAQs

Commonly used bacterial E.coli growth media FAQs

  1. How do you select a commonly used growth medium for a research project?
  2. Why is LB broth the most used medium in many academic labs?
  3. What is the difference between the LB Miller and LB Lennox broths?
  4. Will magnesium (Mg2+) increase the cell densities for commonly-used bacterial E.coli growth media?
  5. Will agitation increase the cell density for commonly used bacterial E.coli growth media?
  6. What is the best commonly used E.coli growth medium for plasmid production?
  7. What is the best commonly used E.coli growth medium for protein production?
  8. What is the best commonly used E.coli growth medium for phage production?
  9. What is the best commonly used E.coli growth medium for making competent cells?
  10. What is the highest cell density obtainable from commonly used bacterial E.coli growth media?

High density bacterial E.coli growth media FAQs...>

FAQ 1. How do you select a commonly used growth medium for a research project?

It will depend on the research project. The following table lists the commonly used media and their applications

Medium Name

Applications

LB Miller Broth

E.coli growth and propagation; plasmid DNA and protein production

LB Lennox Broth E.coli growth and propagation; plasmid DNA and protein production
SOB

Prepare high efficiency competent cells; plasmid DNA and protein production

SOC Plasmid transformation and growth of competent cells
2x YT Phage DNA production
Terrific Broth (TB) High yield protein and plasmid DNA production
Super Broth (SB) High yield plasmid DNA and protein production

FAQ 2. Why is LB broth the most used medium in many academic labs?

LB broth is the most commonly used medium in molecular biology labs for E.coli cell culture. Easy to make, readily available and simple compositions contribute the popularity Of LB broth. In addition, LB broth also has rich nutrient contents. Its osmolarity is close to optimal for E.coli cell growth at early log phase. E.coli strains often grow reasonably fast in LB broth at early log phase. Furthermore, LB broth is sufficient for most molecular biology applications at laboratory scale such as E.coli cell growth and propagation. It is also sufficient for most plasmid, phage, and protein productions at laboratory scales.

FAQ 3. What is the difference between LB Miller and LB Lennox broths?

The LB Miller Broth and LB Lennox Broth contain higher and lower sodium chloride levels respectively. There are minimal differences between the two formulations in most molecular biology studies with commonly used E.coli strains. Different bacteria strains may require different salt concentrations. The low salt formula is more often used in salt-sensitive antibiotic selections. The LB broth used in most labs is LB Miller broth which contains 10 grams NaCl per liter of medium.

FAQ 4. Will magnesium (Mg2+) increase the cell densities for commonly used bacterial E.coli growth media?

Yes. Mg2+ can increase the cell density for LB, 2x YT, TB, and SB. The commonly used Mg2+ concentration is 10 to 20 mM. Either MgCl2, MgSO4, or both of them may be used. In addition, the aeration or the shaking speed of the incubator has to be increased at the same time. The normal shaking speed range of 150 - 250 rpm should be increased to a range of 350 - 400 rpm to obtain significant cell density increase.

SOB and SOC contain sufficient amounts of Mg2+. Adding Mg2+ to these media will not further increase cell density.

FAQ 5. Will agitation increase the cell density for commonly used bacterial E.coli growth media?

Yes. Agitation will increase the aeration of the E.coli cell growth. Oxygen is required for high density growth of E.coli cells. Agitation is controlled by the shaking speed of a shaker incubator. Agitation alone can increase the cell density for all commonly used bacterial E.coli growth media. Cell density will be further increased with combination of increased agitation and added Mg2+ in the common media.

FAQ 6. What is the best commonly used E.coli growth medium for plasmid production?

Super Broth (SB) is the best commonly used E.coli growth medium for plasmid production.

FAQ 7. What is the best commonly used E.coli growth medium for protein production?

Terrific Broth is the best commonly used E.coli growth medium for protein production.

FAQ 8. What is the best commonly used E.coli growth medium for phage production?

