All methods described here regarding animal handling have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Genome Institute of Singapore and Biological Resource Center of the Agency for Science, Technology and Research, Singapore.
1. Mouse Infection
2. Bladder Epithelial Cell Harvesting to Obtain a Cell Suspension
3. Intracellular Bacterial Community (IBC) isolation: Mouth Pipetting of IBCs
NOTE: All methods described in this section have undergone an institutional risk assessment. Mouth pipetting carries the inherent risk of ingestion of the solution that is being transferred. This risk is largely mitigated by the nanoliter volumes that this protocol uses, and we recommend that all users of the protocol pay heed to the precautionary and practice notes listed here and in the discussion.
Apart from confirmation (Figure 3D) of the presence of a single isolated IBC in the collection tube via the dissecting microscope, the purity of the isolated IBC can also be confirmed by confocal microscopy. As shown in Figure 4A, the isolated cells should stain for both E. coli and uroplakin, and are the expected size for IBCs (50-120 µm)17. Furthermore, E. coli staining is not present in the surrounding liquid. Based on our data, more than 90% of cells isolated with this technique are IBCs18. After isolation, the presence and viability of the bacterial cells in the individual IBC can be confirmed through colony forming unit (CFU) enumeration (Figure 4B) or quantitative polymerase chain reaction (qPCR) for genomic equivalents (Figure 4C). Figure 4C also demonstrates that uninfected epithelial cells isolated with the same protocol do not have quantifiable amounts of bacteria. Based on these data, we estimate that the range of CFUs in a single IBC is 102-103 in the murine model of urinary tract infection. One of the main goals of single IBC isolation is to perform downstream analyses such as RNA sequencing. To verify that our isolation method is able to obtain RNA from bacteria in IBCs for analysis, we performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) quantification of three genes (16S, cyoB, and frdA) for a range of individually isolated and pooled IBCs (Figure 4D). All the data shown in Figure 4 has been adapted with permission from Duraiswamy et al.18. An overview schematic of our IBC isolation protocol can be seen in Figure 5, which is reproduced from Duraiswamy et al.18.
Figure 1: Hand-pulled capillaries retain narrow openings. (A) Samples of hand-pulled capillary tubes are displayed on a black background for contrast. From bottom to top, an unpulled capillary, a capillary that was not pulled to a sufficient extent, a capillary that can be used for single bladder epithelial cell harvesting, and a capillary that was pulled too thin (and thus separated into two pieces) are shown. A 15 cm ruler is placed at the bottom of the image for scale. The estimated point for snapping off the usable capillary is indicated on the figure by the red arrow. (B) Image taken with a dissecting microscope confirming the hollow internal diameter of a pulled capillary (bottom). An unpulled capillary is positioned above to demonstrate the relative size difference of the two capillaries. Scale bar = 4.0 mm. Please click here to view a larger version of this figure.
Figure 2: Dissection of mouse to harvest bladder epithelial cells. (A) An image of a mouse with white lines added to indicate the estimated location and angle of incisions to expose the murine peritoneal cavity and bladder. (B) An image of the exposed mouse peritoneal cavity post-incision. (C) An image of the exposed bladder (red arrow) protruding from between the fat pads. (D) An image of the murine bladder with the tip of the forceps inserted into the lumen, with arrows to indicate the direction of motion needed to invert the bladder. The bladder is first pulled slightly outwards, then around and off the first shaft of the forceps. The directions of motion for both actions are as indicated by white arrows numbered 1 and 2. (E) An image showing the final position of the inverted bladder inserted onto the second shaft of the forceps. The shafts of the forceps are labeled in both panels D and E with red arrows and text. Please click here to view a larger version of this figure.
Figure 3: IBC harvesting from bladder cells. (A) An infected and inverted bladder in cold PBS solution before cell scraping. (B) An image showing scraped bladder cells as seen under a microscope. IBCs can be identified as large green fluorescent aggregates in both images (see red arrows). (C) An image of the completed mouth micropipetting apparatus. The aspirating pipette, pipette tip, aspirator tube, and pulled capillary tube are identified with numbered arrows as indicated on the right. (D) An image of a single isolated IBC within a 1.5 mL collection tube (outlined in red). Scale bars (as indicated) are represented by white lines in panels A, B, and D. Please click here to view a larger version of this figure.
