TITLE: SFGR Isolation from Clinical Diagnostic Material: Small volume inoculum
AUTHORS: Marah Condit, Cecilia Kato

[DESCRIPTION]
Rickettsiae are small obligate intracellular gram-negative bacteria that are grown in a Biological Safety Level 3 (BSL-3) laboratory setting.This protocol uses antibiotic-free medium, and therefore cultures are susceptible to contamination by undesirable bacteria or fungi.This procedure provides guidance for the inoculation and growth of primary isolates derived from small volumes of clinical samples including blood and blood products in a small volume culture and includes topics of growth monitoring, changing media, freezing primary isolates, and troubleshooting in the event of contamination. This technique utilizes Vero E6 cells growth in small conical culture flasks.These flasks have a growth area of 10cm2 and can accommodate up to 5 mL of media.All techniques are to be performed in a biological safety cabinet (BSC) in a BSL-3 laboratory.Proper training and extreme caution is of the utmost importance when working with live rickettsial cultures.

[BEFORE_START]
Ensure 5% Complete Media is warmed to room temperature.
When freezing ensure SPG and freezing tubes are chilled.
Ensure all flasks, vials, and tubes are labeled with appropriate identifiers and dates.

[GUIDELINES]
All work is conducted in a BSL-3 laboratory. 
Take care to ensure reagents are not contaminated or expired.
Use proper sterile technique when working in a BSL-3 laboratory.

