STOCK CULTURE PREPARATION

Effective Date: 
Thu, 08/01/2013
Reviewed: 
Tue, 01/10/2017
Policy: 

 

Purpose and Scope:

LYFO DISK®, KWIK-STIK™ and KWIK-STIK™ Plus Microorganisms are lyophilized, reference stock culture preparations containing a single strain of a microorganism. These microorganism preparations are intended to be used for quality control of culture media, educational/instructional programs and industrial applications. The microorganism preparations are traceable to the American Type Culture Collection (ATCC®) or other authentic reference culture collections.

 

Principle of the method:

 

LYFO DISK®, KWIK-STIK™ and KWIK-STIK™ Plus Microorganisms incorporate a lyophilization method reported by Obara et.al. which uses a suspending medium consisting of gelatin, skim milk, ascorbic acid, dextrose, and charcoal. The gelatin serves as a carrier for the microorganism. Skim milk, ascorbic acid, and dextrose protect the microorganism by preserving the integrity of the cell wall during freeze-drying and storage. The charcoal is included to neutralize any toxic substances formed during the lyophilization process.

 

 

Reagents and Supplies:

 

A. KWIK-STIK™ Microorganisms Each KWIK-STIK™ unit contains a lyophilized pellet of a single microorganism strain, a reservoir of hydrating fluid and an inoculating swab. Each device is sealed within a laminated pouch that contains a desiccant to prevent adverse moisture accumulation. The KWIK-STIK™ Microorganisms offer an additional feature.

• Each lyophilized microorganism preparation is less than or equal to four (4) passages from a reference culture.

B. KWIK-STIK™ Plus Microorganisms The packaging of the KWIK-STIK™ Plus Microorganism is identical to the KWIK-STIK™ Microorganisms. The KWIK-STIK™ Plus Microorganisms offer two additional features.

• Each lyophilized microorganism preparation is two (2) passages from a reference culture.

• A Certificate of Assay is provided. It offers documentation regarding the identity and traceability of the microorganism preparation to a reference culture and the number of passages the microorganism preparation has been removed from the reference culture.

C. LYFO DISK® Microorganisms LYFO DISK® Microorganisms are packaged in a resealable vial that contains ten (10) lyophilized pellets of a single microorganism strain and a desiccant to prevent adverse accumulations of moisture. The LYFO DISK® Microorganisms offer an additional feature.

• Each lyophilized microorganism preparation is less than or equal to four (4) passages from a reference culture.

 

LYFO DISK® Microorganisms require sterile tubes and 0.5 ml of sterile liquid such as, Tryptic Soy Broth or sterile saline to hydrate the lyophilized preparation. Sterile swabs or inoculating loops are needed to transfer the hydrated preparation to an agar plate.

• LYFO DISK®, KWIK-STIK™ and KWIK-STIK™ Plus Microorganisms require non-selective agar media (blood agar plates).

 

Storage and Expiration:

 

Store the LYFO DISK®, KWIK-STIK™ and KWIK-STIK™ Plus Microorganisms at 2°C to 8°C in the original, sealed vial or pouch containing the desiccant. Stored as directed, the lyophilized microorganism preparation will retain, until the expiration date stated on the device label, its specifications and performance within the stated limits.

The LYFO DISK®, KWIK-STIK™ and KWIK-STIK™ Plus Microorganisms should not be used if:

• Stored improperly;

• There is evidence of excessive exposure to heat or moisture; or,

• The expiration date has passed.

 

Procedure

A. KWIK-STIK™ and KWIK-STIK Plus™ Microorganism Procedure

1. Allow the unopened KWIK-STIK™ pouch to equilibrate to room temperature. Tear open pouch at notch and remove the KWIK-STIK™ unit.

2. Tear off Pull-Tab portion on the label and attach it to the primary culture plate or QC record. Do not disassemble the device during hydration.

3. Pinch (once only) the ampoule at the top of the KWIK-STIK™ (just below the fluid meniscus of the ampoule) found in the cap to release the hydrating fluid.

4. Hold vertically and tap on a hard surface to facilitate flow of fluid through shaft into bottom of unit containing pellet. Allow the hydrating fluid to flow through the swab shaft and into the bottom portion of the unit containing the pellet.

5. Using a pinching action on the bottom portion of the unit, crush the pellet in the fluid until the pellet suspension is homogenous.

6. IMMEDIATELY heavily saturate the swab with the hydrated material and transfer to agar medium.

7. Inoculate the primary culture plate(s) by gently rolling the swab over one-third of the plate.

8. Using a sterile loop, streak to facilitate colony isolation. Label agar with organism name, ATCC # and inoculation date.

9. Using proper biohazard disposal, discard the KWIK-STIK™.

10. IMMEDIATELY incubate the inoculated primary culture plate(s) at temperature and conditions appropriate to the microorganism.

B. LYFO DISK® Microorganism Procedure

1. Remove the unopened LYFO DISK® vial from 2°C to 8°C storage and allow the unopened vial to equilibrate to room temperature.

2. Aseptically remove one (1) pellet with sterile forceps from the vial. Do not remove desiccant.

3. Place the pellet in 0.5 mL of sterile fluid ( saline or TSB). Label tube with organism name, ATCC # and date inoculated.IMMEDIATELY stopper and recap vial and return the resealed vial to 2°C to 8°C storage.

4. Crush the pellet with a sterile swab until the suspension is homogenous. IMMEDIATELY heavily saturate the same swab with the hydrated material and transfer to agar medium.

5. Inoculate the primary culture plate(s) by gently rolling the swab over one-third of the plate.

6. Using a sterile loop, streak to facilitate colony isolation. Label agar with organism name, ATCC # and inoculation date.

7. Using proper biohazard disposal, discard the remaining hydrated material.

8. IMMEDIATELY incubate the inoculated media at temperature and conditions appropriate

 

C. Stock Cultures

Examine agar plates after 24 hrs incubation. Should be well isolated colonies free from contamination.

  1. Isolated colonies can be used for QC for various tests identification tests and Kirby Bauer method QC.
  2. Subculture organisms weekly to a fresh BAP plate.
  3. Reconstitute fresh organisms from KWIK-STIK or LYFO DISK every 30 days.

REFERENCES

Microbiologics KWIK STIK and LYFO DISK Instructions for Use Package Insert Rev A 10/2011

Y. Obara, S. Yamai, T. Nikkawa, Y. Shimoda, and Y. Miyamoto. 1981. J. Clin. Microbiol. 14:61-66.