# US 8,541,224 B2 · Serum-free growth medium for Acholeplasma laidlawii and methods for retention testing sterilizing grade filters

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- Number: US 8,541,224 B2
- Document ID: US8541224B2
- Status: Granted
- Kind: B2
- Application number: 13/732,870
- Filed: 2013-01-02
- Published: 2013-05-23
- Granted: 2013-09-24
- Earliest claimed: 2007-08-29
- Assignees: EMD Millipore Corporation
- Applicants: EMD Millipore Corporation
- Inventors: Shawn Bates; Kerry Roche Lentine; Nada Bsat
- Web page: https://patentgrep.com/patent/US8541224B2

## CPC

- C12N 1/20: Bacteria; Culture media therefor
- C12Q 1/02: involving viable microorganisms
- C12Q 1/22: Testing for sterility conditions
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

The serum-free growth medium of claim 1, wherein the medium comprises about 0.4% BSA.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 3)

The serum-free growth medium of claim 3, wherein the medium comprises about 0.5% Trizma base.

### Claim 5 (depends on 1)

The serum-free growth medium of claim 1, wherein the medium comprises about 0.7% glucose anhydrous.

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 6)

The serum-free growth medium of claim 6, wherein the one or more fatty acids are oleic acid and palmitic acid.

### Claim 8 (depends on 7)

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.

### Claim 9 (depends on 1)

The serum-free growth medium of claim 1, further comprising about 1.2% agar.

### Claim 10 (depends on 1)

The serum-free growth medium of claim 1, wherein the pH of the growth medium is adjusted to about 7.8.

### Claim 11 (depends on 9)

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.

### Claim 12 (depends on 1)

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.

### Claim 13 (depends on 12)

The serum-free growth medium of claim 12, wherein the nutrient is about 4% tryptose.

### Claim 14 (depends on 12)

The serum-free growth medium of claim 12, wherein the nutrient is 4% polypeptone.

### Claim 15 (depends on 12)

The serum-free growth medium of claim 12, wherein the nutrient is 2% Mycoplasma Broth Base.

### Claim 16 (depends on 12)

The serum-free growth medium of claim 12, further comprising about 1.2% agar.

### Claim 17 (depends on 13)

The serum-free growth medium of claim 13, further comprising about 1.2% agar.

### Claim 18 (depends on 14)

The serum-free growth medium of claim 14, further comprising about 1.2% agar.

### Claim 19 (depends on 15)

The serum-free growth medium of claim 15, further comprising about 1.2% agar.

### Claim 20 (depends on 12)

The serum-free growth medium of claim 12, wherein the pH of the growth medium is adjusted to about 7.8.

### Claim 21 (depends on 16)

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.

### Claim 22 (depends on 17)

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.

### Claim 23 (depends on 18)

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.

### Claim 24 (depends on 19)

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.

### Claim 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.

### Claim 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.

### Claim 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.

### Claim 28 (depends on 27)

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.