cells in water and soil with a high sensitivity, but this technique
requires time-consuming DNA extraction and qPCR [4]. Other
enumeration tools have been developed such as determination of
optical density or measurement of electric resistance using a Coulter counter [5], but none of them quantify viability.
Flow cytometry (FCM) has been extensively used over the past
years for cellular biology applications, in particular in the immunology field. Lately, constructors commercialized new generation
devices capable of detecting smaller events such as bacteria and
viruses of 70 nm [6]. For this reason, FCM has been increasingly
used in diverse fields of microbiology like medicine, environmental
studies, food and pharmaceutical industries. In 2007 and 2010,
FCM was used to efficiently enumerate the spirochetes Borrelia
burgdorferi [7] and Treponema denticola [8]. Recently, we demonstrated that enumeration of Leptospira spp. by this technology is
also effective [9]. We showed that this enumeration technique that
quantifies viability is instantaneous, with only 5 min necessary for a
complete analysis, highly reproducible, sensitive, and statistically
equivalent to the standard Petroff-Hausser method for different
strains and at different physiologic states.
This procedure describes the protocol for enumerating Leptospira spp. by FCM.
2 Materials
2.1 Culture
of Leptospira
1. Ellinghausen-McCullough-Johnson-Harris (EMJH) medium:
Difco Leptospira Medium Base EMJH (Becton Dickinson),
Difco Leptospira Enrichment EMJH (Becton Dickinson). For
1 L of EMJH liquid medium, dissolve 2.3 g of Leptospira
Medium Base EMJH into 900 mL distilled water and sterilize
by autoclaving. Add 100 mL of Leptospira Enrichment EMJH.
2. L. interrogans or L. biflexa strain.
2.2 Dilution
of Leptospira Prior
to Flow Cytometry
Analysis
1. Spectrophotometer for OD 450 nm measurement.
2. Dark-field microscope.
3. 12 Â 75 mm or microcentrifuge tubes.
4. Saline solution: 0.9% NaCl in distilled water, pH 7.5 (see Note
1).
2.3 Staining
of Leptospira
1. SYTO
® 9 and propidium iodide (PI) fluorescent dyes: SYTO
®
9 stains DNA of all cells, and PI only stains DNA of altered
membrane cells corresponding to dying or dead cells. Maximal
wavelengths of excitation and emission are, respectively,
485 and 498 nm for SYTO
® 9 and 535 and 617 nm for PI
(Fig. 1). Both dyes are supplied in the LIVE/DEAD
® BacLight™ Bacterial Viability kit (Life Technologies) (see Note 2).
32
Ce ´ lia Fontana and Jean Reyes
requires time-consuming DNA extraction and qPCR [4]. Other
enumeration tools have been developed such as determination of
optical density or measurement of electric resistance using a Coulter counter [5], but none of them quantify viability.
Flow cytometry (FCM) has been extensively used over the past
years for cellular biology applications, in particular in the immunology field. Lately, constructors commercialized new generation
devices capable of detecting smaller events such as bacteria and
viruses of 70 nm [6]. For this reason, FCM has been increasingly
used in diverse fields of microbiology like medicine, environmental
studies, food and pharmaceutical industries. In 2007 and 2010,
FCM was used to efficiently enumerate the spirochetes Borrelia
burgdorferi [7] and Treponema denticola [8]. Recently, we demonstrated that enumeration of Leptospira spp. by this technology is
also effective [9]. We showed that this enumeration technique that
quantifies viability is instantaneous, with only 5 min necessary for a
complete analysis, highly reproducible, sensitive, and statistically
equivalent to the standard Petroff-Hausser method for different
strains and at different physiologic states.
This procedure describes the protocol for enumerating Leptospira spp. by FCM.
2 Materials
2.1 Culture
of Leptospira
1. Ellinghausen-McCullough-Johnson-Harris (EMJH) medium:
Difco Leptospira Medium Base EMJH (Becton Dickinson),
Difco Leptospira Enrichment EMJH (Becton Dickinson). For
1 L of EMJH liquid medium, dissolve 2.3 g of Leptospira
Medium Base EMJH into 900 mL distilled water and sterilize
by autoclaving. Add 100 mL of Leptospira Enrichment EMJH.
2. L. interrogans or L. biflexa strain.
2.2 Dilution
of Leptospira Prior
to Flow Cytometry
Analysis
1. Spectrophotometer for OD 450 nm measurement.
2. Dark-field microscope.
3. 12 Â 75 mm or microcentrifuge tubes.
4. Saline solution: 0.9% NaCl in distilled water, pH 7.5 (see Note
1).
2.3 Staining
of Leptospira
1. SYTO
® 9 and propidium iodide (PI) fluorescent dyes: SYTO
®
9 stains DNA of all cells, and PI only stains DNA of altered
membrane cells corresponding to dying or dead cells. Maximal
wavelengths of excitation and emission are, respectively,
485 and 498 nm for SYTO
® 9 and 535 and 617 nm for PI
(Fig. 1). Both dyes are supplied in the LIVE/DEAD
® BacLight™ Bacterial Viability kit (Life Technologies) (see Note 2).
32
Ce ´ lia Fontana and Jean Reyes