SFGR Isolation from Clinical Diagnostic Material: Small volume inoculum

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.

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.)

Wipe all reagent bottles down with 70% ethanol before placing in the BSC.

To prepare ~600 mL of Complete Media, pipet the following into a new 500 mL bottle of MEM:

6.15 mL of NEAA

6.15 mL of HEPES

6.15 mL of L-Glutamine

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.

61.5 mL of Sodium Pyruvate

30 mL of Heat Inactivated TET Tested (low antibiotic) fetal bovine serum

Filter sterilize with a 0.22 µm Nalgene Filter Unit

Label with the media type, preparation date, and expiration date and store at 4°C until use.

Confirm 10cm² culture flasks are at least 99% confluent.

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.)

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.

Label with appropriate sample identifiers and date of inoculation.

Remove the spent media and replace with 3 mL of 5% Complete Media.

Obtain clinical blood or blood product and place in the BSC

Either 50 µl of fresh clinical blood or blood product or

100 µl of previously frozen samples in a 1:1 ratio with SPG.

i.Quick thaw any frozen sample in a 36°C water bath, do not over heat.

Pipette the total volume of the inoculum directly into the 3 mL of media in a 10cm² culture flask.

Close the flask and gently mix the inoculum so it evenly spreads over the monolayer, careful not to wet the interior filtered lid.

Place the inoculated 10cm² culture flask in a 34°C 5% CO₂water jacketed incubator.

Incubate the inoculated 10cm² culture flask for 3 to 6 days, and sample the supernatant for DNA extraction.Do not disturb the monolayer.

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.)

To sample, carefully place the 10cm² culture flask upright in a 50ml conical tube rack in the BSC and gently decontaminate with 70% ethanol.

Prepare a 2 mL sterile tube labeled with appropriate sample identifiers, day of growth (day 0 is the day of inoculation), and date.

Using sterile technique, take a 200 µL supernatant sample and place into the labeled

mL sterile tube.Be sure to not disrupt the monolayer!

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% CO₂water jacketed incubator.

Decontaminate the exterior of the 2 mL sterile tube with 5% quaternary ammonium solution followed by 70% ethanol prior to removal from the BSC.

Freeze the supernatant sample at -80°C if it cannot be extracted immediately.

Repeat steps 2 to 8 once more within days 7 to 14 post inoculation.

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.

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.

Scan the monolayer for areas of cell lifting or the absence of cells caused by cell death with the 10x optical.

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.

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.

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.)

Carefully place the 10cm² culture flask upright in a 50ml conical tube rack in the BSC and gently decontaminate with 70% ethanol.

Remove the spent media without disturbing the monolayer and discard into a waste bin containing 5% quaternary ammonium solution to decontaminate.

Add

mL of fresh 5% Complete Media to the 10cm² culture flask.Ensure media is added slowly so the monolayer is not disturbed.

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% CO₂water jacketed incubator.

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.

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.)

Carefully place the 10cm² culture flask upright in a 50ml conical tube rack in the BSC and gently decontaminate with 70% ethanol.

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.)

Add sterile glass beads to the 10cm² culture flask and seal the flask.

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.

Place the 10cm² culture flask back in the rack so that the beads and media settle to the bottom.

Transfer cell suspension to the labeled sterile conical tube. Seal the tube. Decontaminate the tube with 5% quaternary ammonium solution followed by ethanol.

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.

Make an accurate balance for the centrifuge (by volume) if needed.

In the BSC, place high-speed conical tubes in a fixed angle biosafe rotor.Make sure centrifuge is balanced appropriately!

Decontaminate the rotor before removing from the BSC with 5% quaternary ammonium solution followed by 70% ethanol.

Centrifuge at 17,000 x g for 30 min at 4˚C.

Remove rotor from the centrifuge and open in the BSC.

Remove samples from the rotor and place in a rack.

Decontaminate the rotor with 5% quaternary ammonium solution followed by 70% ethanol and remove from the BSC.

Remove the supernatant from the tube without disturbing the pellet and discard into a waste bin containing 5% quaternary ammonium solution to decontaminate.

Resuspend the cell pellet in 2 mL of ice cold SPG.

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.

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.

Place container in a -80°C freezer for at least 24 hours before long term storage in liquid nitrogen.

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 Rickettsia however, 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.

Ammerman N. et al. “Laboratory Maintenance of Rickettsia rickettsia ”. Curr Protoc Microbiol. 2008 November; CHAPTER: Unit-3A-5.

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

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.