# US 7,745,168 B2 · Method for selectively staining chitin-containing organisms

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 7,745,168 B2
- Document ID: US7745168B2
- Status: Granted
- Kind: B2
- Application number: 11/537,665
- Filed: 2006-10-02
- Published: 2007-02-08
- Granted: 2010-06-29
- Earliest claimed: 2002-04-29
- Assignees: University of South Florida
- Inventors: My Lien Dao
- Web page: https://patentgrep.com/patent/US7745168B2

## CPC

- C12Q 1/04: Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)}
- C12Q 2304/00: Chemical means of detecting microorganisms
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention provides a method for the staining of fungi and microsporidia for observation with a light microscope based upon the presence of chitin in the composition of these organisms. With the method of the present invention a sample to be analyzed is treated with a solution of Ponceau S and Stains-all dye. The sample is then selectively decolorized and rinsed. The resulting sample is examined with a light microscope, or photographed for a permanent record, to identify the presence of a variety of microorganisms, to include fungi and microsporidia.

## Claims

### Claim 1

A method for determining the presence or absence of a chitin-containing microorganism in a sample suspected of containing a chitin-containing microorganisms, the method comprising the steps of:
providing a sample suspected of containing a chitin-containing microorganisms to be analyzed;
treating the sample with 3-hydroxy-4((2-sulfo-4-((-4-sulfophenyl(azo)phenyl)azo) 2,7 naphthalenedisulfonic acid tetrasodium salt stain to stain the chitin of the chitin-containing microorganisms;
treating the sample with a cationic carbocyanine 1-Ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphto[1,2-d]thiazolium bromide dye, the cationic carbocyanine dye binding to the 3-hydroxy-4((2-sulfo-4-((-4-sulfophenyl(azo)phenyl)azo) 2,7 naphthalenedisulfonic acid tetrasodium salt stain to form a light-stable complex;
applying three decolorizing solutions to the sample;
rinsing the sample after each application of decolorizing solution; and
examining the sample with a light microscope to identify the presence of chitin-containing microorganisms in the sample, wherein the stained chitin-containing microorganisms are observable in the light microscope.

### Claim 2 (depends on 1)

The method of claim 1, wherein the decolorizing solution is about 0.25% sodium dodecyl sulfate in phosphate buffered saline.

### Claim 3 (depends on 1)

The method of claim 1, wherein the step of rinsing the sample comprises the application of a solution of acid, methanol, and water in the ratio of about 10:40:50, respectively.

### Claim 4 (depends on 1)

The method of claim 1, wherein the decolorizing solution is 6M guanidine hydrochloride.

### Claim 5 (depends on 1)

The method of claim 1, wherein the decolorizing solution is about 0.3% Tween 20.

### Claim 6

A method for determining the presence or absence of chitin-containing fungi or microsporidia, the method comprising the steps of:
providing a sample suspected of containing a chitin-containing fungi or microsporidia to be analyzed;
treating the sample with 3-hydroxy-4((2-sulfo-4-((-4-sulfophenyl(azo)phenyl)azo) 2,7 naphthalenedisulfonic acid tetrasodium salt stain to stain the chitin of the chitin-containing fungi or microsporidia;
treating the sample with 1-Ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphto[1,2-d]thiazolium bromide dye, which dye binds to 3-hydroxy-4((2-sulfo-4-((-4-sulfophenyl(azo)phenyl)azo) 2,7 naphthalenedisulfonic acid tetrasodium salt stain to form a light-stable complex;
applying three decolorizing solutions to the sample;
rinsing the sample after each application of decolorizing solution; and
examining the sample with a light microscope to identify the presence of chitin-containing fungi or microsporidia in the sample, wherein the stained chitin-containing fungi or microsporidia are observable in the light microscope.

### Claim 7 (depends on 6)

The method of claim 6, wherein the sample is selectively decolorized using about 0.25% sodium dodecyl sulfate in phosphate buffered saline.

### Claim 8 (depends on 6)

The method of claim 6, wherein the sample is selectively decolorized using 6M guanidine hydrochloride.

### Claim 9 (depends on 6)

The method of claim 6, wherein the sample is selectively decolorized using about 0.3% Tween 20.

### Claim 10 (depends on 6)

The method of claim 6, wherein the step of rinsing the sample comprises the application of a solution of acid, methanol, and water in the ratio of about 10:40:50, respectively.