QUE-BACT MICROORGANISMS

Technical Data #MICROa / 2006.04.12

QUE-BACT Microorganisms are viable reference stock culture preparations containing a single strain of a microorganism. These microorganism preparations are traceable to authentic reference culture collection. QUE-BACT Microorganisms are designed and manufactured to support microbiology quality assurance and quality control functions. Microorganisms with known and predicable characteristics are also used in education and proficiency programs.


FORMULA

Reference Cultures: Origin and traceability.
The notation “Derived from…” identifies QUE-BACT Microorganisms, the source of which, is an authentic reference culture purchased from a culture type collection. Receipt of each reference culture initiates record keeping and documentation. The receipt date and lot number of each reference culture is recorded. Reference culture number and lot number can trace the documentation of all subsequent passages and subcultures through records from the receipt and identification of each reference culture to the sale and distribution of reference stock cultures.

Primary growth: Processing, Inoculation and Incubation.
The reference culture is hydrated or thawed according to instructions. A portion of the hydrated/thawed reference culture is inoculated to non-selective agar media. The inoculated media is incubated under conditions that optimize the growth of the reference culture. This function and the microorganism growth is identified and labelled as “Primary Growth”.

Subculture # 1: Strain Production and Authenticity.
Established growth on “Primary Growth” is selected and subcultured to a selection of nutrient, enriched, selective and differential media contained in small plastic sterile vial with screw cap. The inoculated slope media are incubated under different conditions to establish and document essential growth requirements and growth characteristics. Following incubation, the Subculture media are examined for: (a) growth requirements (E.g. nutrient, enriched, incubation temperature, aerobic, 5% to 10% carbon dioxide dependence, microaerophilic, anaerobic, etc.); (b) selective and differential characteristic; (c) macroscopic colony features; and , (e) microscopic morphology features. Test results are recorded to document reference culture authenticity. The microorganism preparation, designated as a “Subculture # 1” is labelled, packaged and stored at -70ºC according to FDA: Quality System regulation.


PRECAUTIONS
This product is for laboratory use only. These devices, and subsequent growth of these microorganisms on culture media are considered to be biohazard material which has to be disposed as a clinical waste. These devices contain viable microorganisms that may, under certain circumstances, produce disease. Proper techniques must be employed to avoid exposure and contact with any microorganism growth.


STORAGE
Store refrigerated or frozen or in liquid nitrogen container.


SIGN OF DETERIORATION
This product should not be used if stored improperly, there is evidence of excessive exposure to heat or the expiration date has passed.


INSTRUCTIONS FOR USE

  1. Upon reception the Subculture # 1 is aseptically inoculated to a selection of nutrient, enriched, selective and differential media. Using a sterile loop, streak to facilitate colony isolation.
  2. Immediately incubate the inoculated culture plate(s) under the required growth conditions.
  3. After 18-24 hours examine plate for growth and contamination. If necessary do a Gram stain to confirm pure colonies.
  4. The microorganism growth designed as “Subculture # 2” is examine for growth requirements, selective and differential characteristics, macroscopic colony features and microscopic morphology features.
  5. Test results are recorded to document reference culture authenticity.
  6. Select representative well-isolated colonies for indicated transfers.


PROCEDURE

MAINTENANCE OF CONTROL STRAINS

  1. The Subculture # 2 is aseptically inoculated into 10 ml preserving solution like BHI Glycerol Broth making a heavy cloudy suspension of the microorganism equivalent to 1.0 Mc Farland Standard turbidity.
  2. Mix well and aseptically transfer 1 ml into sterile cryovials.
  3. Identify the vials and immediately place them in a -20ºC to -70ºC freezer to preserve the culture.

Note: Alternatively the Subculture # 2 may be aseptically inoculated into CRY-O-BACT vial which contains a preserving solution. Follow the manufacturer instructions.

RECOVERY

  1. Take out the vial from the freezer and place it in warm water for few minutes.
  2. Using a sterile pipet transfer few drops of the thawed suspension over the surface of a solid medium of choice and incubate under the required growth conditions.

    Note: If CRY-O-BACT vials have been used, take one vial from the freezer and open it. Using sterile inoculating needle, retrieve one bead from the preserving solution. Roll the bead over the surface of a solid medium of choice and incubate under the required growth conditions. Immediately return the CRY-O-BACT vial with the remaining beads to the freezer to prevent thawing of the other beads.

PREPARATION OF THE WORKING SUSPENSION

  1. Following incubation examine for contamination. If necessary do a Gram stain to confirm pure colonies.
  2. For AEROBES using sterile loop, pick and suspend well-isolated colonies into 2 ml of Tryp Soy Broth. Incubate aerobically at 35ºC for 4-5 hours until 0.5 Mc Farland Standard turbidity is present. This suspension should contains 1x107 to 1x108 CFU/ml.