Most scientists use 2x YT for phage production.

FAQ 9. What is the best commonly used E.coli growth medium for making competent cells?

SOB is the best commonly used E.coli growth medium for making competent cells.

FAQ 10. What is the highest cell density obtainable from commonly-used bacterial E.coli growth media?

TB can support the highest E.coli cell density when Mg2+ is added and agitation is increased. The highest cell density may be obtained from TB is about OD600 = 18. Some cell strains may reach OD600 = 20, but this result is often inconsistent and cells lose protein production ability at this density or higher.

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High density bacterial E.coli growth media FAQs

  1. Why should a high density growth medium be used for a project?
  2. How do you select a high density growth medium?
  3. Can DNAGroTM growth medium increase the yield for any plasmid?
  4. Can DNAGroTM growth medium be used for protein expression?
  5. Can chloramphenicol be used in DNAGroTM growth medium?
  6. Can ProGroTM, AutoXTM, and InduXTM growth media increase the yield for any recombinant protein?
  7. Can DetoXTM growth medium reduce the toxicity of any toxic protein to the host cells?
  8. Why can these high density growth media increase the solubility of a recombinant protein?
  9. Will these high density growth media increase the solubility for any insoluble protein?
  10. Can these high density growth media increase protein stability? How about protein activity?
  11. What is the typical conditions used for the high density growth media?
  12. What are the highest OD600 values from different shake flask containers?
  13. What are the OD600 values at the normal shaking speed?
  14. Can high density growth media be used at low temperatures?
  15. What are the differences between glass and plastic flasks? How about different plastics?
  16. What are the differences between baffled and regular flasks?
  17. What are the differences among conical, round- bottom, and square-bottom tubes?
  18. Why can't I reach OD600 = 30 to 50 even though the high density media were shaken at 350 to 450 rpm?
  19. Can we use the high density media in deep-well plates? What will be the OD?
  20. Why do I sometimes get OD higher than 50?
  21. Can all high density growth media reach OD600 = 30 to 50?
  22. How do these high density growth media compare to other special media?
  23. What are the benefits of using high density growth media?

FAQ 1. Why should a high density growth medium be used for a project?

Common media cannot support sustained high density cell growth. With increase aeration and added Mg2+, LB broth can support E.coli cell density to OD600 = 10. Since LB broth contains no buffer, the pH of the medium will quickly change to pH 9 or higher. At this pH, E.coli cells will be unable to express protein. TB can support E.coli cell density to OD600 = 18 with increased aeration and added Mg2+. The medium pH also quickly changes to pH 9 or higher. The time window that allows protein expression is still short. It often does not allow induction of three hours or longer.

All high density growth media can support E.coli cell density OD600 = 30 to 50 depending on the containers used. The high density growth media are buffered by phosphates and organic acids/salts. At OD600 = 30, the pH of all high density growth media is at about 7, which is optimal for most protein expressions. The time windows allowing protein expression in these media are sufficient for most protein productions.

High density growth media allow the yield of plasmid and protein productions to reach the levels of fed-batch fermentation in the shake flask containers. This increase plasmid and protein yields 10 times more than common media such as LB broth. The amounts of mini-prep plasmid and protein will be sufficient for many biochemical analyses. The amounts of large prep plasmid and protein will be comparable with those that may be obtained from a fed-batch fermentor. High density growth media increase efficiency and save time.

High density growth media contains trace metals, minerals, and vitamins which may serve as prosthetic groups, co-factors, or ligands for recombinant proteins. These prosthetic groups, co-factors, or ligands are often critical for protein folding, solubility, stability, and activity. Therefore, many proteins are more soluble, stable, and functional when expressed in these media.

FAQ 2. How do you select a high density growth medium?

The following table lists the high density growth media and their applications.

