TITLE: Isolation of Chlamydia from crocodile tissue samples
AUTHORS: Somjit Chaiwattanarungruengpaisan,  Metawee Thongdee, ladawan.sar 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for isolation of Chlamydia from crocodiles</div></div>

[STEPS]
?. Dissect tissue sample (approximate 50-100 mg) into small pieces.
?. Homogenize the small pieces of tissue sample with a pestle in 5 ml sucrose/phosphate/glutamate (SPG) buffer containing 500 ug/ml streptomycin, 500 ug/ml vancomycin, 50 ug/ml gentamycin, and 2.5 ug/ml fungizone and left at 4°C for 72 h.
?. Centrifuge the homogenized tissue sample at 250×g for 10 min and collect supernatant for cell culture inoculation.
?. Prepare monolayers of McCoy cells in 12-well plates by seeding 3.5105 cell per well in cell culture medium (M199 medium (Gibco BRL Life Technologies Inc., NY, USA) supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL Life Technologies Inc., NY, USA), 0.2% NaHCO3, 1× L-glutamine, 100 g/ml streptomycin).
?. Inoculate 400 l of supernatant into each well of monolayers cells.
?. Centrifuge 12-well plate containing infected cells at 1,000×g and 25°C for 1 h.
?. After centrifugation, incubate the infected cultures at 30°C in an atmosphere of 5% CO2 for an additional 1 h.
?. Replace the inocula with fresh incubation medium (M199 medium (Gibco BRL Life Technologies Inc., NY, USA) containing 0.5 g/ml cycloheximide and supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL Life Technologies Inc., NY, USA), 0.2% NaHCO3, 10% glucose, 1× L-glutamine, 100 g/ml streptomycin, 100 g/ml vancomycin, 10 g/ml gentamycin, and 1 g/ml fungizone).
?. Further incubate the infected cultures at 30°C in an atmosphere of 5% CO2 for 4–5 days.
?. Observe daily for the cytopathic effect (CPE) under an inverted microscope.
?. [Chlamydia propagation]
?. [Chlamydia propagation]
Prepare monolayers of McCoy cells in 12-well plates by seeding 3.5105 cell per well in cell culture medium (M199 medium (Gibco BRL Life Technologies Inc., NY, USA) supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL Life Technologies Inc., NY, USA), 0.2% NaHCO3, 1× L-glutamine, 100 g/ml streptomycin).
?. [Chlamydia propagation]
Thaw frozen stock of Chlamydia culture and place on ice.
?. [Chlamydia propagation]
Dilute culture stock to 1:10 in cell culture medium.
?. [Chlamydia propagation]
Inoculate 200 l of diluted culture stock into each well of monolayers cells and add 200 l of cell culture medium.
?. [Chlamydia propagation]
Centrifuge 12-well plate containing infected cells at 1,000×g and 25°C for 1 h.
?. [Chlamydia propagation]
After centrifugation, incubate the infected cultures at 30°C in an atmosphere of 5% CO2 for an additional 1 h.
?. [Chlamydia propagation]
Replace the inocula with fresh incubation medium (M199 medium (Gibco BRL Life Technologies Inc., NY, USA) containing 0.5 g/ml cycloheximide and supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL Life Technologies Inc., NY, USA), 0.2% NaHCO3, 10% glucose, 1× L-glutamine, 100 g/ml streptomycin, 100 g/ml vancomycin, 10 g/ml gentamycin, and 1 g/ml fungizone).
?. [Chlamydia propagation]
Further incubate the infected cultures at 30°C in an atmosphere of 5% CO2 for 4–5 days.
?. [Chlamydia propagation]
Observe daily for the CPE under an inverted microscope.
?. [IFA]
?. [IFA]
Prepare monolayer McCoy cells on glass coverslip (∅ 15 mm) in 12-well plates by seeding 3.5105 cell per well in cell culture medium (M199 medium (Gibco BRL Life Technologies Inc., NY, USA) supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL Life Technologies Inc., NY, USA), 0.2% NaHCO3, 1× L-glutamine, 100 g/ml streptomycin).
?. [IFA]
Thaw frozen stock of Chlamydia culture and place on ice.
?. [IFA]
Dilute culture stock to 1:20 in cell culture medium.
?. [IFA]
Inoculate 400 l of diluted culture stock into each well of monolayers cells.
?. [IFA]
Centrifuge 12-well plate containing infected cells at 1,000×g and 25°C for 1 h.
?. [IFA]
After centrifugation, incubate the infected cultures at 30°C in an atmosphere of 5% CO2 for an additional 1 h.
?. [IFA]
Replace the inocula with fresh incubation medium (M199 medium (Gibco BRL Life Technologies Inc., NY, USA) containing 0.5 g/ml cycloheximide and supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL Life Technologies Inc., NY, USA), 0.2% NaHCO3, 10% glucose, 1× L-glutamine, 100 g/ml streptomycin, 100 g/ml vancomycin, 10 g/ml gentamycin, and 1 g/ml fungizone).
?. [IFA]
Further incubate the infected cultures at 30°C in an atmosphere of 5% CO2 for 3-4 days.
?. [IFA]
Observe daily for the CPE under an inverted microscope.
?. [IFA]
When CPE in cell culture occur approximately 10%, fix the infected cells with 1 ml of 4% paraformaldehyde for 24 h.
?. [IFA]
Remove 4% paraformaldehyde and wash fixed samples 3 times with 1 ml of 1 phosphate-buffered saline (PBS).
?. [IFA]
Add 1 ml of 0.2% Triton X-100 in PBS and incubate at 37°C for 30 min for permeabilization.
?. [IFA]
Wash fixed samples 3 times with 1 ml of 1PBS.
?. [IFA]
Add 1 ml of 1% BSA and incubate at 37°C for 30 min for blocking of non-specific binding.
?. [IFA]
Transfer the coverslip to a humidity chamber and add 30 l of 1:100 diluted primary antibody (Chlamydiaceae family-specific mouse monoclonal antibody directed against the chlamydial lipopolysaccharide (cat. no. sc-58106, Santa Cruz Biotechnology, Inc., TX, USA). Incubate the humidity chamber at 37°C for 1 h in the dark.
?. [IFA]
Transfer the coverslip to a 12-well plate and wash coverslip 3 times with 1 ml of 1PBS.
?. [IFA]
Transfer the coverslip to the humidity chamber and add 30 l of 1:200 diluted secondary antibody (Alexa Fluor 594-conjugated secondary goat anti-mouse antibody (cat. no. A11005, Life Technologies, CA, USA) and 2 g/ml 4′,6-diamidino-2′-phenylindole dihydrochloride (DAPI) (Molecular Probes, OR, USA). Incubate the humidity chamber at 37°C for 1 h in the dark.
?. [IFA]
Transfer the coverslip to a 12-well plate and wash coverslip 3 times with 1 ml of 1PBS.
?. [IFA]
Mount the coverslip onto microscope glass slides using 50% glycerol.
?. [IFA]
Seal the mounted coverslip with nail polish.
?. [IFA]
Visualize the image under an inverted fluorescence microscope.