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 serum-free growth medium for cultivating or storing Acholeplasma laidlawii having a small, deaggregated and spherical morphology, comprising:
(i) a nutrient selected from the group consisting of about 4% tryptose, about 4% polypeptone, and about 2% Mycoplasma Broth Base;
(ii) bovine serum albumin (“BSA”);
(iii) one or more pH stabilizers;
(iv) glucose; and
(v) one or more fatty acids.
2. The serum-free growth medium of claim 1 , wherein the medium comprises about 0.4% BSA.
3. The serum-free growth medium of claim 1 , wherein the one or more pH stabilizers are selected from the group consisting of Tris, HEPES, and a combination thereof.
4. The serum-free growth medium of claim 3 , wherein the medium comprises about 0.5% Trizma base.
5. The serum-free growth medium of claim 1 , wherein the medium comprises about 0.7% glucose anhydrous.
6. The serum-free growth medium of claim 1 , wherein the one or more fatty acids are selected from the group consisting of oleic, palmitic, myristic acids, and combinations thereof.
7. The serum-free growth medium of claim 6 , wherein the one or more fatty acids are oleic acid and palmitic acid.
8. The serum-free growth medium of claim 7 , where the medium comprises about 75 μM of oleic acid and about 75 μM of palmitic acid.
9. The serum-free growth medium of claim 1 , further comprising about 1.2% agar.
10. The serum-free growth medium of claim 1 , wherein the pH of the growth medium is adjusted to about 7.8.
11. The serum-free growth medium of claim 9 , further comprising a chromogenic agent or dye selected from the group consisting of triphenyl tetrazolium chloride, Dienes stain and crystal violet.
12. The serum-free growth medium of claim 1 , wherein the medium comprises:
(i) a nutrient selected from the group consisting of about 4% tryptose, about 4% polypeptone, and about 2% Mycoplasma Broth Base;
(ii) about 4% BSA;
(iii) about 0.5% Trizma base;
(iv) about 0.7% glucose anhydrous; and
(v) about 75 μM of oleic acid and about 75 μM of palmitic acid.
13. The serum-free growth medium of claim 12 , wherein the nutrient is about 4% tryptose.
14. The serum-free growth medium of claim 12 , wherein the nutrient is 4% polypeptone.
15. The serum-free growth medium of claim 12 , wherein the nutrient is 2% Mycoplasma Broth Base.
16. The serum-free growth medium of claim 12 , further comprising about 1.2% agar.
17. The serum-free growth medium of claim 13 , further comprising about 1.2% agar.
18. The serum-free growth medium of claim 14 , further comprising about 1.2% agar.
19. The serum-free growth medium of claim 15 , further comprising about 1.2% agar.
20. The serum-free growth medium of claim 12 , wherein the pH of the growth medium is adjusted to about 7.8.
21. The serum-free growth medium of claim 16 , further comprising a chromogenic agent or dye selected from the group consisting of triphenyl tetrazolium chloride, Dienes stain and crystal violet.
22. The serum-free growth medium of claim 17 , further comprising a chromogenic agent or dye selected from the group consisting of triphenyl tetrazolium chloride, Dienes stain and crystal violet.
23. The serum-free growth medium of claim 18 , further comprising a chromogenic agent or dye selected from the group consisting of triphenyl tetrazolium chloride, Dienes stain and crystal violet.
24. The serum-free growth medium of claim 19 , further comprising a chromogenic agent or dye selected from the group consisting of triphenyl tetrazolium chloride, Dienes stain and crystal violet.
25. A serum-free growth medium for cultivating or storing Acholeplasma laidlawii having a small, deaggregated and spherical morphology, comprising:
(i) about 4% tryptose or about 4% polypeptone;
(ii) about 4% BSA;
(iii) about 0.5% Trizma base;
(iv) about 0.7% glucose anhydrous; and
(v) about 75 μM of oleic acid and about 75 μM of palmitic acid.
26. A serum-free growth medium for cultivating or storing Acholeplasma laidlawii having a small, deaggregated and spherical morphology, comprising:
(i) about 2% Mycoplasma Broth Base;
(ii) about 4% BSA;
(iii) about 0.5% Trizma base;
(iv) about 0.7% glucose anhydrous; and
(v) about 75 μM of oleic acid and about 75 μM of palmitic acid.
27. A serum-free growth medium for cultivating or storing Acholeplasma laidlawii having a small, deaggregated and spherical morphology, comprising:
(i) one or more nutrients consisting of about 4% tryptose, about 4% polypeptone, or about 2% Mycoplasma Broth Base;
(ii) bovine serum albumin (“BSA”);
(iii) one or more pH stabilizers;
(iv) glucose; and
(v) one or more fatty acids.
28. The serum-free growth medium of claim 27 , wherein the medium comprises:
(i) one or more nutrients consisting of about 4% tryptose, about 4% polypeptone, or about 2% Mycoplasma Broth Base;
(ii) about 4% BSA;
(iii) about 0.5% Trizma base;
(iv) about 0.7% glucose anhydrous; and
(v) about 75 μM of oleic acid and about 75 μM of palmitic acid.