United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-LABOP-08 Page 1 of 6

Title: Procedure for Testing and Maintaining Control Strains

Revision: 04 Replaces: Rv 03 05/01/06 Effective: 04/01/2009

1. Purpose

To provide standard procedures for testing and maintaining all control strains.

2. Scope

This Standard Operating Procedure (SOP) shall be followed by all laboratories

conducting microbiological studies for the USDA/AMS Microbiological Data

Program (MDP), including support laboratories conducting non-routine activities that

may impact the program. This SOP represents minimum MDP requirements and is

presented as a general guideline. Each laboratory shall have written procedures that

provide specific details concerning how the procedure has been implemented in that

laboratory.

All MDP laboratories shall use the control strains specified in SOP MDP-QA-03.

Strains are preserved as master stock cultures for maintenance and working stock

cultures for testing and distribution. If problems are encountered with any control

strain, MPO shall be notified. The MDP (Ohio) Culture Repository will ship a new

stock to replace the problem stock.

3. Outline of Procedures

Equipment and Materials 5.1

Revival and Growth of Control Strains (by MDP Culture Repository only) 5.2

Maintenance, Storage and Testing of Cultures 5.3

(by MDP (Ohio) Culture Repository and MDP participating laboratories)

Revival and Growth of Control Strains (by MDP participating laboratories) 5.4

4. References

4.1 Bacteriological Analytical Manual (BAM) Online, 8th edition. 2001. US

FDA/CFSAN. http://www.cfsan.fda.gov/~ebam/bam-toc.html (last accessed 3/09)

4.1. Monday, SR, Weagant, SD and Feng, P. 2003. Use of endogenous host plasmids

for generation of Escherichia coli O157:H7 and Shigella sonnei that stably

express the green fluorescent protein. Plasmid. 50: 161-167.

4.2. Rang, C, et.al. 2003. Fitness cost of the green fluorescent protein in

gastrointestinal bacteria. Can. J. Microbiol. 49: 531-537.
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-LABOP-08 Page 2 of 6

Title: Procedure for Testing and Maintaining Control Strains

Revision: 04 Replaces: Rv 03 05/01/06 Effective: 04/01/2009

4.3. SOP MDP-QA-03, Quality Assurance (QA) Controls

4.4. MicrobankTM Microorganism Storage System user Instructions

5. Specific Procedures

5.1. Equipment and Materials

5.1.1. Positive and negative control bacterial strains for each test method, as

detailed in SOP MDP-QA-03

5.1.2. Materials, media and reagents required for testing routine MDP produce

samples

5.1.2.1. For cultural media and growth conditions pertaining to individual

strains, refer to method SOPs listed in the Reference section

5.1.2.2. Incubator set at 35 ± 2oC. Refer to individual method SOPs as

listed in the Reference section.

5.1.2.3. Long wavelength UV light source

5.1.2.4. Microbank storage beads, Pro-Lab Diagnostics, http://www/prolab.com,

5.1.2.5. Kanamycin, Sigma Catalog # K1377 and Isopropyl-β-Dthiogalactopyranoside (IPTG), Sigma Catalog # I5502.

5.1.2.5.1. Kanamycin: Prepare stock solution, filter sterilize using a 0.2

μm syringe filter (B-D # 309602 - Fisher Catalog # 09-719C

or equivalent), and store at 2-8°C (for 3 weeks maximum) or

-20°C or lower for long-term storage. Add appropriate

amount to sterile culture medium (after cooling to ~ 50oC)

for a 50 μg/mL final concentration.

5.1.2.5.2. Isopropyl-β-D-thiogalactopyranoside (IPTG): Add 0.24 gram

of IPTG to sterile culture medium (after cooling to ~ 50oC)

for a 1 mM final concentration.

5.1.2.6. Nutrient agar plates containing 50 μg/mL kanamycin

5.2. Revival and Growth of Control Strains (by MDP Culture Repository only)

5.2.1. For strains sent on discs (e.g., 1 cm diameter discs): Place the disc on a

Nutrient agar plate or suitable rich media. Wet the disc with a small

volume (0.1 mL) of sterile solution such as saline, Phosphate Buffered
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-LABOP-08 Page 3 of 6

Title: Procedure for Testing and Maintaining Control Strains

Revision: 04 Replaces: Rv 03 05/01/06 Effective: 04/01/2009

Saline, or any rich broth using a sterile pipette or a micropipette with a

sterile tip. Using a sterile loop, streak the solution from the disc across the

agar surface for colony isolation. Invert the plate and incubate for at 35 ±

2oC 18-24 hours.

5.2.2. For strains sent in stab-vials: Using a sterile loop, remove cells from the

stab and streak on a Nutrient agar plate for colony isolation. Invert the

plate and incubate at 35 ± 2oC for 18-24 h.

5.2.3. For strains sent on slants: Using a sterile loop, streak on a Nutrient agar

plate for colony isolation. Invert the plate and incubate at 35 ± 2oC for 18-

24 h.

5.2.4. For strains sent on MicrobankTM beads: Aseptically remove one bead.

Immediately close the vial tightly and return it to low temperature storage

as soon as possible. Streak on a Nutrient agar plate or drop into a rich

broth and incubate at 35 ± 2oC for 18-24 h.

5.3. Maintenance, Storage and Testing of Cultures (by MDP Culture Repository and

MDP participating laboratories)

5.3.1. Upon receipt, control strains that carry the kanamycin resistance (kanR)

gene should be grown on a non-specific rich media containing kanamycin

to ensure that the plasmid is maintained or the genes are integrated into the

chromosome. Strains that do not carry genes coding for kanRcan be grown

in the non-specific rich medium without kanamycin and inoculated into

the specific pre-enrichment media.

