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5.14.1 McFarland Standards
I. PRINCIPLE
McFarland turbidity standards are used to
standardize the approximate number of
bacteria in a liquid suspension by visually
comparing 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 these
two reagents, standards of varying degrees
of turbidity can be prepared to represent
several different concentrations of bacte-
ria. The standard most commonly used in
the clinical microbiology laboratory for
routine antimicrobial susceptibility tests
(1, 2) is 0.5, which represents 1.5 10
8
(generally, range is 1.0 10
8
to 2.0
10
8
) bacteria/ml. McFarland standards are
commercially available from several
sources. As an alternative to the traditional
barium sulfate standards, McFarland stan-
dards prepared from latex particles have
recently become commercially available.
II. MATERIALS A. Reagents
1. Sulfuric acid, 1% (vol/vol) (H
2
SO
4
)
a. Add approximately 90 ml of
deionized water to a 100-ml vol-
umetric ask.
b. Using a 1.0-ml volumetric pi-
pette, add 1.0 ml of concen-
trated H
2
SO
4
to the ask.
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)
(BaCl
2
2H
2
O)
a. Weigh out 1.175 g of BaCl
2

2H
2
O and place in a 100-ml vol-
umetric ask.
b. Add approximately 50 ml of
deionized water, and mix well to
dissolve.
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.
3. Sterile deionized reagent-grade wa-
ter
Store in a screw-cap glass bottle for
up to 1 year at 25C.
B. Supplies
1. Sterile 1.0-liter and 10.0-ml sero-
logical pipettes and pipette bulb
2. Acid-washed glass screw-cap tubes
of a diameter comparable to that
used for inoculumpreparation (e.g.,
13 by 100 mm)
3. Paralm or parafn
C. Equipment
1. 100-ml volumetric asks
2. 0.5- and 1.0-ml volumetric pipettes
for 0.5 standards
See Appendix 5.14.11 for sizes of
volumetric pipettes needed for
preparation of other standards.
BaCl
2
2H
2
O should be dispensed
with a volumetric pipette.
3. Magnetic stirrer and stirring rod
4. Vortex
5. Spectrophotometer
III. QUALITY CONTROL
A. At time of preparation of standards
Check 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 on
reagent container and in
QC records.
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 lled. A
drop 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% H
2
SO
4
and 1.175% BaCl
2

2H
2
O 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% H
2
SO
4
to a 100-ml volumetric ask.
2. Using a 0.5-ml volumetric pipette, add 0.5 ml of 1.175% BaCl
2
2H
2
O
dropwise to the H
2
SO
4
while constantly swirling the ask.
3. Bring to 100 ml with 1% H
2
SO
4
.
4. Place magnetic stirring bar in ask 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 Paralm or parafn.
9. Store in the dark at room temperature for 3 months or longer (see item III
above).
V. USE OF MCFARLAND
STANDARDS
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 difcult 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)
5.14.1.3 Antimicrobial Susceptibility Testing
VI. PROCEDURE NOTES
A. Test suspension and McFarland standard should be contained in tubes of the
same 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 Paralm or parafn.
C. Standards in use clump and evaporate more rapidly than those in storage, and
in-use standards must be examined regularly. Discard standards with evidence
of 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 rst dilution tube is labeled 10
2
; plating 0.1 ml from the suspension
standardized to the McFarland 0.5 would yield the 10
1
dilution). (TNTC,
too numerous to count.)
Dilution
Expected no. of colonies
(with 0.1-ml aliquots)
10
1
TNTC
10
2
TNTC
10
3
TNTC
10
4
TNTC
10
5
TNTC
10
6
150
10
7
15
2. Plate 0.1-ml aliquots of dilutions expected to yield approximately 30 to 300
colonies (in this example, plate 10
6
and 10
7
) 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 estimatednumber
of bacteria only if high-quality McFarland standards are used. Standards that have
clumped or evaporated will yield unsatisfactory results.
McFarland Standards 5.14.1.4
REFERENCES
1. NCCLS. 2003. Performance Standards for
Antimicrobial Disk Susceptibility Tests, 8th ed.
Approved standard M2-A8. NCCLS, Wayne,
Pa.
2. NCCLS. 2003. Methods for Dilution Antimi-
crobial Susceptibility Tests for Bacteria That
Grow Aerobically, 6th ed. Approved standard
M7-A6. NCCLS, Wayne, Pa.
SUPPLEMENTAL READING
Barry, A. L. 1976. The Antimicrobic Susceptibil-
ity Test: Principles and Practices, p. 7274. Lea
and Febiger, Philadelphia, Pa.
McFarland, J. 1907. Nephelometer: an instru-
ment for estimating the number of bacteria in sus-
pensions used for calculating the opsonic index
and for vaccines. JAMA 14:11761178.
Washington, J. A., E. Warren, and A. G. Karl-
son. 1972. Stability of barium sulfate turbidity
standards. 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/ml
Standard no.
BaCl
2
2H
2
O (1.175%) H
2
SO
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.