This document provides two plans for maintaining quality control microorganisms from Microbiologics to use in quality control testing for up to a month. Plan A involves subculturing the microorganism weekly from the working stock culture. Plan B prepares a new working stock culture weekly instead of monthly and uses daily plates for quality control panels, subculturing the reference stock culture every 7 days. General tips are provided for storage and maintenance of the microorganisms.

1. Maintenance of Quality Control Strains
With proper maintenance, many microorganisms produced by Microbiologics can be used in quality control tests for
up to a month after reconstitution. In this technical bulletin, Microbiologics provides two plans for maintaining qual‐
ity control microorganisms. In the first plan there are fewer passages for the microorganism. In the second plan, the
working stock culture is prepared weekly instead of monthly. When choosing a plan, take into consideration regula‐
tory guidelines and test manufacturer’s directions for maintaining microorganisms.
Tips for maintaining microorganisms can be found in the boxes below.
Getting Started
• A KWIK‐STIK™ or a LYFO DISK® should be started on a non‐selective agar such as Tryptic Soy Agar or Sheep
Blood Agar. Do not use broth.
• A working QC stock culture cannot be frozen to assume reference culture status.
Tips For Best Performance
• Do not test the original pellet plate. This is the plate on which the lyophilized pellet was started. The organisms
growing on this plate are not fully resuscitated.
• Always use fresh microorganisms for testing. Many tests require that the microorganisms are not more than 24
hours old.
• Select isolated colonies for the test.
• It may be necessary to start microorganisms used for quality control of antibiotic susceptibility tests every two
weeks because some microorganisms lose resistance over time. Examples are: P. aeruginosa ATCC® 27853™*
(Catalog # 0353), E. faecalis ATCC® 51299™* (Catalog # 0959), and S. pneumoniae ATCC® 49619™* (Catalog #
0947).
• Do not test colonies from a contaminated plate.
Storage of Microorganisms
• Fastidious microorganisms have shorter survival periods than aerobic bacteria. They will need to be subcul‐
tured every few days. For example, Streptococcus pneumoniae and Neisseria gonorrhoeae need to be subcul‐
tured every third day.
• Store aerobic bacteria at 2‐8°C. Exceptions are a few species of Bacillus which remain viable for a longer period
when stored at room temperature. Store microorganisms which require CO2 at room temperature in a candle
jar or a container with a CO2 packet. Store yeast and fungus at room temperature. Store anaerobes in anaero‐
bic conditions at 2‐8°C. Store Campylobacter at 35°C in microaerophilic conditions.
Wrapping Up
• After the fourth week, dispose of plates and start the process over with a new lyophilized pellet.
• A microorganism may be used beyond expiry date if (one) the lyophilized pellet is grown before expiry date and
(two) the microorganism is not used beyond week 4 of the maintenance program.
TIB.246 REVISION 2011.JAN.4 Page 1 of 3
*The ATCC Licensed Derivative Emblem, the ATCC Licensed Derivative word mark and the ATCC catalog marks are trademarks of ATCC.
Microbiologics, Inc. is licensed to use these trademarks and to sell products derived from ATCC® cultures.

2. Microorganism Maintenance Plan A1
1. Reference: CLSI M22‐A3 Vol. 24 No.19, Quality Control for Commercially Prepared Microbiological Culture Media: Approved Standard ‐ Seventh Edition, April 2004.
2. Do not test colonies from the pellet plate. If it is necessary to the test the microorganism on Day 1, start the reference stock culture one day earlier and subculture it for
the first day of testing.
3. Alternatively, subculture the day before testing.
TIB.246 REVISION 2011.JAN.4 Page 2 of 3
Reconstitute microorganism
(see product insert PI.194)
Start on non‐selective agar or slant
(see TIB.081 “Recommended Growth
Requirements”)
Monthly Working Stock Culture “Pellet Plate”2
Week 1/Day 1
Weekly Working Culture
Reference Stock Culture
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Each day of Week 1,
subculture colonies from
this plate to a fresh plate3
Day 9
Day 10
Day 11
Day 12
Day 13
Day 14
Each day of Week 2,
subculture colonies from
this plate to a fresh plate3
Day 16
Day 17
Day 18
Day 19
Day 20
Day 21
Each day of Week 3,
subculture colonies from
this plate to a fresh plate3
Day 23
Day 24
Day 25
Day 26
Day 27
Day 28
Each day of Week 4,
subculture colonies from
this plate to a fresh plate3
Subculture Weekly to Non‐selective Agar
Week 2/Day 8
Weekly Working Culture
Week 3/Day 15
Weekly Working Culture
Week 4/Day 22
Weekly Working Culture

3. Microorganism Maintenance Plan B1
Week 1/ Pellet Plate/Day 12
TIB.246 REVISION 2011.JAN.4 Page 3 of 3
Reconstitute microorganism
(see product insert PI.194)
Start on non‐selective agar or slant
(see TIB.081 “Recommended Growth
Requirements”)
Reference Stock Culture
Day 2 Day 3 Day 4 Day 6
Day 5 Day 7
Use Daily Plates to set up panels for QC.3
Sub to new slant or
plate every 7 days
Day 9 Day 10 Day 11 Day 13
Day 12 Day 14
Week 2/Day 8
Weekly Working Culture
Day 16 Day 17 Day 18 Day 20
Day 19 Day 21
Week 3/Day 15
Weekly Working Culture
Day 23 Day 24 Day 25 Day 27
Day 26 Day 28
Week 4/Day 22
Weekly Working Culture
1. CLSI M07‐A8 Vol. 29 No. 2, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard‐Eighth Edition, January 2009.
CLSI M02‐A10 Vol. 29 No. 1, Performance Standard for Antimicrobial Disk Susceptibility Tests; Approved Standard‐Tenth Edition, January 2009.
2. Do not test colonies from the pellet plate. If it is necessary to the test the microorganism on Day 1, start the reference stock culture one day earlier and subculture it for
the first day of testing.
3. Alternatively , subculture the day before testing.