United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No. MDP-LABOP-03 Page 1 of 8

Title - Microbiological Media, Reagents, Kits and Identification Systems

Revision 04 Replaces Rev 03 04/01/09 Effective 09/01/11

1. Purpose

To provide standard procedures to ensure that media and reagents used in analyses of 

Microbiological Data Program (MDP) samples meetsthe performance requirements specified by 

their intended use.

2. Scope

This standard operating procedure (SOP) shall be followed by laboratories conducting 

microbiological studies for 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 internal procedures 

that provide specific details concerning how the procedures have been implemented in that 

laboratory.

3. Outline of Procedure

6.1 Reference Organisms

6.2 Dehydrated Media

6.3 Internally prepared media and reagents

6.4 Commercial pre-prepared media and reagents

6.5 Kits and Identification systems

6.6 Storage and Disposal

6.7 Records

4. References

4.1 AOAC INTERNATIONAL Guidelines for Laboratories Performing Microbiological and 

Chemical Analyses of Food and Pharmaceuticals, Appendix B: Food Microbiology, 

ALACC Criteria, March 2010
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No. MDP-LABOP-03 Page 2 of 8

Title - Microbiological Media, Reagents, Kits and Identification Systems

Revision 04 Replaces Rev 03 04/01/09 Effective 09/01/11

5. Definitions

5.1 Batch - A batch refers to all tubes, plates or containers of media prepared at the

same time, on the same date, sterilized on the same date and/or with the same lot 

number when ordered pre-prepared from a manufacturer.

5.2 Selective organism – An organism which, grown on a specific media type, 

produces growth and/or a specific biochemical reaction on the media.

5.3 Non-selective organism – An organism which, grown on a specific media type, 

produces no growth on the media or an atypical biochemical reaction on the

media.

5.4 Sterility – After incubation no bacterial growth (visible turbidity) is observed in 

the media or reagent.

5.5 MSDS – Material Safety Data Sheet. A form with data regarding the properties 

and safety precautions for a particular substance.

6. Specific Procedures:

6.1 Reference Organisms

6.1.1 Microbiological media shall be verified using reference cultures or wellcharacterized isolates. See Table 1 for recommended selective and nonselective organisms used to test media commonly used by MDP 

participating laboratories. 

6.1.2 Laboratory shall have internal procedures for storage and handling of 

organisms.

6.1.3 Laboratories shall have internal written procedures for inoculating

media. A dilute inoculum is recommended since it will give greater 
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No. MDP-LABOP-03 Page 3 of 8

Title - Microbiological Media, Reagents, Kits and Identification Systems

Revision 04 Replaces Rev 03 04/01/09 Effective 09/01/11

assurance that the medium will support growth of a small number of 

colonies. Refer to manufacturer’s product sheet for recommended 

organisms not listed. 

NOTE: Laboratories may use any suitable organism(s) according to their own internal written 

procedures.

Table 1- Selective/Non-selective QC Organism Recommendations for Media 

Media Selective Organism Non-selective Organism

Modified EC + Novobiocin E. coli O157:H7 (MDP-004) Non-O157:H7 E. coli (MDP-017)

Levine Eosin Methylene Blue Agar E. coli (MDP-017) E. aerogenes or E. coli (MDP-005)

MacConkey Agar E. coli (MDP-017) E. aerogenes or E. coli (MDP-005)

Sorbitol MacConkey Agar E. coli O157:H7 (MDP-004) Non-O157:H7 E. coli (MDP-017)

Bismuth Sulfite Agar Salmonella (MDP-014) E. coli (MDP-017)

Hektoen Enteric Agar Salmonella (MDP-014) E. coli (MDP-017)

XLD Agar Salmonella (MDP-014) E. coli (MDP-017)

Lysine Iron Agar Salmonella (MDP-014) E. coli (MDP-017)

Triple Sugar Iron Agar Salmonella (MDP-014) E. coli (MDP-017)