[STEPS]
SECTION: Media Preparation
1. All reagents should be thawed, well mixed and not expired prior to use.Reagents should be stored at the appropriate temperatures recommended by the manufacturer.Prepared media expires 1 month after preparation date.
SECTION: Media Preparation
2. Clean the BSC with 5% quaternary ammonium solution followed by 70% ethanol. (NOTE: do not spray quaternary ammonium solution directly on the BSC surface as residual solution may cause rust.Instead spray on a lint free wipe and carefully decontaminate the surface, followed by removal with 70% ethanol.)
SECTION: Media Preparation
3. Wipe all reagent bottles down with 70% ethanol before placing in the BSC.
SECTION: Media Preparation
4. To prepare ~600 mL of Complete Media, pipet the following into a new 500 mL bottle of MEM:
SECTION: Media Preparation
4.1. 6.15 mL of NEAA 
SECTION: Media Preparation
4.2. 6.15 mL of HEPES 
SECTION: Media Preparation
4.3. 6.15 mL of L-Glutamine 
SECTION: Media Preparation
4.4. Note: L-Glutamine upon thaw from -20ºC will be cloudy.Bring solution up to room temperature and vigorously mix until the reagent is clear with no particulate.Do not use if reagent is not properly mixed.
SECTION: Media Preparation
4.5. 61.5 mL of Sodium Pyruvate 
SECTION: Media Preparation
4.6. 30 mL of Heat Inactivated TET Tested (low antibiotic) fetal bovine serum
SECTION: Media Preparation
5. Filter sterilize with a 0.22 µm Nalgene Filter Unit 
SECTION: Media Preparation
6. Label with the media type, preparation date, and expiration date and store at 4°C until use.
SECTION: Small Volume Inoculation: Fresh clinical blood or blood products can be used as an inoculum or blood or blood products frozen at a 1:1 ratio with SPG can be thawed and used as an inoculum.
7. Confirm 10cm2 culture flasks are at least 99% confluent.
SECTION: Small Volume Inoculation: Fresh clinical blood or blood products can be used as an inoculum or blood or blood products frozen at a 1:1 ratio with SPG can be thawed and used as an inoculum.
8. Clean the BSC with 5% quaternary ammonium solution followed by 70% ethanol. (NOTE: do not spray quaternary ammonium solution directly on the BSC surface as residual solution may cause rust.Instead spray on a lint free wipe and carefully decontaminate the surface, followed by removal with 70% ethanol.)
SECTION: Small Volume Inoculation: Fresh clinical blood or blood products can be used as an inoculum or blood or blood products frozen at a 1:1 ratio with SPG can be thawed and used as an inoculum.
9. Carefully place the 10cm2 culture flask of Vero E6 cells upright in a 50ml conical tube rack in the BSC and gently decontaminate with 70% ethanol.
SECTION: Small Volume Inoculation: Fresh clinical blood or blood products can be used as an inoculum or blood or blood products frozen at a 1:1 ratio with SPG can be thawed and used as an inoculum.
10. Label with appropriate sample identifiers and date of inoculation.
SECTION: Small Volume Inoculation: Fresh clinical blood or blood products can be used as an inoculum or blood or blood products frozen at a 1:1 ratio with SPG can be thawed and used as an inoculum.
11. Remove the spent media and replace with 3 mL of 5% Complete Media.
SECTION: Small Volume Inoculation: Fresh clinical blood or blood products can be used as an inoculum or blood or blood products frozen at a 1:1 ratio with SPG can be thawed and used as an inoculum.
12. Obtain clinical blood or blood product and place in the BSC
SECTION: Small Volume Inoculation: Fresh clinical blood or blood products can be used as an inoculum or blood or blood products frozen at a 1:1 ratio with SPG can be thawed and used as an inoculum.
12.1. Either 50 µl of fresh clinical blood or blood product or
SECTION: Small Volume Inoculation: Fresh clinical blood or blood products can be used as an inoculum or blood or blood products frozen at a 1:1 ratio with SPG can be thawed and used as an inoculum.
12.2. 100 µl of previously frozen samples in a 1:1 ratio with SPG.
SECTION: Small Volume Inoculation: Fresh clinical blood or blood products can be used as an inoculum or blood or blood products frozen at a 1:1 ratio with SPG can be thawed and used as an inoculum.
12.3. i.Quick thaw any frozen sample in a 36°C water bath, do not over heat.
SECTION: Small Volume Inoculation: Fresh clinical blood or blood products can be used as an inoculum or blood or blood products frozen at a 1:1 ratio with SPG can be thawed and used as an inoculum.
13. Pipette the total volume of the inoculum directly into the 3 mL of media in a 10cm2 culture flask.
SECTION: Small Volume Inoculation: Fresh clinical blood or blood products can be used as an inoculum or blood or blood products frozen at a 1:1 ratio with SPG can be thawed and used as an inoculum.
