isolation be in the alkaline range. To fulfill this condition, a mixture of two sodium
salts, carbonate, and/or bicarbonate in various ratios is added to the medium. This is
the main difference between an alkaline medium and the “usual” one that is used for
neutralophiles. The mixture of both salts (sometimes is referred to as a sesquicarbonate solution) forms an alkaline buffer, and, depending on their ratios, it is possible to
get different pH values in the range of 8.3–11.5. While sodium ions will be present in
the carbonate buffer, it is still recommended to include sodium chloride in the
formulation at least at the initial step of isolation. Chloride ions are inherent in
many habitats of alkaliphiles and are absolutely necessary for the isolation of
haloalkaliphiles and can significantly enhance the growth for natronophiles.
When selecting substrates for an alkaline medium, it should always be remembered that when heated at an elevated temperature and high pH condition, very few
substances remain unchanged. These are mainly low molecular organic acids, alcohols, most of amino acids (but not all), sugar alcohols, and already hydrolyzed
substrates like peptone or casamino acids. All complex organic substances such as
polymers (pectin, casein, starch, etc., except for cellulose) and sugars will irreversibly
decompose by thermoalkaline hydrolysis during autoclaving, and, thus, the convenient “all-in-one” principle does not always fit. There are two possible solutions for
this problem. The first one is the well-known approach, labile substance prepared as
concentrated solution which is sterilized separately either in the same autoclave but as
neutral solution or by filtrating through membrane filter with a pore size of 0.2 μm.
Then, separately sterilized component is poured into the bulk of the medium.
However, not all substances can be prepared as concentrated solutions (e.g., most
polymers, poorly soluble or insoluble substances), so another approach called the
“double concentration technique” is used. In this case, sodium carbonate and sodium
bicarbonate (sometimes sodium chloride is also included) are dissolved in a separate
screw or crimp cap closed bottle with concentration double of that required
(an alkaline solution I), and all the other components, including the labile one, are
also dissolved with a double concentration in the flask with a cotton plug (a neutral
solution II). After separate autoclaving, both solutions are mixed together (solution I
is poured into solution II), and the final solution will have the given concentrations of
all components. When mixing, the volumes of both solutions must be the same.
A closed bottle is necessary, since sodium bicarbonate reversibly decomposes on
heating. In the case of extreme haloalkaliphiles (Na
+
> 2 M), it may be necessary to
divide solution I into two parts: a mixture of carbonates and a separate NaCl solution.
In case an anaerobic medium is prepared, solution II should also be prepared in a
closed bottle, and both bottles must be previously purged with nitrogen prior to
autoclaving. A reducing agent that is needed for strict anaerobes can be added either
in solution I or solution II, depending on its stability toward pH. We found that the
redox indicator resazurin used in working with strict anaerobes proved to be stable to
pH 10 and destroyed above this value. When several labile substances are used, as in
the case of testing of the spectrum of substrates used, individual solutions II can be
prepared in test tubes (or in Hungate tubes for anaerobes), and solution I is used as the
stock solution. In case of doubt, whether the particular substance is resistant to
thermoalkaline hydrolysis, the common advice will be either to search for its
Isolation and Cultivation of Alkaliphiles
69
salts, carbonate, and/or bicarbonate in various ratios is added to the medium. This is
the main difference between an alkaline medium and the “usual” one that is used for
neutralophiles. The mixture of both salts (sometimes is referred to as a sesquicarbonate solution) forms an alkaline buffer, and, depending on their ratios, it is possible to
get different pH values in the range of 8.3–11.5. While sodium ions will be present in
the carbonate buffer, it is still recommended to include sodium chloride in the
formulation at least at the initial step of isolation. Chloride ions are inherent in
many habitats of alkaliphiles and are absolutely necessary for the isolation of
haloalkaliphiles and can significantly enhance the growth for natronophiles.
When selecting substrates for an alkaline medium, it should always be remembered that when heated at an elevated temperature and high pH condition, very few
substances remain unchanged. These are mainly low molecular organic acids, alcohols, most of amino acids (but not all), sugar alcohols, and already hydrolyzed
substrates like peptone or casamino acids. All complex organic substances such as
polymers (pectin, casein, starch, etc., except for cellulose) and sugars will irreversibly
decompose by thermoalkaline hydrolysis during autoclaving, and, thus, the convenient “all-in-one” principle does not always fit. There are two possible solutions for
this problem. The first one is the well-known approach, labile substance prepared as
concentrated solution which is sterilized separately either in the same autoclave but as
neutral solution or by filtrating through membrane filter with a pore size of 0.2 μm.
Then, separately sterilized component is poured into the bulk of the medium.
However, not all substances can be prepared as concentrated solutions (e.g., most
polymers, poorly soluble or insoluble substances), so another approach called the
“double concentration technique” is used. In this case, sodium carbonate and sodium
bicarbonate (sometimes sodium chloride is also included) are dissolved in a separate
screw or crimp cap closed bottle with concentration double of that required
(an alkaline solution I), and all the other components, including the labile one, are
also dissolved with a double concentration in the flask with a cotton plug (a neutral
solution II). After separate autoclaving, both solutions are mixed together (solution I
is poured into solution II), and the final solution will have the given concentrations of
all components. When mixing, the volumes of both solutions must be the same.
A closed bottle is necessary, since sodium bicarbonate reversibly decomposes on
heating. In the case of extreme haloalkaliphiles (Na
+
> 2 M), it may be necessary to
divide solution I into two parts: a mixture of carbonates and a separate NaCl solution.
In case an anaerobic medium is prepared, solution II should also be prepared in a
closed bottle, and both bottles must be previously purged with nitrogen prior to
autoclaving. A reducing agent that is needed for strict anaerobes can be added either
in solution I or solution II, depending on its stability toward pH. We found that the
redox indicator resazurin used in working with strict anaerobes proved to be stable to
pH 10 and destroyed above this value. When several labile substances are used, as in
the case of testing of the spectrum of substrates used, individual solutions II can be
prepared in test tubes (or in Hungate tubes for anaerobes), and solution I is used as the
stock solution. In case of doubt, whether the particular substance is resistant to
thermoalkaline hydrolysis, the common advice will be either to search for its
Isolation and Cultivation of Alkaliphiles
69