Figure 4: Harvested IBCs are pure and can be used for downstream analysis. This figure has been modified with permission from Duraiswamy et al18. (A) Images of two isolated GFP-positive cells that were stained with anti-uroplakin and anti-E.coli antibodies. The first cell (IBC 1) has images of individual channels (at low-magnification) on the left, and a high-magnification merged image is on the right. The second cell (IBC 2) is shown in high magnification in merged and individual channels. Scale bars are as indicated. DNA is stained with 4′,6-diamidino-2-phenylindole (DAPI) and represented in the blue channel. Anti-E. coli is stained with a secondary antibody conjugated to fluorescein isothiocyanate (FITC) and represented in the green channel. Anti-uroplakin is stained with a secondary antibody conjugated to tetramethylrhodamine isothiocyanate (TRITC) and represented in the red channel. (B) Bacterial CFUs from isolated IBCs. IBCs were processed immediately, or incubated in 0.1% Triton-X for 10 or 30 min. Pooled CFU counts of individual IBCs isolated from n = 3 separate experiments are shown. Limit of detection = 0.7 log10 CFUs/IBC. Red dots plotted at the limit of detection indicate samples for which no colonies were recovered. All IBC-containing samples are not significantly different (p > 0.05, Mann-Whitney test); the uninfected epithelial cells are significantly different from the IBC (10 min) data (p < 0.001, Mann-Whitney test). (C) qPCR quantification of bacteria on individual IBCs and uninfected epithelial cells after a 10 min incubation in 0.1% Triton-X (*, p < 0.0001, Mann-Whitney test, n = 4). Limit of detection = 1.18 log10 bacterial genome equivalents/IBC. Red dots indicate samples for which no colonies were recovered on titering in panel B. (D) Quantification of the 16S rRNA, cyoB, and frdA genes for varying numbers of individually isolated and pooled IBCs (n = 1 experiment; each point indicates the mean of 3 technical replicates). NC = no DNA negative control. Please click here to view a larger version of this figure.
Figure 5: A schematic and its associated photographs representing the isolation of IBCs via mouth micropipetting from infected mice bladders. This figure is reproduced from Duraiswamy et al.18. (A) A harvested whole bladder; (B) an inverted whole bladder exposing the GFP expressing IBCs; (C) a close-up of the edge of a scraped bladder showing individual IBCs in suspension in the adjacent buffer; (D) a single isolated IBC pipetted into a tube. Red arrows in panel B indicate examples of GFP-positive IBCs on the luminal surface of the bladder. The red dotted line in panel C indicates the right border of the inverted bladder (indicated as "BL"); red arrows in panel C indicate apparent individual GFP-positive epithelial cells that have been scraped off the bladder surface. White dotted line in panel D indicates a micropipetted sub-microliter droplet containing an isolated IBC, which is indicated by a white arrow. Scale bars = 2 mm. Please click here to view a larger version of this figure.
1.5ml eppendorf tube | For static bacterial culture and OD measurement | ||
100% ethanol | For Alcohol Burner | ||
15 ml conical tube | For static bacterial culture and OD measurement | ||
1ml Tuberculin Syringe | BD Biosciences | 302100 | |
3% Bacterial Agar | For static bacterial culture and OD measurement | ||
70% ethanol | For static bacterial culture and OD measurement | ||
Aesculap anatomic forceps | Braun/Kruuse | BD222R | For initial dissection of mouse (skin, fascia) |
Alcohol Burner | Wheaton | 237070 | |
Aspirating pipette | BD Biosciences | 357558 | |
Aspirator tube | Sigma-Aldrich | A5177 | |
Bacterial loops | For static bacterial culture and OD measurement | ||
Benchtop centrifuge | Eppendorf | 5424 | Any centrifuge for 1.5ml eppendorf tubes |
Conical flasks | For static bacterial culture and OD measurement | ||
Digital camera for microscope | Olympus | DP71 | For image capture and harvesting of IBCs. Any other fluorescent microscope with a GFP channel will suffice |
Glass Capillaries | Kimax | 6148K07 | |
Iris Scissors STR SS 110MM | Braun | BC110R | |
Isoflurane (Isothesia) | Henry Schein Animal Health | 29405 | |
Kanamycin Sulfate | Calbiochem | 420311 | For static bacterial culture and OD measurement |
LB broth (Miller) | Thermo/Gibco | 10855021 | For static bacterial culture and OD measurement |
Light source unit for microscope | Olympus | LG-PS2 | For image capture and harvesting of IBCs. Any other fluorescent microscope with a GFP channel will suffice |
Lubricant | KY | Any similar commercial medical lubricant will suffice | |
Macro fluorescence microscope | Olympus | MVX10 | For image capture and harvesting of IBCs. Any other fluorescent microscope with a GFP channel will suffice |
Micropipette + micropipette tips | For static bacterial culture and OD measurement | ||
PBS 1x | For static bacterial culture and OD measurement | ||
Pipette controller + Pipettes | For static bacterial culture and OD measurement | ||
Polyethylene Tubing | BD Intramedic | 427401 | |
Precision Glide needle 30G | BD Biosciences | 305107 | Possibly under new catalogue number (305106) |
Splinter forceps curved | Braun | BD312R | |
Spray bottle (for ethanol) | For static bacterial culture and OD measurement | ||
Square cuvettes | Elkay | 127-1010-400 | For static bacterial culture and OD measurement |
Sterilgard III Advance Safety Cabinet | Baker | SG403 | Any biosafety cabinet with a UV irridiator |
Sterilin 90mm Standard Petri Dish | Thermo | 101VR20 | Any sterile petri dish |
Stevens, vascular and tendon scissors, curved, delicate, 110 mm | Braun | OK366R | Recommended for harvesting of bladder |
Surgical Scissors STR S/B 105MM | Braun | BC320R | |
Tabletop Centrifuge | Eppendorf | 5810R | Any refridgerated centrifuge for 15ml conicals |
WPA C08000 cell density meter | Biowave (Biochrom) | 80-3000-45 | For static bacterial culture and OD measurement |
In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.
In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.
In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.