Viagram red + bacterial gram stain and viability kit

ViaGram™ Red + Bacterial Gram Stain and Viability Kit (V-7023) $ DAPI solution (in water) (Component A), 40 µL, blue-
$ SYTOX Green solution in anhydrous DMSO (Component
B), 40 µL, green-fluorescent dead-cell stain $ Texas Red-X conjugate of wheat germ agglutinin,
lyophilized (Component C), 1.0 mg, red-fluorescent gram-positive stain $ Reconstitution buffer (Component D), 1.0 mL of 0.1 M so-
dium bicarbonate, pH 8.3, for use with Component C $ BacLight™ mounting oil (Component E), 10 mL, for bacte-
ria immobilized on membrane filters. Refractive index at25°C is 1.517 ± 0.003. DO NOT USE FOR IMMERSION Molecular Probes’ ViaGram™ Red+ Bacterial Gram Stain and Viability Kit provides a simple fluorescent staining protocolthat differentially stains many gram-positive and gram-negative At the recommended reagent dilutions and volumes, the bacterial species and, at the same time, discriminates live from ViaGram Red+ Gram Stain and Viability Kit contains sufficient dead cells on the basis of plasma membrane integrity. The kit reagents for 200 individual slide preparations.
contains three reagents: two nucleic acid stains for viability de-termination and a fluorescently labeled wheat germ agglutinin (WGA) for gram-sign determination. The nucleic acid stains dif- $ BSA–saline solution: 0.25% bovine serum albumin (BSA), fer from one another in their spectral characteristics and in their ability to penetrate bacterial cell membranes. Bacteria with in- $ Spin filters, 0.2 µm–pore size (such as Molecular Probes’ tact cell membranes stain fluorescent blue with 4′,6-diamidino- 2-phenylindole (DAPI), whereas bacteria with damaged mem-branes stain fluorescent green with SYTOX® Green nucleic acid stain. The background remains virtually nonfluorescent. The Upon receipt, the kit should be stored at -20°C, upright and Texas Red®-X dye–labeled WGA component selectively binds to protected from light. Allow reagents to warm to room tempera- the surface of gram-positive bacteria1,2 and stains them fluores- ture and centrifuge briefly before opening the vials. Before re- cent red, effectively distinguishing them from gram-negative freezing, tightly seal all vials. The BacLight mounting oil may bacteria, even in the presence of the viability stains. Thus, with be stored at room temperature. When stored properly, the com- three fluorescent colors, the four possible combinations — live ponents of this kit are stable for at least one year. The wheat vs. dead cells and gram-positive vs. gram-negative cells — are germ agglutinin conjugate (Component C) is supplied as a lyo- discriminated. Live bacteria are stained fluorescent blue; dead philized powder. Once reconstituted (see below), store the solu- cells, fluorescent green; and gram-positive cells, fluorescent red tion at 4°C or, for longer storage, divide into aliquots and freeze at -20°C. PROTECT FROM LIGHT. AVOID REPEATEDFREEZING AND THAWING. It is a good practice to centrifugethe protein conjugate solution briefly in a microcentrifuge beforeuse; only the supernatant solution should be used in experiments.
Table 1. Staining pattern for the ViaGram Red+ Kit.
This step will eliminate any protein aggregates that may have Gram-Negative
Gram-Positive
formed in solution, thereby reducing nonspecific backgroundstaining.
Live Cells
Caution: DAPI and SYTOX Green stains (Components A
and B) bind to nucleic acids. DAPI is a known mutagen, and we Dead Cells
have no data addressing the mutagenicity or toxicity of SYTOX Green nucleic acid stain. Both stains should be used with appro- ViaGram™ Red+ Bacterial Gram Stain and Viability Kit priate care. The DMSO stock solution of SYTOX Green stain several times. If there is a problem observing a difference between should be handled with particular caution as DMSO is known to gram-negative and gram-positive cells, then it may be necessary to facilitate the entry of organic molecules into tissues. We strongly reduce the amount of WGA conjugate added to the cells.
recommend using double gloves when handling the DMSO stocksolutions. As with all nucleic acid stains, solutions containing 1.7 Incubate for 5–15 minutes at room temperature.
these reagents should be poured through activated charcoalbefore disposal. The charcoal must then be incinerated to destroy 1.8 Centrifuge at 2000 rpm for 1–2 minutes to remove the WGA
1.9 Resuspend in 50 µL BSA–saline.
1.10 Add 2.5 µL of the DAPI stain/SYTOX Green working
The following protocol is provided to guide researchers in the solution from step 1.2, mix and incubate for 10 minutes at room development of their own bacterial staining procedures. Molecu- lar Probes has used this procedure and found it effective in dis-criminating bacteria with respect to viability and gram sign. The 1.11 Transfer about 10 µL of the sample to a slide, apply a glass
reliability of WGA staining for assessing gram sign in fixed bac- coverslip, seal and observe immediately in the fluorescence mi- teria has been discussed by Sizemore and co-workers.1,2 Care should be taken when comparing bacterial viability determinedby plasma membrane integrity with determinations made by other techniques. For example, formaldehyde or glutaraldehyde If desired, the stained bacteria may be immobilized on mem- fixation will usually kill bacteria; however, when aldehyde-fixed brane filters and mounted in BacLight mounting oil (Component bacteria are stained with the ViaGram Red+ Kit, the staining pat- E) before examination in the fluorescence microscopy. Filters tern for viability is frequently identical to that of unfixed cells. In with low dye binding and superior flatness should be used.
