Laboratory name
Location
Head/Responsible person / Standard Operating Procedure (SOP)
Preparation of MacFarland no.1 standard / Code:
Version: no.
Date: of release
Page: 2 of 4
1. Preparation of McFarland No. 1 Standard
2. Objectives and scope
This SOP describes the procedure for preparation of McFarland No.1 Standard.
The McFarland standard is intended to standardize the mycobacterium suspension at a known concentration of bacilli. The bacterial suspension with turbidity similar to McFarland No1 is assumed to be approximately 3x108 CFU/mL (or approximately 1 mg of bacilli/mL). This suspension is the starting point of any drug susceptibility testing on Löwenstein-Jensen media.
This SOP is applicable to all technical laboratory employees of the laboratory.
3. Abbreviations, definitions and terms
• N.A. Not applicable
• LJ Löwenstein-Jensen
• DW Distilled water
• RT room temperature
• QC Quality control
• SOP Standard Operating Procedure
4. Tasks, responsibilities and accountabilities
Preparation of 1% Bacl2 and 1% H2SO4 / Techs in LJ DST section / Lab Manager
Preparation of McFarland / Techs in LJ DST section / Lab Manager
5. Safety and environment
1. Sulphuric acid is corrosive on contact with skin. It causes burns.
Oral exposure: If swallowed, wash out mouth with water provided person is
conscious. Call a physician immediately.
Inhalation exposure: If inhaled, remove to fresh air.
Skin exposure: In case of skin contact, flush with copious amounts of water.
Eye exposure: In case of contact with eyes, flush with copious amounts of
water for at least 15 minutes. Assure adequate flushing by separating the eyelids with fingers. Call a physician.
2. Always wear a lab coat and gloves in the lab.
6. Procedure
6.1 Reagents and Materials
1. 1 mL graduated pipette
2. 10 mL graduated pipette
3. 100 mL flask
4. 100 mL graduated cylinder
5. Balance sensitive to 0.001g
6. Barium chloride BaCl2
7. 14 mL Bijou bottles (same as used for mycobacterial suspension)
8. Distilled water
9. Spatula
10. Sulphuric acid H2SO4
11. Weighing paper
6.2 Stepwise procedure
6.2.1 Preparation of primary solutions
1. 1% BaCl2 w/v
a. Weigh 1g of Barium Chloride (BaCl2) into a clean conical flask/suitable container.
b. To 1g of BaCl2, add 100 mL of Distilled/de-ionized water.
c. Mix well so that BaCl2 completely dissolves in water.
d. Label properly and store at Room Temperature up to 6 months
2. 1% H2SO4
a. Measure 99 ml of Distilled Water into a suitable container.
b. Pipette and add 1.0 ml of concentrated sulphuric acid to the water.
c. Swirl to mix well
d. Label properly and store at Room Temperature up to 6 months
6.2.2 Preparation of McFarland Standard No. 1
1. Measure 9,9 mL of 1% H2SO4 solution from 6.2.1 (2) above into a suitable container.
2. Add to it 0,1 mL (100 µl) of 1% BaCl2 from 6.2.1 (1) above.
3. Mix well.
4. Transfer all the resultant 10 ml into a 14ml bijou bottle.
5. Label properly and store at Room Temperature up to 4 weeks.
6.3 Storage
Store properly labeled bottles of 1% Barium chloride and 1% sulphuric acid at room temperature up to 6 months. Store McFarland Standard 1 solution at room temperature for 4 weeks.
6.4 Quality Control
1. McFarland Standard 1 is stable at room temperature for up to 4 weeks. Make fresh McFarland every 4 weeks to avoid errors.
2. Label the container with the date prepared and date of expiration (add 28 days to the date prepared).
3. Discard the 1% BaCl2 and 1% Sulphuric acid after 6 months. Do not use expired solutions to constitute McFarland Standard.
4. Agitate Bijou bottle with McFarland Standard before matching its turbidity with bacterial suspensions.
5. If necessary use a dark background when comparing turbidity of suspensions.
7. Related documents
SOP “Preparation and dilution of Bacterial suspensions”
8. Related forms
N.A.
9. References
N.A.
10. Attachments/Annexes
Appendix 1, Preparation of McFarland No.1 Standard
Source: GLI Stepwise Process towards TB Laboratory Accreditation
Preparation of McFarland No.1 Standard
Component/ Reagent / Lot No: / Expiration dateSulphuric acid
Barium Chloride
Date Prepared: ______Prepared by: ______
Ingredients / Batch No: / Expiration date / Amount Used1% Sulphuric acid
1% Barium chloride
Amount of McFarland No. 1 Standard prepared: ______ml
Quality Control Check:
Not Applicable
McFarland No. 1 Standard BATCH NO: ______Expiry Date: ______
NB: Storage at Room Temp for 4 weeks.
Reviewed by:______Date:______
Source: GLI Stepwise Process towards TB Laboratory Accreditation