HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) is a hydrogen ion buffer with good pH buffering capacity and can effectively buffer the pH changes of the system. It is often indispensable in the process of cell culture. Let's take a look at what effects it has played in the culture of Mycoplasma pneumoniae?

Mycoplasma pneumoniae

Mycoplasma pneumoniae is one of the important pathogenic microorganisms of human respiratory tract infections. Conventional Mycoplasma pneumoniae culture uses liquid medium (PH 7.0-7.4) glucose as an important carbon source for its growth and metabolism. However, as the metabolite lactic acid accumulates during the culture process, the pH value of the medium will drop rapidly. Too low pH value is not good for the growth of Mycoplasma pneumoniae, which makes it enter the decline phase. Therefore, during the culture of Mycoplasma pneumoniae, it is necessary to pay close attention to its growth and pH changes to prevent the rapid death of Mycoplasma pneumoniae due to the drop in pH during the culture process and the inability to isolate live Mycoplasma.

HEPES buffer

Low-toxic HEPES is a commonly used cell culture buffer. The final concentration is generally 10-50 mmol/L. Normally, the culture medium contains 20 mmol/L HEPES to serve as a buffer. The usual concentration for cell culture is 10 mmol/L . HEPES buffer has a buffering effect in this process, and the greater the concentration, the more obvious the buffering effect. When the pH of the medium is around 6.8, the cultured Mycoplasma pneumoniae has entered the mid-late logarithmic growth period; the pH of the medium is between 6.5 and 6.0, the content of Mycoplasma pneumoniae in the medium reaches the plateau, and the bacterial population is maintained at a large level.

As the concentration of HEPES in the medium increases, the buffering capacity of the medium is correspondingly enhanced. Theoretically, the amount of cultured Mycoplasma pneumoniae can be increased, and the plateau period should be correspondingly extended. However, in the actual culture process, only the amount of Mycoplasma pneumoniae cultured in 10 mmol/L HEPES buffer increased significantly, and HEPES may have a certain inhibitory effect on the growth of Mycoplasma pneumoniae. As the concentration of HEPES increases, this inhibitory effect retards the increase in the amount of bacteria in the culture process to a certain extent, and only manifests as the extension of the plateau period of the culture process. 10 mmol/L HEPES has little inhibitory effect on Mycoplasma pneumoniae and still has a certain buffering capacity. However, due to the limited buffering capacity, the excessive acidic substances produced by it cannot be effectively buffered, and the pH value of the medium drops rapidly, causing its growth and growth during the decay period. Culture without HEPES is similar.

Therefore, adding HEPES at different concentrations of 10 to 80 mmol/L to the medium can affect the growth of Mycoplasma pneumoniae. 10 mmol/L HEPES can increase the maximum CCU for liquid culture of Mycoplasma pneumoniae. For Mycoplasma pneumoniae, which is a small, difficult-to-cultivable microorganism with a low culture concentration, it can save money when a large number of cultures need to be obtained for protein extraction, antigen analysis, strain preservation, etc. Cultivation cost, improve the collection efficiency of mycoplasma. Although 50 mmol/L HEPES can not significantly improve the liquid culture CCU, it can prolong the growth plateau during the culture of Mycoplasma pneumoniae, making it less difficult to judge the culture and passage, which is beneficial to observe the growth status of Mycoplasma pneumonia and avoid the lack of experience. Cultivation failure caused by improper passaging time.