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5.14.1.1

5.14 P R M R U A S T

5.14.1 McFarland Standards

I. PRINCIPLE

McFarland turbidity standards are used tostandardize the approximate number ofbacteria in a liquid suspension by visuallycomparing the turbidity of a test suspen-sion with the turbidity of a McFarland

standard. McFarland standards are pre-pared by adding barium chloride to sul-furic acid to obtain a barium sulfate pre-

cipitate. By adjusting the volumes of thesetwo reagents, standards of varying degreesof turbidity can be prepared to representseveral different concentrations of bacte-ria. The standard most commonly used in

the clinical microbiology laboratory forroutine antimicrobial susceptibility tests(1, 2) is 0.5, which represents 1.5 108

(generally, range is 1.0 108 to 2.0 108) bacteria/ml. McFarland standards arecommercially available from severalsources. As an alternative to the traditionalbarium sulfate standards, McFarland stan-

dards prepared from latex particles haverecently become commercially available.

II. MATERIALS A. Reagents1. Sulfuric acid, 1% (vol/vol) (H2SO4)

a. Add approximately 90 ml ofdeionized water to a 100-ml vol-umetric flask.

b. Using a 1.0-ml volumetric pi-pette, add 1.0 ml of concen-trated H2SO4 to the flask.

c. Bring to 100 ml with deionized

water, and mix.d. Store in a screw-cap glass bottle

for up to 1 year at 25C.2. Barium chloride, 1.175% (wt/vol)

(BaCl2 2H2O)a. Weigh out 1.175 g of BaCl2

2H2O and place in a 100-ml vol-umetric flask.

b. Add approximately 50 ml ofdeionized water, and mix well todissolve.

c. Bring to 100 ml with deionizedwater, and mix.

d. Store in a screw-cap glass bottlefor up to 1 year at 25C.

3. Sterile deionized reagent-grade wa-terStore in a screw-cap glass bottle forup to 1 year at 25C.

B. Supplies1. Sterile 1.0-liter and 10.0-ml sero-

logical pipettes and pipette bulb2. Acid-washed glass screw-cap tubes

of a diameter comparable to that

used for inoculum preparation (e.g.,13 by 100 mm)

3. Parafilm or paraffinC. Equipment

1. 100-ml volumetric flasks2. 0.5- and 1.0-ml volumetric pipettes

for 0.5 standardsSee Appendix 5.14.11 for sizes ofvolumetric pipettes needed forpreparation of other standards.BaCl2 2H2O should be dispensedwith a volumetric pipette.

3. Magnetic stirrer and stirring rod4. Vortex5. Spectrophotometer

III. QUALITY CONTROL A. At time of preparation of standardsCheck optical density of McFarland standard at wavelength of 625 nm and

record results. The acceptable range for a McFarland 0.5 standard is 0.08 to

0.10. For standards other than 0.5, establish acceptable ranges in-house.

Include QC information onreagent container and inQC records.

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McFarland Standards 5.14.1.2

B. Additional checks

1. Check in-use standards with each use for visible evidence of clumping, and

periodically compare in-use standard with a standard that has not been in

use. If clumping is apparent, discard the standard.

2. Check standards for any evidence of evaporation. This can be accomplished

by drawing a line on the tube at the meniscus when the tubes are filled. Adrop in liquid level below this line indicates that evaporation has occurred.

If evaporation is evident, discard the standard.

3. Check the optical density of a representative standard (that has not been in

use) at 3 months to make certain that the optical density of the lot is still

within control limits. If in control, the shelf life of the lot can be extended

for another month.

4. Repeat optical density check monthly. If in control, shelf life can be extended

for another month for up to 12 months.

IV. PROCEDURE A. Refer to Appendix 5.14.11 for volumes of 1% H2SO4 and 1.175% BaCl2

2H2O needed to prepare different grades of McFarland standards (representing

various concentrations of bacteria), and substitute these volumes into the pro-cedure below when preparing standards other than 0.5.

B. To prepare McFarland 0.5 standards

1. Add approximately 85 ml of 1% H2SO4 to a 100-ml volumetric flask.

2. Using a 0.5-ml volumetric pipette, add 0.5 ml of 1.175% BaCl2

2H2O

dropwise to the H2SO

4while constantly swirling the flask.

3. Bring to 100 ml with 1% H2SO

4.

4. Place magnetic stirring bar in flask and place on magnetic stirrer for ap-

proximately 3 to 5 min.

5. Examine solution visually to make certain it appears homogeneous and free

of visible clumps; check optical density and record on QC sheet.

6. Providing QC is acceptable, dispense 2- to 7-ml volume (depending on vol-

ume routinely used in inoculum suspension preparation) into each tube.

7. Cap tubes tightly.8. Seal with Parafilm or paraffin.

9. Store in the dark at room temperature for 3 months or longer (see item III

above).

V. USE OF MCFARLANDSTANDARDS

A. Mix McFarland standard and test suspension well on a vortex mixer prior to

examination.

B. In the presence of good lighting, visually compare turbidity of test suspension

to the McFarland standard.