Medium Name

Applications
DNAGroTM Increases plasmid DNA yield 10 times over LB
ProGroTM Increases protein yield, solubility, stability and activity
AutoXTM Increases protein yield, solubility, stability and activity; automatic protein expression without adding IPTG
DetoXTM Reduces protein toxicity and support E.coli growth to OD600=30
InduXTM Increases protein yield, solubility, stability and activity
SecProTM Increases secretary protein expression over 100 times than LB or TB

FAQ 3. Can DNAGroTM growth medium increase the yield for any plasmid?

Yes. DNAGroTM growth medium will support E.coli cell density to OD600 = 30 or higher. The results apply to all commonly used E.coli strains such as DH5a, XL1blue, and Top10 cells. It is important to make sure that the amount of appropriate antibiotic is sufficient for plasmid selection at higher cell density.

FAQ 4. Can DNAGroTM growth medium be used for protein expression?

No. DNAGroTM growth medium contains inhibitors for protein expression.

FAQ 5. Can chloramphenicol be used in DNAGroTM growth medium?

Yes. Chloramphenicol can be used to amplify relaxed plasmid copy number in E.coli cells, but not the stringent plasmids. This applies to common media such as LB as well as high density growth medium DNAGroTM.

FAQ 6. Can ProGroTM, AutoXTM, and InduXTM growth media increase the yield for any recombinant protein?

Yes. ProGroTM and AutoXTM growth media increase the cell density for all bacterial strains such as BL21. Protein expression will increase proportionally with the cell density. It is important to make sure that the amount of appropriate antibiotic is sufficient for plasmid selection at higher cell density.

FAQ 7. Can DetoXTM growth medium reduce the toxicity of any toxic protein to the host cells?

No. DetoXTM growth medium can only reduce the toxicity of proteins induced by sugars such as lactose, IPTG, and arabinose. It cannot reduce the toxicity of proteins induced by heat, salts, or anything other than sugars. In addition, it can only reduce the pre-induction toxicity. It cannot reduce the post-induction toxicity.

FAQ 8. Why can these high density growth media increase the solubility of a recombinant protein?

High density growth media contains trace metals, minerals, and vitamins which may serve as prosthetic groups, co-factors, or ligands for recombinant proteins. These prosthetic groups, co-factors, or ligands are often critical for protein folding and solubility. Therefore, many proteins are more soluble when expressed in these media.

FAQ 9. Will these high density growth media increase the solubility for any insoluble protein?

No. The high density growth media will only increase the solubility of the proteins which required trace metals, minerals, and vitamins.

FAQ 10. Can these high density growth media increase protein stability? And how about protein activity?

High density growth media contain trace metals, minerals, and vitamins which may serve as prosthetic groups, co-factors, or ligands for recombinant proteins. These prosthetic groups, co-factors, or ligands are often critical for protein stability and activity. Therefore, many proteins are more stable and functional when expressed in these media.

FAQ 11. What are the typical conditions used for the high density growth media?

High density growth media can be used in 14 ml tubes or in any flasks with or without baffles. Both glass and plastic containers may be used. these growth media are designed to grow bacterial cells at 370C. Temperatures lower than 370C may be used for protein expression. Typical shaking speeds for high density growth media in a baffled flask is 350 rpm and 450 rpm in a regular (non-baffled) flask. All containers should be balanced and securely fixed in a shaker incubator. In addition, the incubator and incubation room must be sufficiently ventilated for cultures of 500 ml or more. High density growth media may be used the same as common media except that they need better aeration and will support higher cell density.

FAQ 12. What are the highest OD600 values from different shake flask containers?

The highest OD600 values from the different shake flask containers are listed in the following tables.

