Requires specific bacterial environment
The aim is to provide a culture medium that is physiologically favorable for bacteria that is empirically useful for many nutritional, metabolic, and physiological purposes and sufficient for its extensive transport. Specifically, an environment with the following characteristics is desired: (1) The concentration of each of the major nutrients in the culture medium (phosphorus, nitrogen, carbon and sulfur) must be independently regulated and must be incorporated into the bacterial protoplasm by Set appropriate values. To facilitate isotopic labeling. (B) The growth rate supported by the environment shall be at least as fast as supported by any known minimum. (Iii) The culture medium for Enterobacteria should support growth to cell density useful for biochemical measurements. (Iv) should be easily manufactured and inexpensive. (v) Must be stable and preferably capable of storing in concentrate. (vi) Must set a repetitive growth rate. (vii) shall permit absorption measurement. (viii) Cell growth should be supported indefinitely and should not depend on the advent of micronutrients, and (ix) the concentration of hydrogen ions should be sufficiently buffered.
MOPS culture medium
The MOPS culture medium described here meets all of these specifications, with a few comments on the individual characteristics of the environment, respectively. The pH buffer system In many ways, the selection of a suitable buffer is a key issue in the formulation of a synthetic material for biochemical studies on bacteria or other microorganisms. The physiologist’s desire to achieve high cell density in the culture medium places a heavy demand on the buffering capacity of the pH environment and requires that the concentration of specific ions, especially Fe2 3+, be soluble in pH. Phosphate salts are a classical selective buffer due to their favorable pK and because of the phosphate ion as a metal chelate a useful metal ion storage tank as well as this enterobacterial culture medium for Enterobacteria provide an adventurous source of the various elements required in trace amounts. he does. Unfortunately, the usefulness of 32p [P04] labeling in molecular biology requires that the phosphate concentration be reduced below a level where a hydrogen ion buffer is effective. It has been a common substitute for Tris, but for many species it is toxic to thevulture medium at high concentrations required for undesirable PK. MOPS is one of a series of organic buffers made by Good et al. For use in biological work because of its useful pK (7.2 at 20 ° C) instead of at the trance site. Promised to be non-toxic and relatively inexpensive. At concentrations where a buffer is satisfactory, it has no detectable effect on growth.
Final formulation of Enterobacteriaceae
The formulation selected for the culture medium (40 mm initial mean pH 7.2 at 37 ° C) supports glucose growth over the entire recommended range of cell density for physiologically sensitive studies (up to A420 2.0) with a drop of only 0.2 pH units. And allows exponential growth of up to A420 from 5.0 with a drop of only 0.5 pH units. Since glucose is the most acidic micronutrient known for the bacterial enzyme in the culture medium, it is satisfactory to have any quality organic compounds in the media. MOPS cannot be used as a source of carbon, nitrogen or sulfur by S. typhimurium. The species of E. coli we tested cannot use MOPS as a carbon or nitrogen source, but in the absence of added sulfate they can use MOPS as a sulfur source. This use is completely suppressed by low levels of free sulfate in the bacterial culture medium for Enterobacteria, so isotopic labeling with 35S [SO, I-2] shows no problem. The only way the MOPS media cannot be used is the restricted growth of E. coli. For this purpose only Tris can be replaced with MOPS provided that the pH is carefully controlled. MOPS is a weak metal monitor and therefore does not have the desirable properties that it is unlikely to introduce foreign metal pollutants into the culture medium. However, it is not useful in maintaining the appropriate concentration of Fe2 + at neutral pH. Tricine (6) causes chelate Fe2 3 3+ and the addition of a small amount of this organic buffer not only provides a satisfactory reservoir of dissolved iron in the normal environment but also allows the manufacture of a 10-fold thicker material for use Provides comfortable.
Storage: Carbon and energy source
The use of glucose as a culture medium for the growth of Enterobacteriaceae is so widespread that despite its many unusual metabolic characteristics, aerobic fermentation was selected as a standard carbon and energy source, and the MOPS environment was greatly developed. We identified two concentrations. It is theoretically capable of supporting growth at a cell density of 1.5 mg (dry weight) per ml, which is five times higher than the maximum (Al20 = 2.0 ؛ ۰٫۲۸۶ mg [dry weight] / ml) for physiological studies. Recommended. This level is for saturation growth rate. The lower level was selected to provide overnight culture medium. Supports exponential growth up to 1.05 optical density (0.15 mg / cell). Prior to the measurement of cells from these glucocellulose cultures, inoculation is appropriate for most experiments, until a suitable growth period is allowed (preferably three generations). On the other hand, in many cases it is preferable to inoculate light culture medium with cells that are currently in growth. The size of the inoculum can be adjusted to suit the density at a specified time the next day.
Nutrients in the culture of Enterobacteriaceae
Phosphate and sulfate nutrients were adjusted in the new culture medium for, such as glucose, in amounts equivalent to a cellular product of 1.50 mg (dry weight) per ml (A520 = 10.5) calculated from feeding studies with S. typhimurium NT1. For all practical purposes, these concentrations are saturated with the growth rate. Sulfate levels are also sufficient to suppress the destruction of MOPS by E. coli. The magnesium level in the culture medium had to be adjusted to three times higher concentration because it was not sufficiently known that the level equivalent to a cell product of 1.50 mg / ml was saturated in terms of growth rate. However, it has been specified for S. typhimurium. The levels of these components may be balanced for all practical purposes for E. coli. Specific salts used (K2HPO, K2SO, and MgCl2) were selected because of their availability of potassium or chloride as a counterpart and the relatively desirable properties of these media in terms of stability, solubility, fineness and presence of contaminants.
