SMILE

Johns Hopkins University

Baltimore, MD USA
Preparation of McFarland Standards - Guidelines
Preparation of McFarland Standards Guideline Number Pro67-A-13-
G
Effective Date 9 Nov 2009
Subect
Pre!aration of "c#arland Standard$- Guideline$
Pa%e 1 of 1
Su!ercede$ Ne&
S"'(E )omment$* +,i$ document i$ !rovided a$ an e-am!le onl./ 't mu$t be revi$ed to accuratel. reflect .our lab0$
$!ecific !roce$$e$ and1or $!ecific !rotocol re2uirement$/ 3$er$ are directed to counterc,ec4 fact$ &,en con$iderin%
t,eir u$e in ot,er a!!lication$/ 'f .ou ,ave an. 2ue$tion$ contact S"'(E/

Background Information:
McFarland standards are prepared by adding barium chloride to sulfuric acid in order to obtain a
barium sulfate precipitate and they are used to standardize the quantity of bacteria in a liquid
suspension. Using the appropriate McFarland standard, a visual comparison of the turbidity of a
bacterial suspension with the turbidity of the McFarland Standard is performed. The turbidity of
the test suspension is then adusted until it matches the standard,
Resources
!. "SM Manual of #linical Microbiology $%%&.
$. '.#. #hapin and T. (auderdale. $%%). *eagents, stains, and media+ bacteriology, p. ),-. .n
/. *. Murray, 0. 1. 2aron, 1. 3. 1orgensen, M. ". /faller, and *. 3. 4ol5en 6ed.7, Manual of
Clinical Microbiology, -
th
ed. "SM /ress, 8ashington, 9.#.
Pro6.7-A-13 Preparation of McFarland Standards Version#: 1.0 Page 1 of 6
SMILE
Johns Hopkins University
Baltimore, MD USA
Preparation of McFarland Standards SOP
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Pro6.7-A-13 Preparation of McFarland Standards Version#: 1.0 Page 2 of 6
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Johns Hopkins University
Baltimore, MD USA
I acknowledge that I have read, understand and agree to follow this SOP.
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Pro6.7-A-13 Preparation of McFarland Standards Version#: 1.0 Page 3 of 6
SMILE
Johns Hopkins University
Baltimore, MD USA
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 suspension of bacteria 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!
". #arium $hloride 1.1%&!
Supplies:
1. 'cid washed glass screw(cap tubes comparable to that used for test
". Sterile serological pipettes and pipette bulb
). *arafilm or paraffin
Equipment:
1. 1++ml volumetric flas,s
". -ortex
). Spectrophotometer
.. Magnetic stirrer and stirring rod
Procedure for the Preparation of a 0. McFarland Standard:
1. 'dd approximately /& ml of 1! sulfuric acid 01"S2.3 to a 1++ml volumetric flas,.
". 4sing a volumetric pipette add +.&ml of 1.1%&! anhydrous barium chloride 0#a$l"3
dropwise to the 1! sulfuric acid 01"S2.3 while constantly swirling the flas,.
). #ring the volume to 1++ml with 1! 1"S2..
.. Stir or mix for approximately ) to & minutes while examining visually until the solution
appears homogeneous and free of clumps. ' magnetic stirrer can be used for this step if
available.
&. $hec, optical density following the procedure described in the 5$ section below and
record on 5$ sheet.
6. 7f 5$ is acceptable dispense " to % ml volumes 0depending on volumes routinely used
in test3 into each glass screw( cap tube.
%. 8abel the tubes appropriately including the expiration date and the initials of the person
preparing the standards. Ma,e sure that the labeling is positioned so that it does not
interfere with spectrophotometer readings.
/. $ap the tubes tightly.
9. :raw a line to mar, the meniscus on each tube. This mar, can be used as a guide to
chec, for evaporation at a later time.
1+. Seal the tubes with paraffin or *arafilm.
11. ;epeat the procedure to ma,e additional standards using volumes indicated in 'ppendix
'.
Pro6.7-A-13 Preparation of McFarland Standards Version#: 1.0 Page 4 of 6
SMILE
Johns Hopkins University
Baltimore, MD USA
1". Store the prepared standards in the dar, at room temperature for ) months or longer as
per 5$ acceptability.
!":
1. 't time of preparation chec, the optical density 02:3 of the McFarland standard at a
wavelength of 6"&nm and record results. The acceptable range for a McFarland +.&
standard is +.+/ to +.1+.2:. For standards other than +.& establish
acceptable ranges in(house.
". -isually chec, standards with each use for evidence of clumping and<or evaporation.
:iscard the standard if either is apparent
). $hec, the line drawn to indicate the position of the meniscus during the preparation. 7f
there has been significant evaporation discard the standard.
.. $hec, the optical density of a representative standard 0that has not been in use3 at three
months to determine if the optical density is still within limits. 7f in control the shelf life
can be extended for another month. ;epeat chec, monthly for up to a year.
#se of McFarland Standards:
1. Mix standard and test suspension using a vortex prior to examination.
". =ith good lighting visually compare the turbidity of the test suspension to the McFarland
standard.
). 7f the suspension is too dense in comparison to the standard dilute the suspension until
it is comparable to the McFarland standard.
.. 7f 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.
&. 'll adjustments to the bacterial suspension should be performed using sterile technique.
6. ' =ic,erham $ard 0see 'ppendix #3 can also be used as an additional guide when
adjusting a bacterial suspension to match the appropriate standard.
References:
1. 'SM Manual of $linical Microbiology "++%.
". >.$. $hapin and T. 8auderdale. "++). ;eagents stains and media? bacteriology p.
)&/. 7n *. ;. Murray @. A. #aron A. 1. Aorgensen M. '. *faller and ;. 1. Bol,en 0ed.3
Manual of Clinical Microbiology, /
th
ed. 'SM *ress =ashington :.$.
$ppendices
1. 'ppendix '? Cuide for the *reparation of McFarland Standards
". 'ppendix #? =ic,erham $ard 7nformation
Appendix A: Guide for the Preparation of McFarland Standards
Pro6.7-A-13 Preparation of McFarland Standards Version#: 1.0 Page 5 of 6
SMILE
Johns Hopkins University
Baltimore, MD USA
%olume in m&
Standard 15 6a)(2 15 72S89 Number of 6acteria1
m(1:10
;
< re!re$ented
0/= 0/= 99/= 1/=
1 1/0 99/0 3
2 2/0 9;/0 6
3 3/0 97/0 9
9 9/0 96/0 12
= =/0 9=/0 1=
6 6/0 99/0 1;
7 7/0 93/0 21
; ;/0 92/0 29
9 9/0 91/0 27
10 10/0 90/0 30
.
Appendix B: Wickerham Card Information
A 2 x 3 inch plastic laminated card with thick black and white lines to facilitate the
preparation of bacterial and yeast suspensions when comparing the turbidity to a
McFarland Standard. A handy visual aid to be used for the preparation of inocula for
the disk diffusion and other tests that reuire a specified McFarland turbidity.
!ickerham cards can be ordered from "ardy #iagnostics www.hardydiagnostics.com
or $arolina %iological Supply www.carolina.com
Pro6.7-A-13 Preparation of McFarland Standards Version#: 1.0 Page 6 of 6