Brand Pyrex Pyrex Pyrex Pyrex Nalgene Nalgene Nalgene Nalgene
Material Glass Glass Glass Glass Polypropylene Polypropylene Polycarbonate Polycarbonate
Volume 2L 2L 250 ml 250 ml 2L 250 ml 2L 250 ml
Test ml 500 500 50 50/100 500 50 50 50
Baffled No Yes No Yes No No Yes Yes
rpm 450 350 450 350 450 450 350 350
OD600 30 30 50 40 30 50 30 40

 

Brand Corning Kimax Kimax Bellco UltraYield UltraYield Falcon Falcon
Material Polycarbonate Glass Glass Glass Polypropylene Polypropylene Polypropylene polyethylene
Volume 250 ml 250 ml 250 ml 250 2.5L 250 ml 14 ml 14 ml
Test ml 50 50 50 50 500* 50 1.5 1.5
Baffled No No Yes Yes Yes Yes No No
rpm 450 450 350 350 350** 400 400 400
OD600 50 50 40 40 30 40 40 40

* Larger volume will result in medium spilling out.
** Higher shaking speed will result in medium spilling out.

FAQ 13. What are the OD600 values at the normal shaking speed?

The normally-used shaking speed for E.coli growth is 250 rpm in most labs. At 250 rpm, the high density growth media can only reach about OD600 = 10. The OD values will be lower at speeds slower than 250 rpm. At low shaking speeds (<350 rpm), the oxygen concentration is limited in the medium and the cells will produce large quantity of acids which will make the medium acidic. Therefore, it is not recommended to use high density growth media at low shaking speeds.

FAQ 14. Can high density growth media be used at low temperatures?

Yes. Temperatures lower than 37 0C are often used to express insoluble or toxic proteins. In these cases, the E.coli cells should be grown at 37 0C to reach inducible OD and the temperature should be changed to induce the protein expression rate.

FAQ 15. What are the differences between glass and plastic flasks? How about different plastics?

There are no significant differences between glass and plastic flasks in E.coli cell cultures (see tables in FAQ 12). The differences between polypropylene and polycarbonate are also minimal. Different materials exhibit little difference in E.coli cell culture. The designs of the containers are important for E.coli growth and cell density. Flasks generally give better aeration than the tubes. Smaller flasks allow better aeration than larger ones.

FAQ 16. What are the differences between baffled and regular flasks?

Baffled flasks will generate more agitation. More agitation means better aeration. More agitation also produces more foam. Foams act as oxygen barriers. Foams produced at high shaking speeds offset the benefits of agitation. We found that shaking speed range of 350 - 400 rpm is optimal for E.coli growth in baffled shake flask containers. For regular flasks, the optimal shaking speed range is 400 - 450 rpm. Regular flasks (non-baffled) generate less agitation. Less agitation also produces less foam. These flasks produce no foam with our high density growth media with antifoaming agents. With no foam produced, aeration increases with higher shaking speed. In general, the cell densities in regular flasks are higher than those in baffled flasks, but they require higher shaking speed.

Baffled flasks should be used if the shaker incubator cannot reach a high speed. Regular shake flasks should be chosen if the shaker incubator can reach a high speed. Both regular and baffled flasks can achieve OD600 = 30 to 50 with high density growth media.

FAQ 17. What are the differences among conical, round- bottom, and square-bottom tubes?

Conical tubes allow least aeration. They should be avoided in E.coli cell culture. Round-bottom tubes are most commonly used in E.coli cell cultures. They allow less aeration than flasks. The medium and container volume ratio for round-bottom tubes should be at about 1/10. Square-bottom tubes give best aeration at a given shaking speed. They can be easily fixed on the shaking platform too. Containers tightly fixed on the shaking platform give better aeration than those loosely placed on the holders. Similar as the flasks, different materials do not make significant difference on E.coli growth and cell density.

FAQ 18. Why can't I reach OD600 = 30 to 50 even though the high density media were shaken at 350 to 450 rpm?

