Product Information

Revised: 04–March–2004

BacLight™ Bacterial Stains

B-35000 BacLight™ Green bacterial stain
B-35001 BacLight™ Red bacterial stain

BacLight Green
Quick Facts 200
A
200
B
S. aureus E. coli
160 160

Counts
Storage upon receipt:

Counts
120 120

• ≤–20ºC 80 80

• Desiccate 40 40
• Protect from light
0 0 1 2 3 4 0 0 1 2 3 4
10 10 10 10 10 10 10 10 10 10

Abs/Em: See Table 1 Green fluorescence Green fluorescence

BacLight Red
Solvent for Stock: DMSO
200 200
C S. aureus D E. coli
160 160

Counts
Counts

120 120

80 80

40 40
Introduction
0 0 1 2 3
0 0 1 2 3 4
4
10 10 10 10 10 10 10 10 10 10
The BacLight™ Green and BacLight™ Red bacterial stains Red fluorescence Red fluorescence
are fluorescent, non–nucleic acid labeling reagents for detect-
ing and monitoring bacteria (Table 1). Bacteria stained with the Figure 1. Flow cytometry histograms from a BD FACScan (BD Biosciences, San Jose,
BacLight Green and BacLight Red bacterial stains exhibit bright CA) showing fluorescence of live and dead gram-positive and gram-negative bacteria
green and red fluorescence, respectively, and can be resolved stained with the BacLight bacterial stains. Untreated and alcohol-treated gram-positive
(Staphylococcus aureus, (A and C)) and gram-negative (Escherichia coli, (B and D))
using the appropriate flow cytometric channels. Although these
bacteria were each stained separately with 100 nM of either the BacLight Green (A
dyes were specifically chosen for flow cytometry applications, and B) or the BacLight Red (C and D) bacterial stains in 0.85% NaCl buffer and then
bacteria stained with these BacLight reagents can also be visual- analyzed by flow cytometry. The histograms for the untreated (shaded histogram curve)
ized by fluorescence microscopy with only minor, if any, adjust- and alcohol-treated (unshaded histogram curve) bacteria samples were overlaid for each
ments in the staining concentrations. Furthermore, the BacLight species and BacLight bacterial stain.
bacterial stains are compatible with formaldehyde or alcohol fixa-
tion methods.
These BacLight bacterial stains efficiently label a variety of Storage and Handling
different bacteria species. The intensity of the staining appears to The BacLight bacterial stains are provided in specially pack-
depend on several factors, including gram character, outer mem- aged sets of 20 separate vials, each containing 50 µg of solid for
brane composition and overall membrane integrity. In the species reconstitution as required. Upon receipt, the vials of dye should
tested, gram-positive bacteria generally exhibited brighter fluo- be stored desiccated at ≤–20°C, and protected from light.
rescence than gram-negative bacteria, and cells with compromised Note: Before opening a vial containing a solid or DMSO solu-
membranes accumulated more dye than intact cells (Figure 1). tion, allow the product to warm to room temperature. To avoid
Because the stains are compatible with various labeling schemes, multiple freeze-thaw cycles, divide any unused DMSO stock
the BacLight bacterial stains can also be combined with other flu- solution into single-use aliquots before storing desiccated at
orescent cell probes — including nucleic acid stains, lectin conju- ≤ –20°C. When stored properly, the BacLight bacterial stains are
gates and antibody conjugates — for multiparameter analyses. stable for at least six months.

MP 35000 BacLight™ Bacterial Stains

Table 1. Spectral characteristics of BacLight bacterial stains.
2.2 Add BacLight bacterial stain to the bacteria sample. Stain
Catalog # BacLight Abs * Em * Recommended Optical bacteria by adding 1 µL of the working dye solution prepared in
Bacterial Filters step 1.2 to 1 mL of the bacteria sample prepared in step 2.1.
Stain
B-35000 BacLight 480 516 Filters suitable for 2.3 Incubate the bacteria sample with the stain. Incubate the
Green fluorescein sample for 15 minutes at room temperature.
B-35001 BacLight 581 644 Filters suitable for
Red tetramethylrhodamine 2.4 (Optional) Wash the bacteria sample. Because the BacLight
or Texas Red dye. bacterial stain is nonfluorescent when not associated with cells,
* Fluorescence absorption and emission maxima, in nm, determined in wash and centrifugation steps are not necessary. However, the
methanol; values may vary somewhat in cellular environments. cells can be washed with buffer to remove excess dye if required
for subsequent labeling steps or analysis methods.

