Serum-free growth medium for
View Patent ↗A method for retention testing sterilizing grade filters comprises: a) providing a stock of Acholeplasma laidlawii ; b) growing up the stock of A. laidlawii for about 24 hours or less in a single serum-free growth medium that supports cell growth to a high titer and yields a cellular morphology where the cells are small, deaggregated and spherical, thereby producing a bacterial culture; c) challenging a test filter by filtering the bacterial culture through the test filter at a known challenge level, thereby producing a filtrate downstream of the test filter; and d) detecting concentration of A. laidlawii in the filtrate. Serum-free growth media for cultivating or storing A. laidlawii are also described.
1. A method for retention testing sterilizing grade filters, comprising:
a) providing a stock of Acholeplasma laidlawii;
b) growing up the stock of Acholeplasma laidlawii for about 24 hours or less in a single serum-free growth medium that supports cell growth to a high titer and yields a cellular morphology where the cells are small, deaggregated and spherical, thereby producing a bacterial culture, wherein the serum-free growth medium comprises
(i) a nutrient selected from the group consisting of about 4% tryptose, about 4% polypeptone, about 2% Mycoplasma Broth Base, and combinations thereof;
(ii) bovine serum albumin (“BSA”);
(iii) one or more pH stabilizers;
(iv) glucose; and
(v) one or more fatty acids;
c) challenging a test filter by filtering the bacterial culture through the test filter at a known challenge level, thereby producing a filtrate downstream of the test filter; and
d) detecting concentration of Acholeplasma laidlawii in the filtrate.
2. The method of claim 1 , wherein the stock of Acholeplasma laidlawii is a frozen stock.
3. The method of claim 2 , wherein the stock of Acheloplasma laidlawii is frozen in a solution comprising the same serum-free growth medium that is used to grow up the stock.
4. The method of claim 1 , wherein the one or more pH stabilizers are selected from the group consisting of Tris, HEPES, and a combination thereof.
5. The method of claim 1 , wherein the one or more fatty acids are selected from the group consisting of oleic, palmitic, myristic acids, and combinations thereof.
6. The method of claim 1 , wherein the serum-free growth medium comprises:
about 4% tryptose;
about 0.4% BSA;
about 0.5% Trizma base;
about 0.7% glucose anhydrous;
about 75 μM oleic acid; and
about 75 μM palmitic acid.
7. The method of claim 1 , wherein the serum-free growth medium comprises:
about 4% polypeptone;
about 0.4% BSA;
about 0.5% Trizma base;
about 0.7% glucose anhydrous;
about 75 μM oleic acid; and
about 75 μM palmitic acid.
8. The method of claim 1 , wherein the serum-free growth medium comprises:
about 2% Mycoplasma Broth Base;
about 0.4% BSA;
about 0.5% Trizma base;
about 0.7% glucose anhydrous;
about 75 μM oleic acid; and
about 75 μM palmitic acid.
9. The method of claim 1 , wherein the stock of Acholeplasma laidlawii is grown for about 18 to about 24 hours.
10. The method of claim 1 , wherein the bacterial culture has a titer of at least 1×10 9 cfu/mL.
11. The method of claim 1 , wherein the test filter is a 0.1 μm rated test filter.
12. The method of claim 1 , wherein detecting concentration of Acholeplasma laidlawii in the filtrate uses a growth medium.
13. The method of claim 12 , wherein the growth medium used to detect concentration of Acholeplasma laidlawii comprises agar.
14. The method of claim 12 , wherein the growth medium used to detect concentration of Acholeplasma laidlawii further comprises a chromogenic agent or dye selected from the group consisting of triphenyl tetrazolium chloride, Dienes stain and crystal violet.
15. The method of claim 12 , wherein the growth medium used to detect concentration of Acholeplasma laidlawii is serum-free.
16. The method of claim 15 , wherein the serum-free growth medium used to detect concentration of Acholeplasma laidlawii comprises the same serum-free growth medium that is used to grow up the stock.