University of Texas at San Antonio
SOP No.: 50 ver 1.0
Effective Date: 7/6/2006
Page 1 of 4
Title: Intramacrophage Bacterial Analyses
Approved:
Author: Jieh-Juen Yu/Bernard Arulanandam
Bernard Arulanandam
UTSA PI
Date: 9/19/2008
1. INTRODUCTION
The ability of Francisella spp. to survive and replicate within macrophages is an
important aspect of virulence. Intramacrophage bacterial analyses are useful tools to
assess the virulence/attenuation of a clinical or environmental Francisella isolate or a
genetically engineered mutant.
2. PURPOSE
This SOP describes the procedure to assay the growth (replication) of Francisella
spp. in macrophage cell lines.
3. RESPONSIBILITY
Research Technologist: performs the SOP
Technical Specialist: reviews the data, calculations and interpretations
Principal Investigator: reviews the data and interpretations
4. SCOPE
This procedure is used by the UTSA team under the direction of Dr. Bernard
Arulanandam.
5. PRECAUTIONS
Gloves, safety glasses, and a lab coat should be worn at all times when preparing
media. If the laboratory requires additional PPE, such as the ABSL–3 Facility, these
requirements should be observed as well. Proper tissue culture techniques should be
used to prepare macrophage cultures.
6 MATERIALS AND EQUIPMENT
6.1 DMEM
6.2 D10: DMEM plus 10% fetal bovine serum (heat-inactivated, 560C, 20 min;
HyClone)
6.3 D10 with gentamicin (20 g/ml, GIBCO)
6.4 0.2% Deoxycolate solution in PBS
6.5 Murine macrophage J774 cells (ATCC)
6.6 TSA plates: tryptic soy agar (TSA) supplemented with 0.025% sodium pyruvate,
0.025% sodium metabisulfite, 0.025% ferrous sulfate and 0.1% L-cysteine
6.7 CO2 culture incubator (Model 3110, ThermoForma)
1
University of Texas at San Antonio
SOP No.: 50 ver 1.0
Effective Date: 7/6/2006
Page 2 of 4
Title: Intramacrophage Bacterial Analyses
Approved:
Author: Jieh-Juen Yu/Bernard Arulanandam
Bernard Arulanandam
UTSA PI
Date: 9/19/2008
6.8 Incubator (Innova 4230. New Brunswick)
7. PROCEDURE
7.1 Grow J774 cells in 10 ml of D10 in a 75 cm2 flask at 370C and 5% CO2 till
confluence. Collect cells by gentle scrapping with cell scrappers (BD Falcon Ref
353086).
7.2 Take 50 l of the J774 culture and make 3 serial 2-fold dilution with PBS in
microtubes and, add an equal volume of 0.4% Trypan blue and gently mix, let
stand for 5 minutes at room temperature. Place 10 µL of stained cells in a
hemocytometer and count the number of viable (unstained) and dead (stained)
cells. Make more dilutions if cell density is tooo high to count.
7.3 Seed cells in 96 well tissue culture plates at a concentration of 105 cells per well
(200µL) 2-4 hours prior to infection.
7.4 J744 cells should adhere to well. Remove media supernatant by gentle pipetting.
7.5 Add 200 L of D10 containing 106 (10 MOI) or 107 (100 MOI) bacteria to
desired wells for infection.
7.6 Place 96 well plates in a 37C CO2 incubator for 2 hours.
7.7 Remove supernatant by gentle pipetting and wash once with 200 L DMEM per
well.
7.8 Add 200 L of D10 with gentamicin to each sample well and incubate in CO2
incubator for 1 hour.
7.9 Remove and discard supernatant with a pipettor.
7.10 Wash cells with DMEM three times with gentle pipetting. Since J774 is
adherent to the well, no centrifugation is needed between washing.
2
University of Texas at San Antonio
SOP No.: 50 ver 1.0
Effective Date: 7/6/2006
Page 3 of 4
Title: Intramacrophage Bacterial Analyses
Approved:
Author: Jieh-Juen Yu/Bernard Arulanandam
Bernard Arulanandam
UTSA PI
Date: 9/19/2008
7.11 For 24h samples, add 200 µL D10 per well and return the plate to CO2 incubator
for additional 21 h of incubation. Lyse cells and enumerate CFU as described
below (7.12 to 7.14).
7.12 For 3h samples, add 200 µL of 0.2% deoxycolate solution per well to lyse J774.
7.13 Take 100 µL of the lysate and make 10-fold serial dilutions of the lysate using
PBS and plate 100 l of each diluent on TSA plate to determine the number of
CFU per sample.
7.14 Allow bacteria to grow on the TSA plate for 2-3 day at 37oC and count the CFU.
Colony number between 30 and 300 on each 100x15mm plate is considered to be
accurate.
8. QUALITY CONTROL
8.1 Bacterial and cell line waste generated in BSL2 lab are disinfected with 10 %
bleach for minimal 20 min. before disposal.
8.2 Bacterial and cell line waste generated in BSL3 lab are disinfected with phenolic
disinfectant (Vesphene IIse, Steris # 6461-08) for minimal 20 min and
autoclaved (121oC, 15 psi, 60 min.).
8.3 Use wild type Francisella strain (e.g. U112, SCHU S4) as positive control when
determine intramacrophage growth of the derived mutants.
8.4 Sterility of D10 media are checked by plating 100 l of the medium on TSA and
incubating at 37oC or shaking (225 rpm) 5ml of D10 in a 15 ml tube for at least
2 days prior to the growth of J774.
9. REFERENCES
1. Michael J. Lebffe and B.E. Pierce, 2008. Microbiology laboratory theory &
application. Morton Publishing Company, Englewood, Colorado, USA.
10. CALCULATIONS AND FORMULAS
Sample CFU= 20 x (number of bacterial colonies on plate) x (dilution factor)
Since we only use half (100 l) of the sample (200 l) for staring dilution, and plate
only 1/10 (100 l) of the diluents (1ml), so the factor 20 (2x10) is added to the CFU
calculation.
For example, 60 bacteria are counted on a TSA plated with 100 l of 1:1000 diluted
sample. The sample CFU= 20 x 60 x 1000.
3
University of Texas at San Antonio
SOP No.: 50 ver 1.0
Effective Date: 7/6/2006
Page 4 of 4
Title: Intramacrophage Bacterial Analyses
Approved:
Author: Jieh-Juen Yu/Bernard Arulanandam
Bernard Arulanandam
UTSA PI
Date: 9/19/2008
Use at least 3 bio-replicates to calculate average and standard deviation.
11. GLOSSARY
 mL- milliliter
 µL- microliter
 mm- millimeter
 CFU- colony forming unit
 DMEM- Dulbecco's modified Eagle's medium
 ddH2O- double deionized water
 C- centigrade
 PBS- Phosphate buffered salt solution
12. APPENDICES
None
4