CHROMagar E. coli E. coli (MDP-017) E. coli (MDP-017)

CHROMagar O157 E. coli O157:H7 (MDP-004) Non-O157 E. coli (MDP-017)

6.2 Dehydrated Media

6.2.1 Receipt

Upon receipt of dehydrated media and dehydrated media components,

laboratory shall keep records for the following information at a minimum:

6.2.1.1 Unique identification (laboratory number)

6.2.1.2 Name or description of media/reagent

6.2.1.3 Manufacturer’s lot number

6.2.1.4 Quantity received, (i.e., size and number of containers)
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No. MDP-LABOP-03 Page 4 of 8

Title - Microbiological Media, Reagents, Kits and Identification Systems

Revision 04 Replaces Rev 03 04/01/09 Effective 09/01/11

6.2.1.3 Date received

6.2.1.4 Manufacturer’s Expiration date

6.2.1.5 Date opened

6.2.1.6 Date prepared for quality control (QC)

6.2.1.7 Initials of responsible person

6.2.2 Dehydrated media (containers) shall be labeled with the laboratory 

number, identification, and date received.

6.2.3 The laboratory shall have internal procedures to prevent the accidental use

 of expired or defective dehydrated media.

6.3 Internally prepared media and commercially purchased media 

6.3.1 Each new lot received from manufacturer shall be examined for suitability

before use or concurrently with samples. If concurrent testing with 

samples is performed and QC fails, sample results may be invalid. An 

investigation shall be performed following the laboratory’s internal 

procedures. 

6.3.2 Preparation records shall include:

6.3.2.1 Preparation (recipes)

6.3.2.2 pH

6.3.2.3 Sterilization records

6.3.2.3.1 Autoclave records shall show date, run number,

autoclave number if applicable, nature of material/load, 

time into autoclave, time at desired temperature, time

out of autoclave, responsible person. 

6.3.2.3.2 If other sterilization is used, records shall indicate the

 method and responsible person.

6.3.2.3.3 Autoclaves shall be verified for temperature, time and 
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No. MDP-LABOP-03 Page 5 of 8

Title - Microbiological Media, Reagents, Kits and Identification Systems

Revision 04 Replaces Rev 03 04/01/09 Effective 09/01/11

sterility each day of use; each load sterile control all 

media; weekly spore check.

 

6.3.2.4 Fill volumes (if appropriate)

6.3.2.5 Batch size

6.3.2.6 Quantity

6.3.3 Suitability testing (QC) for internally prepared or purchased prepared 

media shall include:

6.3.3.1 Productivity (+ culture)

6.3.3.2 Sterility

6.3.3.3 Appearance of media (internally prepared)

6.3.3.4 The person who approves or rejects

6.3.4 New lots or batches of commercially pre-prepared media or reagents.

Each new lot received from manufacturer shall be examined for suitability. 

Initial suitability may be satisfied through a Certificate of Analysis (CoA). 

The CoA shall state that the media has been properly evaluated for 

productivity (+ culture); sterility; and appearance. The CoA is a record 

and shall be maintained according to Records Section 6.7. Alternatively, 

in- house laboratory suitability testing may satisfy suitability requirements.

6.3.5 The laboratory shall have internal procedures to prevent the accidental use

of expired or defective commercial pre-prepared media and reagents.

6.4 Kits, Quality Critical Reagents and Identification systems

6.4.1 Every lot of material shall be approved using the laboratory’s internal 

procedures. Records shall be sufficient to establish an audit trail, 

including the date approved and traceability to the person approving or 

rejecting the material.
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No. MDP-LABOP-03 Page 6 of 8

Title - Microbiological Media, Reagents, Kits and Identification Systems

Revision 04 Replaces Rev 03 04/01/09 Effective 09/01/11

6.4.2 Internally prepared reagents shall be labeled with

6.4.2.1 Substance

6.4.2.2 Strength

6.4.2.3 Solvent (if not water)

6.4.2.4 Special precautions or use restrictions

6.4.2.5 Date of preparation

6.4.2.6 Date of expiration

6.4.2.7 The responsible person

6.4.3 Serological tests shall include a positive control and a saline negative

 control.