Ratio of concentration of elements in the environment
The same properties exist for the nitrogen source in the selected enterobacterial culture medium NH4Cl. The latter is deliberately set at the equivalent cellular equivalent level of 1.0 mg / ml (A420 = 7.0) such that, in the presence of excess carbon substrate, the cultures in this culture medium are all at the same density and in a constant phase. The same limitation on the need for added iron is easily demonstrated by cultures such as acetate, which are largely metabolized by oxidative pathways using iron proteins and are readily reduced by glycerol or glucose. With some media containing high concentrations of phosphate salts (eg Davies-Mingoly, M9 and Wormen), an iron requirement is less easily demonstrated. Probably because of the introduction of iron as an adventurous contaminant. We set the iron level in the MOPS culture medium Enterobacteria at 10 seconds per millimeter for the culture media because it is saturated but not yet detectable for carbon sources commonly used, such as glucose and glycerol. To grow on acetate (and possibly saccharinate) the amount of iron must be increased fivefold. At this level iron will be saturated with growth rate and the environment will be yellowish only (A420 = 0.012). An alternative course is recommended to increase the amount of Tricine-isnot chloride chelate due to the unexpectedly optimal range of iron concentrations under these conditions. We have found that FeSO4 hepatidate is a good salt for control. The small amount of sulfate in the bacterial culture medium (less than 4%) that adds to the sulfate supplied as K2SO4 is only a minor breach of our principle of using single sources of essential nutrients. FeCl2 can be easily replaced if needed under specific conditions.
In the absence of sensitive methods to determine the low requirements of Enterobacteriaceae in the culture medium for copper, manganese, cobalt, molybdenum, boron and zinc, a different strategy needs to be adopted. Components of a micronutrient solution (Mackelis) produced to support the growth of fungi were each tested for toxicity to Enterobacter species. Inhibitory effects were observed only at concentrations 1000-fold higher than Machelis solution. Using immunity, a concentration of up to 100-fold lower for the MOPS culture medium can be determined to ensure that bacterial cells are saturated for these elements and laboratory-to-laboratory fluctuations in the level of contamination of other environmental components (including glass and water containers). It will be insignificant. As mentioned. The subsequent discovery of a mutant that responds to the micronutrient supplement in the MOPS environment confirms the need for such elements and justifies their intentional inclusion.
Salt concentration: Strive for the highest growth rate and the lowest concentration of culture medium
The inclusion of 50 mM NaCl in the final formulation of the culture medium Enterobacteria was performed with reluctance. It has been attempted to place the concentration of each of the environmental components at the lowest level consistent with the optimal growth rate and a theoretical cell product of 1.50 mg (dry weight) per ml. This conservatism was based on the tendency to minimize the opportunity for adventurous introduction of stimulants or inhibitors. There is no known bacterial need for sodium and the amount of chloride present in the culture medium appears to be sufficient. There is no indication that the slight but real stimulation of growth was with 50 mM NaCl from the contaminants. We experimentally conclude that it simply contributes to the ionic strength of the media. If you want to use a completely sodium-free culture medium, no doubt KCl can be substituted for NaCl arbitrarily chosen.
Aerobic Growth in Bacteria (Soluble Gases)
This culture medium was produced using aerobic growth as a determining guide. Of course this supports anaerobic growth, but under these conditions we did not make any extensive measurements of its properties. The rotating Erlenmeyer flasks we used provide good aeration. But the difficulty of responding to oxygen demand from the rapidly growing and growing bacterial culture medium is quite evident. For this reason, in physiological studies we have defined the upper limit of cell density with this substance as A420 2.0 (exactly less than 0.3 mg of dry weight per ml). Due to the inconvenience of the problem in standardizing the gas composition from laboratory to laboratory, we considered and rejected the possibility of introducing chemical pollutants as gas pollutants and CO2-induced problems with the use of forced aeration (spraying). Needs cells that grow moderately in glucose.
Cultivation of E. coli bacteria
The latter issue appears to be at the heart of E. coli’s high dilution behavior in glucose-MOPS culture medium. The delay of several hours under these conditions is eliminated for all practical purposes including 10 mm NaHCO in the environment. This result is consistent with previous reports of the need for CO2 to E. coli that grows aerobically in the low glucose culture medium. The problem with S. typhimurium NT1 is less acute and seems unnecessary, including NaHCO.3. It should be noted that there are at least two practical reasons for preparing a suitable culture medium Enterobacteria to dilute a few cells per ml without introducing a lag period. The first is useful if there is continuous automatic dilution to maintain cells in balanced growth for long periods, and the second is the excellent prediction of cell growth that prepares the cultures one day in advance and has exponentials. Phase cells prepared at a specific density at a predetermined time the next day.
New bacterial culture medium
The most convenient feature of the newculture medium is that it can be prepared as a 10-fold concentrate containing only phosphate salt and a carbon source at the dilution site. For many researchers, the least suitable media feature may be the need for nutrients and filter sterilization. Sterilization of any media by autoclave is not recommended due to the difficulty in obtaining reproducible product. Autoclaving this particular environment is not possible due to the lack of buffer heat resistance. Filter sterilization. However, this is easily accomplished with the availability of preset (and preservative) membrane filters, which is not a tolerable requirement. Only the denseculture medium Enterobacteria requires sterile filters. Autoclaved distilled water is commonly used to provide the environment. The care required to provide this environment is very poor given the importance of this aspect of physiological studies.