Preparation of McFarland Standards

 

Purpose:

 McFarland Standards are turbidity standards that are used to gauge approximately how many bacteria are present in a liquid suspension. The standards are used to visually compare the turbidity of a Bacterial suspension with the turbidity of the appropriate standard.  Standards are prepared by adding barium chloride to sulfuric acid to obtain a barium precipitate. The volumes of the two reagents are adjusted to prepare standards of different turbidity that represent different concentrations of bacteria.

 

Reagents:

1.       Sulfuric acid, 1%

2.       Barium Chloride, 1.175%

 

Supplies:

1.       Acid washed glass screw-cap tubes comparable to that used for test

2.       Sterile serological pipettes and pipette bulb

3.       Parafilm or paraffin

 

Equipment:

1.       100ml volumetric flasks

2.       Vortex

3.       Spectrophotometer

4.       Magnetic stirrer and stirring rod

 

Procedure for the Preparation of a 0.5 McFarland Standard:

1.       Add approximately 85 ml of 1% sulfuric acid (H₂SO₄) to a 100ml volumetric flask.

2.       Using a volumetric pipette, add 0.5ml of 1.175% anhydrous barium chloride (BaCl₂) drop wise to the 1% sulfuric acid (H₂SO₄) while constantly swirling the flask.

3.       Bring the volume to 100ml with 1% H₂SO₄.

4.       Stir or mix for approximately 3 to 5 minutes while examining visually, until the solution appears homogeneous and free of clumps. A magnetic stirrer can be used for this step if available.

5.       Check optical density, following the procedure described in the QC section below and record on QC sheet.

6.       If QC is acceptable, dispense 2 to 7 ml volumes (depending on volumes routinely used in test) into each glass screw- cap tube.

7.       Label the tubes appropriately including the expiration date and the initials of the person preparing the standards. Make sure that the labeling is positioned so that it does not interfere with spectrophotometer readings.

8.       Cap the tubes tightly.

9.       Draw a line to mark the meniscus on each tube. This mark can be used as a guide to check for evaporation at a later time.

10.   Seal the tubes with paraffin or Parafilm.

11.   Repeat the procedure to make additional standards using volumes indicated in Appendix A.

12.   Store the prepared standards in the dark at room temperature for 3 months or longer as per QC acceptability.

 

QC:

1.       At time of preparation check the optical density (OD) of the McFarland standard at a wavelength of 625nm and record results. The acceptable range for a McFarland 0.5 standard is                         0.08 to 0.10.OD. For standards other than 0.5 establish acceptable ranges in-house.

2.       Visually check standards with each use for evidence of clumping and/or evaporation. Discard the standard if either is apparent

3.       Check the line drawn to indicate the position of the meniscus during the preparation. If there has been significant evaporation discard the standard.

4.       Check the optical density of a representative standard (that has not been in use) at three months to determine if the optical density is still within limits. If in control, the shelf life can be extended for another month. Repeat check monthly for up to a year.

 

Use of McFarland Standards:

1.       Mix standard and test suspension using a vortex, prior to examination.

2.       With good lighting, visually compare the turbidity of the test suspension to the McFarland standard.

3.        If the suspension is too dense in comparison to the standard dilute the suspension until it is comparable to the McFarland standard.

4.       If the suspension is too dilute in comparison to the standard, inoculate it with additional organism until the concentration matches that of the standard, or prepare a new suspension.

5.       All adjustments to the bacterial suspension should be performed using sterile technique.

6.       A Wickerham Card (see Appendix B) can also be used as an additional guide when adjusting a bacterial suspension to match the appropriate standard.

 

References:

1.       ASM Manual of Clinical Microbiology 2007.

2.       K.C. Chapin and T. Lauderdale. 2003. Reagents, stains, and media: bacteriology, p. 358. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of Clinical Microbiology, 8^th ed. ASM Press, Washington, D.C.

3.       www.google.com

 

Appendices

1.       Appendix A: Guide for the Preparation of McFarland Standards

2.       Appendix B: Wickerham Card Information

 

 

 

 

 

 

Appendix A:  Guide for the Preparation of McFarland Standards

 

Volume in mL
Standard	1% BaCL2	1% H2SO4	Number of Bacteria/ mL/(108) represented
0.5	0.5	99.5	1.5
1	1.0	99.0	3
2	2.0	98.0	6
3	3.0	97.0	9
4	4.0	96.0	12
5	5.0	95.0	15
6	6.0	94.0	18
7	7.0	93.0	21
8	8.0	92.0	24
9	9.0	91.0	27
10	10.0	90.0	30

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