6.5 Laboratory Grade Water

The laboratory shall have written internal procedures to ensure the laboratory 

water is fit for use in microbiological methods.

6.6 Records

6.6.1 Records shall be traceable to the original manufacturer and lot.

6.6.2 Records shall be available for on-site inspections or desk audit reviews 

6.6.3 Records shall be maintained for at least the time specified in MPO 

 ADMIN 5.4.3.

Disclaimer: Reference to brand names (kits, equipment, media, reagents etc.) does not constitute 

endorsement by this agency.
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No. MDP-LABOP-03 Page 7 of 8

Title - Microbiological Media, Reagents, Kits and Identification Systems

Revision 04 Replaces Rev 03 04/01/09 Effective 09/01/11

Carol Larson 29 June 2011

__________________________________________________________

Revised by: Carol Larson Date

Quality Assurance Officer, Washington State Department of Agriculture

Microbiology Laboratory, 3939 Cleveland Ave. SE

Olympia, WA 98501, (360) 586-9561

Maya Achen 27 June 2011

___________________________________________________________

Reviewed by: Maya Achen Date

Chairperson, MDP Technical Advisory Group

Ohio Department of Agriculture, Consumer Analytical Laboratory, Bldg 3

8995 E Main Street, Reynoldsburg, OH 43068

(614) 728-2790

Shanker Reddy 6 July 2011

___________________________________________________________

Reviewed by: Shanker Reddy, PhD Date

Molecular Biologist, Microbiological Data Program

8609 Sudley Road, Suite 206, Manassas, VA 20110

(703) 330-2300 X135

Diana Haynes 7 July 2011

Approved by: Diana Haynes Date

Deputy Director, Monitoring Programs Division

8609 Sudley Road, Suite 206, Manassas, VA 20110

(703) 330-2300 x134

ORIGINAL SIGNATURE PAGE MAINTAINED BY USDA, AMS, SCIENCE & TECHNOLOGY, MONITORING PROGRAMS OFFICE ELECTRONICALLY REPRODUCED 

SIGNATURES
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No. MDP-LABOP-03 Page 8 of 8

Title - Microbiological Media, Reagents, Kits and Identification Systems

Revision 04 Replaces Rev 03 04/01/09 Effective 09/01/11

Revision 04 May 2011 Microbiological Data Program 

 Revised entire SOP to conform to ISO 17025 and AOAC requirements

 Non-selective organism challenge is “optional” in March 2010 ALACC guidelines

 Deleted disposal traceability for media, reagents, kits

 Added requirement for traceability of person approving or rejecting media and reagents.

 Reformatted/renumbered SOP

 Added requirements for sterilization and lab grade water. 

 Provided allowance for in-line testing.

Revision 03 March, 2009 Monitoring Programs Office

 Re-wrote entire SOP to conform to ISO 17025 and AOAC requirements

Revision 02 March, 2006 Monitoring Programs Office

 Added “reagents” in title

 Updated references

 Condensed sections 5.3, 5.4, and 5.5 to one section

 Added sterility testing 

Revision 01 May 16, 2003 Technical Advisory Committee

 Renumbered document for consistency with other SOPs

 Added reference for media efficiency testing procedures to section 4

 Modified subsection 5.1.1 to require inspection for condition of media and reagents

 Revised subsection 5.2 media and reagent storage requirements

 Modified subsection 5.3.2 requirements for maintenance of reference organisms

 Added provision for testing media inline with samples by streaking the controls as subsection 

5.3.4

 Revised subsection 5.3.4 growth performance/efficiency testing requirements

 Modified subsection 5.6 reagent storage and labeling requirements

 Modified subsection 5.7.2 antisera storage requirements