contrast, all bacteria in a suspension treated with 70% isopropyl Blackened polycarbonate filter membranes with a 13 mm diam- alcohol are likely to stain as dead cells. It is therefore very eter and 0.2 µm pores (e.g., Poretics cat. #10532) are typically important to evaluate the efficacy of the kit’s viability discrimina- used in conjunction with drain discBsupport membranes, which tion in reference to a specific experimental procedure.
are placed beneath the filters to promote uniform distribution of Care must be taken to remove traces of growth medium be- bacteria on the filter surface (e.g., Poretics cat. #87481).
fore staining bacteria with these reagents. Nucleic acids andother media components can bind the DAPI and SYTOX Green 2.1 Prepare and stain bacteria as in steps 1.1–1.10. After stain-
stains in unpredictable ways, resulting in unacceptable variations ing, increase the volume to 1 mL by adding 0.9 mL of BSA– in staining. A single wash step is usually sufficient to remove traces of inter-fering media components from the bacterial sus-pension. Phosphate wash buffers are not recommended because 2.2 For vacuum filtration, filter bacteria onto a 13 mm–diameter
they may decrease staining efficiency.
membrane under low vacuum using a stainless steel vacuum fil-tration apparatus. For pressure filtration, filter bacteria using a 13 mm–diameter filter membrane secured in a stainless steel The following protocol describes the preparation of enough Swinney filter holder that is attached to a syringe apparatus.
staining solutions for up to 20 slide preparations.
2.3 Place 4 µL of sterile water on a glass microscope slide.
1.1 Prepare a 2 mg/mL stock solution of Texas Red-X conjugate
of WGA (Component C) by adding 500 µL of buffer from the
2.4 Remove filter and drain disc together and place both, bacteria
sodium bicarbonate solution (Component D). For 20 prepara- side up, on top of the water droplet.
tions, 50 µL will be used; the remainder can be stored in aliquotsat -20°C.
2.5 Add 6–10 µL of BacLight mounting oil to the top of the
filter.
1.2 Prepare a working solution of the viability indicators by add-
ing 3 µL of DAPI stain (Component A) and 3 µL of SYTOX
Green nucleic acid stain (Component B) to 54 µL of water for a
Table 2. Optical filters recommended for use with the ViaGram Red+
1.3 Centrifuge 50 µL of a bacterial suspension (about 5 × 107 cells)
Kit Component
Optical Filters *
in a 0.2 µm–pore size spin filter at 2000 rpm for 1–2 minutes.
1.4 Wash the cells in 50 µL BSA–saline solution by pipetting up
1.5 Recentrifuge as in step 1.3 and resuspend in 50 µL BSA–
* Catalog numbers for recommended bandpass filter sets for fluorescence
microscopy. Omega® filters are supplied by Omega Optical Inc.
1.6 Add 2.5 µL of the WGA conjugate stock solution (for a final
(www.omegafilters.com). Chroma filters are supplied by Chroma concentration of 100 µg/mL) and mix by pipetting up and down ViaGram™ Red+ Bacterial Gram Stain and Viability Kit 2.6 Place an oversized 22 mm square coverslip on top of the
The actual appearance of bacteria in stained preparations de- mounting oil and apply gentle pressure to spread the fluid over pends on several factors, including the optical filters used in the the filter. Do not spread the mounting oil past the edge of the fluorescence microscope, differences in the relative brightness of the fluorescent reagents, the abundance of target sites for dye-binding and the spatial localization of the fluorophores.
2.7 Seal the coverslip with melted paraffin or other suitable
For fluorescence microscopy, it is best to use three separate bandpass optical filter sets to independently view the blue, greenand red fluorescent components of the stained preparation. An 2.8 Observe in a fluorescence microscope equipped with appro-
appropriate longpass optical filter can sometimes be used to simul- priate filter sets, as described below.
taneously visualize the live- and dead-cell staining; however, thegram-positive stain must be viewed with a bandpass optical filter set. Likewise, a DAPI/fluorescein/Texas Red multiple-dye filter The ViaGram Red+ staining reaction is designed to discrimi- set, can be used to observe the live- and dead-cell staining, but nate live from dead cells and, at the same time, gram-negative the gram-positive surface staining is often obscured by the from gram-positive cells. The four possible staining patterns that brighter interior stain. Table 2 presents filter sets recommended may occur with the ViaGram Red+ Kit are described in Table 1.
for visualizing the staining reactions of the ViaGram Red+ Kit.
References
1. U.S. Patent No. 5,137,810; 2. Appl Environ Microbiol 56, 2245 (1990).
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Cat # ViaGramTM Red+ Bacterial Gram Stain and Viability Kit *200 assays* .
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ViaGram™ Red+ Bacterial Gram Stain and Viability Kit

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