For ANAEROBES using sterile loop, pick and suspend well-isolated colonies into a Fastidious Anaerobes broth. Incubate at 35ºC for 3 days with tight screwed cap. Following incubation keep the tube at room temperature in the dark. Before use pre-reduce the medium in a boiling water bath for 15 minutes with loosened cap. After dissolved oxygen drive off screw cap tightly and allow medium to cool without agitation to room temperature. Using sterile loop inoculate an anaerobic media like Columbia Anaerobe 5% Blood. Incubate anaerobically at 35ºC for 48 hours. Using sterile loop, pick and suspend well-isolated colonies into 2 ml of Tryp Soy Broth until 0.5 Mc Farland Standard turbidity is present.

TESTING PROCEDURE OF CULTURE MEDIA

For testing nutritive capacity of a medium, plated or tubed, dilute the cell suspension (0.5 Mc Farland) 1:100 in normal saline. Using sterile 10 ul loop, inoculate test plate and incubate under required growth conditions. This loopful contains 1x 103 to 1x104 CFU/ml. If this loopful does not give isolated colonies on the test plate, use a tenfold lighter inoculum (1 ul instead of 10 ul loop).

For testing inhibitory capacity of a selective medium, dilute the 0.5 Mc Farland Standard 1/10 in normal saline and inoculate test plate with a sterile 10 ul loop. This loopful contains 1x104 to 1x105 CFU/plate. Incubate under required growth conditions.

For testing the performance of tubed media or sensitivity studies, use 0.5 Mc Farland Standard turbidity suspension.


QUALITY CONTROL

This product is produced and distributed in compliance with the 1996 FDA: Quality System Regulation and NCCLS Guidelines and, in conformance with the requirements of ISO 9001:2000, ISO 13485:2003 and CE Mark.

Quality control functions include, but are not limited to:


LIMITATIONS OF METHOD
The microbiology laboratory must be equipped and have the facilities to receive, process, maintain, store and dispose of biohazard material. The laboratory personnel using this product must be trained, experienced and demonstrate proficiency in processing, maintaining, storing and disposing of biohazard material. Each laboratory must be aware of, and comply with, the proper disposal of biohazard material.


REFERENCES

  1. AOAC Compendium of Microbiological Methods.
  2. Clinical Microbiology Procedure Handbook. Vol.1 and 2. 1st Ed.1992 ASM Washington, DC.
  3. FDA Bacteriological Analytical Manual.
  4. Manual of Clinical Microbiology, 4th Ed. 1985. ASM, Washington, DC.
  5. Manual of Quality Control Procedures for Microbiology Laboratories, 3rd Ed., 1981 CDC Atlanta, GA.
  6. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that grow aerobically. NCCLS
  7. Official Methods of Analysis of the Association of Official Analytical Chemists.
  8. Performance Standards for Antimicrobial Disk Susceptibility Tests. NCCLS
  9. Quality Assurance for Commercially Prepared Microbiological Culture Media. NCCLS 1990 Approved Standard Document M22-A Vol.10 No. 14
  10. Methods for Antimicrobial Susceptibility Testing of ANAEROBIC bacteria. NCCLS
  11. Standard Methods for the Examination of Dairy Products.
  12. Standard Methods for the Examination of Water and Wastewater.
  13. US Pharmacopoeia 25 and National Formulary 20.


CATALOGUE NUMBERS
All QUE-BACT MICROORGANISMS catalogue numbers start with the four letters "QB-MI" followed by digits. Example: QB-MI-8001 refers to Streptococcus agalactiae.

Prepared media    
BHI 15% Glycerol Broth, vial 17 x 60 mm, 2 ml 10/pkg 2139
BHI 15% Glycerol Broth, tube 16 x 125 mm, 10 ml 10/pkg 2339
Tryp Soy Broth, tube 13 x 100 mm, 2 ml 10/pkg 2341
Fastidious Anaerobes Broth, tube 16 x 125 mm, 10 ml 10/pkg 2960
Normal Saline, tube 13 x 100 mm, 2 ml 10/pkg 2250
Nutrient Agar, tube 16 x 125 mm, 5 ml 10/pkg 2741
Columbia Anaerobe 5% Sheep Blood, 90 mm 10/pkg 1121
Mc Farland Standard    
No. 0.5 tube 16 x 125 mm, 5 ml 10/pkg 2901
No. 1.0 tube 16 x 125 mm, 5 ml 10/pkg 2586
CRY-O-BACT 25 vials 100 vials
Red Beads COB-25R COB-100R
Green Beads COB-25G COB-100G
Yellow Beads COB-25Y COB-100Y
Blue Beads COB-25B COB-100B
White Beads COB-25W COB-100W
Mixed Colours COB-25M COB-100M


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