Protocol for Antiobiotic Sensitivity Testing Using SYTOX Green

1. Introduction

The Turner BioSystems TD-700 Laboratory Fluorometer in combination with Molecular Probes' SYTOX Green nucleic acid stain provides a rapid and convenient alternative to traditional methods for assessment of bacterial sensitivity to antibiotics and a variety of bactericidal agents. Established methods, such as plate counts, disc diffusion plates, most probable number (MPN) broth dilutions, or direct observation based on sample turbidity, require incubation times of 18-48 hours before results can be obtained. Methods using fluorescent probes reduce the test-to-result period and allow bacterial viability to be continually monitored in real time. (1,2) SYTOX Green penetrates damaged plasma membranes of dead bacteria (both gram-positive and gram-negative), producing green fluorescent staining of nucleic acids. Live bacteria with intact membranes are unstained. This method for discriminating live and dead bacteria has been successfully used to monitor bactericidal action of disinfectants on Pseudomonas and Staphylococcus bacteria.(3)

2. Materials Required

* TD-700 Fluorometer with standard PMT and 10 mm x 10 mm square cuvette adaptor (P/N 7000-009)
* Quartz-halogen lamp (P/N 7000-930)
* 13 mm round test tube adapter (P/N 7000-981)
* 25 mm diameter optical filters for excitation at 485 ± 11 nm, (Omega Optical 485DF22) and emission at 530 ± 15 nm (Omega Optical 530DF30), installed using O-rings (P/N 7000-949).
* 13 mm x 100 mm borosilicate glass test tubes (P/N 10-031)
* SYTOX Green nucleic acid stain (catalog number S-7020), supplied by Molecular Probes, Inc., Eugene, OR. This reagent is supplied as a 5 mM solution in DMSO in units of 250 L. One 250 L unit is sufficient for analysis of about 80 samples using this protocol. Handling, storage and the use of the reagent should be performed in accordance with the product information sheet supplied by Molecular Probes, Inc.
* 5% TSB/saline comprised of Tryptone (17 g/L), Soytone (3 g/L), NaCl (5 g/L), K2HPO4 (2.5 g/L) pH 7.3 ± 0.2, available from Difco Laboratories Inc.
* American Type Culture Collection (ATCC) Preceptrol cultures: Escherichia coli strains 25922 (sensitive) and 35218 (resistant).
* Ampicillin (Sigma Chemical Co.)

3. Experimental Protocol

3.1. Bacterial Staining

3.1.1 Prepare suspensions of ampicillin-sensitive and -resistant E. coli with densities of 2 X 10^7 cfu/mL in 5% TSB/saline (cfu = colony forming units).

3.1.2 Prepare 2X SYTOX Green staining solution by diluting the 5 mM SYTOX Green/DMSO stock solution by a factor of 1:500 in 5% TSB/saline, yielding a final dye concentration of 10 M. To prepare sufficient 2X staining solution for analysis of 10 samples, add 30 L of 5 mM SYTOX Green/DMSO to 15 mL of 5% TSB/saline.

3.1.3 Prepare a 200 g/mL stock solution of ampicillin in 2X SYTOX Green staining solution (step 3.1.2). 5 mL of this stock solution is sufficient for the measurements specified in this protocol.

3.1.4 Prepare dilutions of the ampicillin stock solution to concentrations of 2 g/mL, 20 g/mL and 60 g/mL by addition of further 2X SYTOX Green staining solution (step 3.1.2).

3.1.5 Label a set of 5 glass test tubes "Resistant" and a second set of 5 tubes "Sensitive". Label one member of each set of tubes with the following designations: Reagent blank, 1, 10, 30 and 100 g/mL ampicillin.

3.1.6 Transfer 1.5 mL of TSB/saline/2X SYTOX Green (step 3.1.2) to each reagent blank test tube and make up to 3 mL total volume by adding a further 1.5 mL TSB/saline.

3.1.7 Transfer 1.5 mL of 2 g/mL, 20 g/mL, 60 g/mL and 200 g/mL ampicillin in 2X SYTOX Green staining solution (steps 3.1.3 and 3.1.4) to the tubes labeled 1, 10, 30 and 100 g/mL ampicillin, respectively.

3.1.8 Transfer 1.5 mL of the resistant E. coli suspension (step 3.1.1) to each of the 4 ampicillin-containing test tubes labeled "Resistant".

3.1.9 Transfer 1.5 mL of the sensitive E. coli suspension (step 3.1.1) to each of the 4 ampicillin-containing test tubes labeled "Sensitive".

3.1.10 Incubate all samples at 37° C for 1 hour prior to carrying out fluorescence measurements.

3.2 Fluorescence Measurements

3.2.1 Set-up TD-700 with a quartz halogen lamp, Omega Optical 485DF22 excitation filter, and Omega Optical 530DF30 emission filter.

3.2.2 Calibrate in the Simple Mode according to section VII of the TD-700 Manual using the sample from the antibiotic-sensitive set that contains the highest concentration of ampicillin (100 g/mL) as the calibration standard. This sample should be the most fluorescent among those to be measured.

3.2.3 Read the "Sensitive" and "Resistant" samples in the order of reagent blank followed by the bacterial samples in order of increasing ampicillin concentration.

3.2.4 For each sample, record the fluorescence intensity value shown on the fluorometer display screen. To equalize any photobleaching effects, insert samples into the fluorometer for approximately equal time periods. The data may be corrected for the dye background by subtracting the reagent blank fluorescence value from bacterial sample fluorescence readings.

3.2.5 Plotted data should show increasing fluorescence as a function of ampicillin concentration for antibiotic-sensitive bacteria in contrast to relatively constant low fluorescence for antibiotic-resistant bacteria

4. References

1. FEMS Microbiology Lett 133, 1 1995.
2. J Antimicrobial Chemotherapy 34, 613 1994.
3. J Appl Bacteriol 81, 411 1996.

5. Patent & Trademark Information

SYTOX is a registered trademark of Molecular Probes, Inc. SYTOX Green nucleic acid stain is covered by current or pending U.S. and foreign patents.

6. About Molecular Probes

Orders for Molecular Probes' products may be placed by:

Phone: (541) 465-8338 or Toll-Free (800) 438-2209 in the U.S. and Canada
Fax: (541) 344-6504 or Toll-Free (800) 438-0228 in the U.S. and Canada
Email: order@probes.com
Mail: Molecular Probes, Inc., PO Box 22010, Eugene, OR 97402-0469, USA

Information on the scientific and technical background of Molecular Probes' products is available from the Technical Assistance Department by:

Phone: (541) 465-8353
Fax: (541) 465-4593
Email: tech@probes.com
Internet: http://www.probes.com

7. About Turner Biosystems, Inc.

Orders for Turner BioSystems' products may be placed by:

Phone: (408) 749-0994
Fax: (408) 749-0998

E-mail: sales@turnerbiosystems.com
Internet: www.turnerbiosystems.com

Mailing Address:
Turner BioSystems, Inc.
845 W. Maude Avenue
Sunnyvale, CA 94085

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