14. Close the flask and gently mix the inoculum so it evenly spreads over the monolayer, careful not to wet the interior filtered lid.
SECTION: Small Volume Inoculation: Fresh clinical blood or blood products can be used as an inoculum or blood or blood products frozen at a 1:1 ratio with SPG can be thawed and used as an inoculum.
15. Place the inoculated 10cm2 culture flask in a 34°C 5% CO2 water jacketed incubator.
SECTION: Monitoring Isolate Growth
16. 
SECTION: Monitoring Isolate Growth
17. Incubate the inoculated 10cm2 culture flask for 3 to 6 days, and sample the supernatant for DNA extraction.Do not disturb the monolayer.
SECTION: Monitoring Isolate Growth
18. Prior to sampling clean the BSC with 5% quaternary ammonium solution followed by 70% ethanol. (NOTE: do not spray quaternary ammonium solution directly on the BSC surface as residual solution may cause rust.Instead spray on a lint free wipe and carefully decontaminate the surface, followed by removal with 70% ethanol.)
SECTION: Monitoring Isolate Growth
19. To sample, carefully place the 10cm2 culture flask upright in a 50ml conical tube rack in the BSC and gently decontaminate with 70% ethanol.
SECTION: Monitoring Isolate Growth
20. Prepare a 2 mL sterile tube labeled with appropriate sample identifiers, day of growth (day 0 is the day of inoculation), and date.
SECTION: Monitoring Isolate Growth
21. Using sterile technique, take a 200 µL supernatant sample and place into the labeled
SECTION: Monitoring Isolate Growth
22. mL sterile tube.Be sure to not disrupt the monolayer!
SECTION: Monitoring Isolate Growth
23. Seal the top of the 10cm2 culture flask, decontaminate the exterior of the flask with 70% ethanol, and place back into the 34°C 5% CO2 water jacketed incubator.
SECTION: Monitoring Isolate Growth
24. Decontaminate the exterior of the 2 mL sterile tube with 5% quaternary ammonium solution followed by 70% ethanol prior to removal from the BSC.
SECTION: Monitoring Isolate Growth
25. Freeze the supernatant sample at -80°C if it cannot be extracted immediately.
SECTION: Monitoring Isolate Growth
26. Repeat steps 2 to 8 once more within days 7 to 14 post inoculation.
SECTION: Monitoring Isolate Growth
27. Extract samples and run the PanR8 real-time PCR assay (2).If CT values increase from the first to second sample of≥ then 3.3 CT, indicating ≥ 1 fold increase in DNA copies between timepoints, the inoculated culture should be considered a viable isolate.
SECTION: Monitoring Isolate Growth
28. To visually monitor the inoculated culture, carefully remove the 10cm2 culture flask from the incubator and place under the inverted light microscope.Care must be taken to not wet the interior of the lid.
SECTION: Monitoring Isolate Growth
29. Scan the monolayer for areas of cell lifting or the absence of cells caused by cell death with the 10x optical.
SECTION: Monitoring Isolate Growth
30. If an area of cell lifting is identified, switch the optical to a 40x or 100x optical and scan for extracellular Rickettsia.If identified, take a DNA sample (if 2 have not been taken yet, and confirm growth via the PanR8 real-time PCR assay (2)Monitor 10cm2 culture flask(s) for at least 14 days or until isolation confirmation via real-time PCR.
SECTION: Media Changes
31. If > 2 DNA samples are taken over the 14 day culture incubation and minimal media coverage is not achieved, or the culture is incubated > 14 days, a media change is required.
SECTION: Media Changes
32. Prior to culture manipulation, clean the BSC with 5% quaternary ammonium solution followed by 70% ethanol. (NOTE: do not spray quaternary ammonium solution directly on the BSC surface as residual solution may cause rust.Instead spray on a lint free wipe and carefully decontaminate the surface, followed by removal with 70% ethanol.)
SECTION: Media Changes
33. Carefully place the 10cm2 culture flask upright in a 50ml conical tube rack in the BSC and gently decontaminate with 70% ethanol.
SECTION: Media Changes
34. Remove the spent media without disturbing the monolayer and discard into a waste bin containing 5% quaternary ammonium solution to decontaminate.
SECTION: Media Changes
35. Add
SECTION: Media Changes
36. mL of fresh 5% Complete Media to the 10cm2 culture flask.Ensure media is added slowly so the monolayer is not disturbed.
SECTION: Media Changes
37. Seal the top of the 10cm2 culture flask, decontaminate the exterior of the flask with 70% ethanol, and place back into the 34°C 5% CO2 water jacketed incubator.
SECTION: Freezing Isolate Cultures (1)
38. Once the inoculated culture is confirmed to be a Rickettsia isolate, continue the incubation until 40 – 60% of the monolayer is lifted due to cell death.This will ensure a high concentration of viable bacteria to freeze.
SECTION: Freezing Isolate Cultures (1)
