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 µL. One 250 µL 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 µM. To prepare sufficient 2X staining solution for analysis of 10 samples, add 30 µL of 5 mM SYTOX Green/DMSO to 15 mL of 5% TSB/saline.

3.1.3 Prepare a 200 µg/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 µg/mL, 20 µg/mL and 60 µg/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 µg/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 µg/mL, 20 µg/mL, 60 µg/mL and 200 µg/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 µg/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 µg/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 (Figure 1).

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.