1. Glycerol concentration has to be correct when preparing the medium from powder. Glycerol is viscous. Measurement will be accurate if it is made into 50% solution in water.
2. Additives must be added just before use with antibiotics. Adding Additives when making the medium will cause precipitation. Medium with precipitation will only reach OD600 = 20.
3. Medium volume should not exceed the recommended volume:

Flask Regular Baffled tube
Volume* 1/8 to 1/4 1/4 to 1/2 1/10
rpm 400 t0 450 350 to 400 350 to 400

*The recommended volume is the medium volume divided by container volume. For example, the recommended volume is 250 to 500 ml if the container volume is 2 liters and it does not have baffles. 500 ml is recommended for the containers with volume larger than 2 liters.
4. All containers MUST be securely fixed on the shaker incubator. This is especially critical for when the tubes are used in the culture. If the tubes are loosely placed on a rack with large holders, the tubes will rotate in the holders and the media inside the tubes will not be sufficiently agitated. Racks with small holders should be used. Rubber bands may be used to fix the tubes onto the rack. Balanced loading is also important when the medium volumes are greater than 50 ml. Fixing containers securely is not only for safety but also for aeration.
5. The container cover should allow the best aeration possible. In no case should the container cover be completely closed. Use the cover that allows the best ventilation possible. After the OD600 reaches 10, the container cover may be removed to maximize aeration. We never encountered any cross-contamination at this or higher cell density.
6. The inoculation ratio is incorrect. The recommended inoculation ration is 1:100 with the seed culture prepared in a high density growth medium. With this inoculation ratio, the E.coli cells will reach OD600 = 30 or higher in 12 to 16 hours (overnight). Any ratio lower than the recommended inoculation ratio will require longer growth time. Any ratio higher than the recommended inoculation ration will result in over-growth.
7. OD measurement is more accurate at values less than 0.5 for most spectrometers. For common media, this means to dilute the culture 10 times. For high density growth media, this means a 100-time dilution. Distilled or deionized water should be used as a blank and in dilution. Rich media often have color by themselves. OD measurement will not be accurate if a medium is used as a blank or in dilution.

The high density growth media cannot reach expected cell density if any of the recommended operations above cannot be followed. However the cell density in the high density growth media will be significantly higher than what may be obtained from common media such as LB or TB.

FAQ 19. Can we use the high density media in deep-well plates? What will be the OD?

Yes. The OD600 will be 50 or higher. Use caution to prevent the medium from desiccating or dry out.

FAQ 20. Why do I sometimes get OD higher than 50?

1. The medium may desiccate during incubation. Open water may be placed in the incubator to increase the humidity and to avoid medium desiccation.
2. Incubation  longer than recommended time may result in OD600 higher than 50.
3. OD600 up to 200 may be reached with our high density growth media in a fed-batch fermentor. In these cases, extra oxygen should be supplied and the medium pH should be adjusted. The cells must be diluted 500 to 1000 times to get an accurate OD reading. Only water should be used both as a blank and in dilution for OD reading.

FAQ 21. Can all high density growth media reach OD600 = 30 to 50?

Yes. All these high density growth media can reach OD600 = 30 to 50 in a shake flask container. Most wild-type cell strains can reach OD600 = 20 to 30 in SecProTM medium. Some cell strains with some toxic proteins do not grow well in SecProTM medium. In these cases, the cells may be grown in DetoXTM or ProGroTM medium to reach inducible OD and induce the cells in SecProTM medium to get high yield secretary expression.

FAQ 22. How do these high density growth media compare to other special media?

We tested ON Express, Circular Grow, and Magic media. The highest cell density may be reach in these media is about OD600 = 20. The plasmid and protein yields are lower in these media than those in high density growth media. Some proteins are not soluble and functional when expressed in ON Express and Magic media indicating these media lack required trace metals, minerals, and vitamins for protein solubility and activity.

FAQ 23. What are the benefits of using high density growth media?

1. High density growth media will increase the yields of both plasmid DNA and recombinant proteins. The yields are often increased 10 times or more. The yields will increase over 100 times in secretary and toxic protein expression. They will save time and labor and therefore increase the efficiency of your work.
2. Since the high density growth media contain trace metals, minerals, and vitamins, they may serve as prosthetic groups, co-factors or ligands for recombinant proteins and therefore increase the solubility, stability, and activity of a recombinant protein.

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