39. Prior to culture manipulation, clean the BSC with 5% quaternary ammonium solution followed by 70% ethanol. (NOTE: do not spray quaternary ammonium solution directly on the BSC surface as residual solution may cause rust.Instead spray on a lint free wipe and carefully decontaminate the surface, followed by removal with 70% ethanol.)
SECTION: Freezing Isolate Cultures (1)
40. Carefully place the 10cm2 culture flask upright in a 50ml conical tube rack in the BSC and gently decontaminate with 70% ethanol.
SECTION: Freezing Isolate Cultures (1)
41. Remove about half of the media, and place into a labeled sterile high-speed conical tube.Be sure to use a conical tube appropriate for high-speed centrifugation. (NOTE: never centrifuge a T10 culture tube at high speed.)
SECTION: Freezing Isolate Cultures (1)
42. Add sterile glass beads to the 10cm2 culture flask and seal the flask.
SECTION: Freezing Isolate Cultures (1)
43. Shake or swirl the flask until all the monolayer is lifted.Do not shake so hard that bubbles and foam is formed.Do not wet the interior culture lid.
SECTION: Freezing Isolate Cultures (1)
44. Place the 10cm2 culture flask back in the rack so that the beads and media settle to the bottom.
SECTION: Freezing Isolate Cultures (1)
45. Transfer cell suspension to the labeled sterile conical tube. Seal the tube. Decontaminate the tube with 5% quaternary ammonium solution followed by ethanol.
SECTION: Freezing Isolate Cultures (1)
46. It is recommended to take ~10-50ul aliquot of the cell suspension for mycoplasma evaluation in a labeled 2 ml cryo-tube.Follow the steps 4-8 of Monitoring Isolate Growth procedure above.
SECTION: Freezing Isolate Cultures (1)
47. Make an accurate balance for the centrifuge (by volume) if needed.
SECTION: Freezing Isolate Cultures (1)
48. In the BSC, place high-speed conical tubes in a fixed angle biosafe rotor.Make sure centrifuge is balanced appropriately! 
SECTION: Freezing Isolate Cultures (1)
49. Decontaminate the rotor before removing from the BSC with 5% quaternary ammonium solution followed by 70% ethanol.
SECTION: Freezing Isolate Cultures (1)
50. Centrifuge at 17,000 x g for 30 min at 4˚C.
SECTION: Freezing Isolate Cultures (1)
51. Remove rotor from the centrifuge and open in the BSC.
SECTION: Freezing Isolate Cultures (1)
52. Remove samples from the rotor and place in a rack.
SECTION: Freezing Isolate Cultures (1)
53. Decontaminate the rotor with 5% quaternary ammonium solution followed by 70% ethanol and remove from the BSC.
SECTION: Freezing Isolate Cultures (1)
54. Remove the supernatant from the tube without disturbing the pellet and discard into a waste bin containing 5% quaternary ammonium solution to decontaminate.
SECTION: Freezing Isolate Cultures (1)
55. Resuspend the cell pellet in 2 mL of ice cold SPG.
SECTION: Freezing Isolate Cultures (1)
56. Aliquot 1 mL volumes of the cell suspension into 2 chilled sterile labeled 2 mL screw top cryotubes.Cryotubes should be labeled with the appropriate identifiers, isolate passage #, cell type, cell passage #, freezing media type, date, and initials.
SECTION: Freezing Isolate Cultures (1)
57. Ensure tubes are sealed tight and decontaminate the tubes with quaternary ammonium solution followed by 70% ethanol. Place cold cell aliquots in a room temperature cell freezing container.
SECTION: Freezing Isolate Cultures (1)
58. Place container in a -80°C freezer for at least 24 hours before long term storage in liquid nitrogen.
SECTION: Troubleshooting Contamination
59. Isolates are grown in media without antibiotics.This makes them susceptible to contamination with unwanted and aggressive bacterial and fungal species.None of the reagents listed below will kill intracellular Rickettsiahowever, they may deter growth.It is best to act preemptively to stop contamination before it takes over a culture. If contamination is seen the culture media should be changed with media containing an antibiotic or antimycotic and incubated for 24 hours.It is important to observe contamination early so multiple doses are not needed.

See table below for reagents and concentrations.Serial dilution may be required to achieve correct final concentrations from the stock solutions.
SECTION: References
60. Ammerman N. et al. “Laboratory Maintenance of Rickettsia rickettsia”. Curr Protoc Microbiol. 2008 November; CHAPTER: Unit-3A-5.

SECTION: References
61. Ogawa M. et al. “Decontamination of mycoplasma-contaminated Orientia tsutsugamushi strains by repeating passages through cell cultures with antibiotics”. BMC Microbiology. 2013; 13:32.

SECTION: References
62. Kato CY, Chung IH, Robinson LK, Austin AL, Dasch GA, Massung RF. Assessment of real-time PCR assay for detection of Rickettsia spp. and Rickettsia rickettsii in banked clinical samples. J Clin Microbiol. 2013;51(1):314-7.