Caution: No data are available addressing the mutagenicity or 2.5 (Optional) Fix the bacteria sample. If fixation is desired,
toxicity of these reagents. The DMSO stock solutions should be centrifuge the bacteria sample and resuspend the pellet in 1–4%
handled with particular caution, as DMSO is known to facilitate formaldehyde or 70% alcohol. Alternatively, prepare a 2X form-
the entry of organic molecules into tissues. Please dispose of the aldehyde solution and then mix equal volumes of the bacteria
stains in compliance with all pertaining local regulations. sample in the staining solution and the concentrated formalde-
hyde solution. Incubate the bacteria in the formaldehyde solution
for 10–15 minutes at room temperature. Analyze the fixed bacteria
sample directly by flow cytometry or fluorescence microscopy.
Experimental Guidelines Alternatively, centrifuge the sample, resuspend the pellet in PBS,
The following protocols are provided as examples to guide 0.85% NaCl or other appropriate buffer, and then proceed with
researchers in the development of their own staining procedures. the analysis.
Researchers at Molecular Probes have used these procedures and
found them to be simple and reliable for both gram-positive and Analyzing the Stained Bacteria by Flow Cytometry
gram-negative bacteria, including Bacillus subtilis, Escherichia Instrument capabilities may vary considerably, but the tech-
coli, Klebsiella pneumoniae, Micrococcus luteus and Staphylo- niques and parameters established here should aid considerably
coccus aureus. To thoroughly optimize the staining of a particular in setting up similar analyses in the majority of flow cytometers
bacteria population, experimenting with a range of concentrations now in use, both in research and clinical environments. Stained
of BacLight bacterial stains and then evaluating the staining pat- bacteria can be assayed in a flow cytometer equipped with a laser
tern using the desired instrumentation is recommended. emitting at 488 nm. Fluorescence is collected in the green- and
red-fluorescence channels for the BacLight Green and BacLight
Reagent Preparation Red bacterial stains, respectively. Filters used for detecting fluo-
rescein are generally suitable for analyzing bacteria stained with
1.1 Prepare a stock solution of the BacLight bacterial stain. the BacLight Green bacterial stain, whereas filters suitable for
Allow a vial of the BacLight bacterial stain to warm to room tem- detecting tetramethylrhodamine or the Texas Red dye are gener-
perature before opening. Prepare a 1 mM stock solution of dye by ally suitable for analyzing bacteria stained with the BacLight Red
dissolving the vial contents in DMSO — 74 µL for the BacLight bacterial stain. The forward scatter, side scatter and fluorescence
Green bacterial stain or 69 µL for the BacLight Red bacterial should be collected with logarithmic signal amplification.
stain.
3.1 Adjust the flow cytometer. Instrument adjustments are es-
1.2 Prepare a working solution of the BacLight bacterial pecially critical for detecting relatively small particles such as
stain. Prepare a 100 µM working solution of the BacLight bacte- bacteria. To avoid contamination of the data by electronic noise,
rial stain by adding 2 µL of the 1 mM stock solution prepared in the following procedure for instrument setup is recommended.
step 1.1 to 18 µL of DMSO in a microcentrifuge tube and mix The appropriate controls — including unstained bacteria, known
well. Note: For most experimental samples, a 100 µM working gram-positive and gram-negative bacteria or untreated and treated
concentration for a 1:1000 final dilution is generally adequate, bacteria — can be used to locate bacteria populations and deter-
however, the optimal staining concentration may depend on the mine optimal compensation settings. Using an unstained bacteria
specific application and species. Adjust the working solution sample, acquire signals with the amplifiers set to logarithmic
concentration and volume accordingly to achieve the desired final amplification. Use the side scatter as the parameter for setting
staining concentration. the acquisition trigger. Set the amplification of the signals from
forward and side scatter such that the bacteria are in the middle
Staining Protocol of the data space. Adjust the trigger level (also named “threshold
level” on some instruments) to minimize electronic noise appear-
2.1 Prepare the bacteria sample. Grow a bacteria culture or ing on the monitor. Gate on the bacteria in forward versus side
collect a bacteria sample in appropriate medium or buffer. If com- scatter, and set the amplification of the green- or red-fluorescence
parisons among samples are being performed, normalize the cell channel such that the signals from the unstained bacteria appear
number in each sample. Perform dilutions in phosphate-buffered in the lowest quadrant of the logarithmic scale. Briefly run se-
saline (PBS), 0.85% NaCl or other appropriate buffer. lected stained bacteria samples to ensure that the fluorescence

Bac Light™ Bacterial Stains 2

signal is on scale. If necessary, adjust the fluorescence amplifica- 4.1 Prepare a sample for fluorescence microscopy. Transfer
tion to bring the stained samples on scale. 5-10 µl of the stained bacteria to a clean, glass microscope slide,
and place a clean coverslip over the sample. If appropriate, seal
3.2 Analyze a sample by flow cytometry. After adjusting the the edges of the coverslip.
flow cytometer as described in step 3.1, apply an experimental
sample containing stained bacteria. Gate on the bacteria in for- 4.2 Visualize a sample by fluorescence microscopy. The fluo-
ward versus side scatter, and collect stained populations in the rescence from the BacLight Green and BacLight Red bacterial
appropriate fluorescence channel. stains may be viewed separately using bandpass filter sets appro-
priate for fluorescein and tetramethylrhodamine (or the Texas
Visualizing the Stained Bacteria by Fluorescence Microscopy Red dye), respectively (Table 1). Alternatively, a mixed popula-
Although the BacLight bacterial stains were selected based on tion of bacteria stained with both of these bacterial stains may be
their utility for staining bacteria analyzed by flow cytometry, the viewed simultaneously using a longpass filter set appropriate for
reagent preparation and staining protocol described above may fluorescein.
also be useful for visualizing bacteria by fluorescence microscopy.
However, further optimization the staining conditions to enhance
differences in gram character, membrane permeability or other
attributes may be necessary.

Product List Current prices may be obtained from our Web site or from our Customer Service Department.

Cat # Product Name Unit Size

B-35000 BacLight™ Green bacterial stain *special packaging* .................................................................................................................................... 20 x 50 µg
B-35001 BacLight™ Red bacterial stain *special packaging*........................................................................................................................................ 20 x 50 µg

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Molecular Probes’ products are high-quality reagents and materials intended for research purposes only. These products must be used by, or directly under the
supervision of, a technically qualified individual experienced in handling potentially hazardous chemicals. Please read the Material Safety Data Sheet provided for
each product; other regulatory considerations may apply.

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Copyright 2004, Molecular Probes, Inc. All rights reserved. This information is subject to change without notice.

Bac Light™ Bacterial Stains 3