5.3.2. Storage of the cultures: For making master and working stocks of strains,

follow MicrobankTM beads manufacturer’s directions (refer to product

insert for additional details):

5.3.2.1. Label appropriate MicrobankTM vials with the name of organism,

date, vial number (i.e. 1 of 5) and initials.

5.3.2.2. To prepare stocks, follow manufacturer’s instructions to inoculate

MicrobankTM vials. Inoculate desired number of cryovials

(minimum requirement: 1 vial for reference and 2 vials as working

stocks).

5.3.2.3. Store at -70o C or lower.

5.3.3. Tests
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-LABOP-08 Page 4 of 6

Title: Procedure for Testing and Maintaining Control Strains

Revision: 04 Replaces: Rv 03 05/01/06 Effective: 04/01/2009

Test control strains when they are received from the Culture Repository or

other sources, and when stocks for long term storage are prepared

5.3.3.1. For detection of control strains that carry genes coding for GFP in

broth or on agar, the cultures can be exposed to long wavelength

UV at 365 nm. These cultures will show green to bluish-green

fluorescence, indicating the expression of the gene coding for GFP.

Occasionally, the intensity can be enhanced by holding the cultures

at room temperature for 1-2 hours following routine incubation

followed by examination with UV light. Cultures grown on blood

agar plates may fluoresce better than cultures grown on selective

agar plates or Nutrient agar plates. For control strains (e.g. MDP004, E. coli O157:H7 carrying the gene coding for GFP) in which

the expression of the gene coding for GFP is under the control of

lac or tac promoter, addition of 1 mM IPTG to cultures will

enhance the fluorescence.

5.3.3.2. For additional strain characteristics, refer to SOP MDP-QA-03.

5.3.3.3. Laboratories should verify the purity of control strains prior to use

and periodically according to internal laboratory procedures.

5.4. Revival and Growth of Strains (by MDP participating laboratories)

5.4.1. Control strains are revived from MicrobankTM long-term storage on a

monthly basis during routine testing.

5.4.1.1 Aseptically remove one MicrobankTM bead and transfer to Nutrient

media with or without kanamycin (refer to 5.3.1). Non-specific rich

medium can be used for subsequent transfers.

5.4.2. To minimize contamination risk and genetic drift, cultures should not be

subjected to more than four passages after their revival from long term

storage.

5.4.3. Each laboratory shall have internal written procedures for storage,

handling and maintenance of QC cultures.
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-LABOP-08 Page 5 of 6

Title: Procedure for Testing and Maintaining Control Strains

Revision: 04 Replaces: Rv 03 05/01/06 Effective: 04/01/2009

Kristi McCallum 4/1/09

Revised and Approved by: Kristi McCallum Date

MDP Technical Advisory Group Chairperson

Colorado Department of Agriculture

Inspection & Consumer Services Division

Laboratory Section

2331 West 31st Avenue

Denver, CO 80211-3859

(303) 477-0014

Mary Tijerina 1 April 09

Approved by: Mary Tijerina Date

Microbiologist, Monitoring Programs Office

8609 Sudley Road, Suite 206

Manassas, VA 20110

(703) 330-2300 x111

Diana Haynes 4/1/09

Approved By: Diana Haynes Date

Deputy Director, Monitoring Programs Office

8609 Sudley Road, Suite 206

Manassas, VA 20110

(703) 330-2300

ORIGINALSIGNATURE PAGE MAINTAINED BY USDA, AMS,SCIENCE & TECHNOLOGY, MONITORING PROGRAMSOFFICE

ELECTRONICALLYREPRODUCED SIGNATURES
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-LABOP-08 Page 6 of 6

Title: Procedure for Testing and Maintaining Control Strains

Revision: 04 Replaces: Rv 03 05/01/06 Effective: 04/01/2009

Revision 04 March 2009 Monitoring Program Office

 Added 4.4 and 4.5 to the Reference section.

 Deleted archived SOPs and non-referenced SOPs from the Reference section.

 Added 5.1.2.4 to section 5.1

 Added, “such as” to 5.2.1.

 Changed “scoop out” to “remove” in 5.2.2.

 Changed “scrape cells using” to “Using” in 5.2.3.

 Added, “Upon receipt” to 5.3.1.

 Changed “cryo” to “appropriate” in 5.3.2.1 and changed “passage number” to

“vial number (i.e. 1 of 5)” to 5.3.2.1.

 Deleted “cryo” from 5.3.2.2.

 Changed, “other” to “additional” in 5.3.3.2.

 Deleted 5.3.3.3 and added, “Laboratories should verify the purity of control

strains prior to use and periodically according to internal laboratory procedures”.

 Deleted “This can be achieved by removing a bead from the Microbank vials, or

inoculating from cryovials with preserved cultures under aseptic conditions” from

5.4.1.

 Revised 5.4.1.1 to read, “Aseptically remove on Microbank bead and transfer”.

 Added 5.4.3.

Revision 03 March 2006 Monitoring Program Office

 Updated References (4.0), Equipment and Materials (5.1)

 Revised instructions for the preparations of kanamycin and Isopropyl-β-Dthiogalactopyranoside (IPTG) to reflect current practices

 Deleted Control Maintenance for MDP Control Strains table

 Revised Maintenance and Testing of Cultures (5.3) and Revival and Growth of

Strains (5.4) sections

Revision 02 January 2005 Monitoring Programs Office

 Updated specific strains

 Changed concentration of kanamycin as antibiotic supplement

Revision 01 July 2004 Monitoring Programs Office

 Eliminated tetracycline stock preparation instructions

 Deleted unused control strains, MDP-006 and MDP-007