1. Hold both tubes in front of a Wickerham card (Appendix 5.14.12).

2. If it is more difficult to see lines on Wickerham card through test suspension

than through the McFarland standard, the suspension is too dense and must

be diluted.

3. If dilution is necessary, use a sterile pipette to add additional sterile broth or

saline to the tube to obtain turbidity comparable to that of the McFarland

standard.

4. Before subsequent examinations, vortex test suspension and McFarland stan-

dard well.

5. If test suspension is too light, inoculate with additional organisms or rein-

cubate suspension (depending on inoculum preparation protocol) until tur-

bidity reaches that of the standard.

III. QUALITY CONTROL(continued)

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5.14.1.3 Antimicrobial Susceptibility Testing

VI. PROCEDURE NOTES A. Test suspension and McFarland standard should be contained in tubes of thesame diameter.

B. McFarland standards are more likely to clump and evaporate if exposed to air.

Thus, it is imperative that caps be tightly sealed with Parafilm or paraffin.

C. Standards in use clump and evaporate more rapidly than those in storage, and

in-use standards must be examined regularly. Discard standards with evidenceof clumping or evaporation regardless of stated shelf life.

D. When glass tubes are used, acid washing helps minimize clumping.

E. A practical storage container for the McFarland standards is a test tube mailing

container. Alternatively, wrap tube in aluminum foil.

F. An alternative to using a spectrophotometer to QC McFarland standards is per-

forming colony counts on bacterial suspensions (e.g., Escherichia coli ATCC

25922) that have been standardized against a particular lot of standards.

1. Prepare six successive 10-fold dilutions of test suspension in 0.85% NaCl

(the first dilution tube is labeled 102; plating 0.1 ml from the suspensionstandardized to the McFarland 0.5 would yield the 101 dilution). (TNTC,too numerous to count.)

DilutionExpected no. of colonies

(with 0.1-ml aliquots)

110 TNTC210 TNTC310 TNTC410 TNTC510 TNTC610 150710 15

2. Plate 0.1-ml aliquots of dilutions expected to yield approximately 30 to 300

colonies (in this example, plate 106 and 107) to BAP (in duplicate) and

spread evenly to distribute using a loop or sterile

hockey stick.

3. Colony counts should fall within 10% of expected numbers.

G. Actual numbers of viable bacteria present in McFarland standardized-suspen-

sions may vary depending on size, viability, and clumping of the particular

bacteria tested.

H. When using commercially available McFarland standards prepared from latex

particles, follow manufacturers directions for use and storage.

I. Other devices are satisfactory for standardizing bacterial suspensions. These

include the following:

1. Wand-type inoculum devices that pick up a standardized volume of cell paste

(from colonies grown overnight on an agar plate) that is subsequently sus-

pended in a standardized volume of liquid (BBL Prompt inoculation system,

[Becton Dickinson Microbiology Systems, Sparks, Md., or Dade MicroScan,

West Sacramento, Calif.).2. Photometers or colorimeters such as the following

a. Vitek colorimeter (bioMerieux Vitek, Inc., Durham, N.C.)

b. MicroScan turbidity meter (Dade MicroScan)

VII. LIMITATIONS Suspensions standardized to McFarland standards will contain the estimated numberof bacteria only if high-quality McFarland standards are used. Standards that have

clumped or evaporated will yield unsatisfactory results.

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McFarland Standards 5.14.1.4

REFERENCES 1. NCCLS. 2003. Performance Standards forAntimicrobial Disk Susceptibility Tests, 8thed.Approved standard M2-A8. NCCLS, Wayne,Pa.

2. NCCLS. 2003. Methods for Dilution Antimi-crobial Susceptibility Tests for Bacteria ThatGrow Aerobically, 6th ed. Approved standardM7-A6. NCCLS, Wayne, Pa.

SUPPLEMENTAL READING Barry, A. L. 1976. The Antimicrobic Susceptibil-ity Test: Principles and Practices, p. 7274. Leaand Febiger, Philadelphia, Pa.

McFarland, J. 1907. Nephelometer: an instru-ment for estimating the number of bacteria in sus-pensions used for calculating the opsonic indexand for vaccines. JAMA 14:11761178.

Washington, J. A., E. Warren, and A. G. Karl-son. 1972. Stability of barium sulfate turbiditystandards. Appl. Microbiol. 24:1013.

APPENDIX 5.14.11 Preparation of McFarland Standards

Adapted from K. C. Chapin and T. Lauderdale. 2003. Reagents, stains, and media: bac-

teriology, p. 358. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H.

Yolken (ed.), Manual of Clinical Microbiology, 8th ed. ASM Press, Washington, D.C.

Vol (ml)

No. of bacteria/mlStandard no.BaCl

2 2H

2O (1.175%) H

2SO

4(1%) (10

8) 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

APPENDIX 5.14.12 Wickerham Card

L. J. Wickerham. 1951. Taxonomy of yeasts, p. 11. In Technical bulletin no. 1029. U.S.

Department of Agriculture, Washington, D.C.