MacFarland Standard

7/25/2019 MacFarland Standard

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SMILEJohns Hopkins University

Baltimore, MD USA

Preparation of McFarland Standards - Guidelines

Preparation of McFarland Standards Guideline Number Pro67-A-13-

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Effective Date 9 Nov 2009

Subect

Pre!aration of "c#arland Standard$- Guideline$

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Su!ercede$ Ne&

S"'(E )omment$* +,i$ document i$ !rovided a$ an eam!le onl./ 't mu$t be revi$ed to accuratel. reflect .our lab$

$!ecific !roce$$e$ andor $!ecific !rotocol reuirement$/ $er$ are directed to counterc,ec4 fact$ &,en con$iderin%

t,eir u$e in ot,er a!!lication$/ 'f .ou ,ave an. ue$tion$ contact S"'(E/

Background Information:

McFarland standards are prepared by adding barium chloride to sulfuric acid in order to obtain abarium sulfate precipitate and they are used to standardize the quantity of bacteria in a liquidsuspension. Using the appropriate McFarland standard, a visual comparison of the turbidity of abacterial suspension with the turbidity of the McFarland Standard is performed. The turbidity ofthe test suspension is then adusted until it matches the standard,

Resources

!. "SM Manual of #linical Microbiology $%%&.2. '.#. #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,-thed. "SM /ress, 8ashington, 9.#.

Pro6.7-A-13 Preparation of McFarland Standards Version#: 1.0 Page 1 of 6


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Baltimore, MD USA

Preparation of McFarland Standards SOP

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I acknowledge that I have read, understand and agree to follow this SOP.

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Baltimore, MD USA

Preparation of McFarland Standards

Purpose:

McFarland Standards are turbidity standards that are used to gauge approximatelyhow many bacteria are present in a liquid suspension. The standards are used tovisually compare the turbidity of a suspension of bacteria with the turbidity of theappropriate standard. Standards are prepared by adding barium chloride to sulfuricacid to obtain a barium precipitate. The volumes of the two reagents are adjusted toprepare standards of dierent turbidity that represent dierent concentrations ofbacteria.

Reagents:

!. Sulfuric acid" !#2. $arium %hloride" !.!&'#

Supplies:!. (cid washed glass screw)cap tubes comparable to that used for test2. Sterile serological pipettes and pipette bulb*. +ara,lm or para-n

Equipment:

!. !ml volumetric /as0s2. 1ortex*. Spectrophotometer. Magnetic stirrer and stirring rod

Procedure for the Preparation of a 0. McFarland Standard:

!. (dd approximately 3' ml of !# sulfuric acid 452S67 to a !ml volumetric/as0.

2. 8sing a volumetric pipette" add .'ml of !.!&'# anhydrous barium chloride

4$a%l27 dropwise to the !# sulfuric acid 452S67while constantly swirling the

/as0.*. $ring the volume to !ml with !# 52S6.. 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.'. %hec0 optical density" following the procedure described in the 9% section

below and record on 9% sheet.

:. ;f 9% is acceptable" dispense 2 to & ml volumes 4depending on volumesroutinely used in test7 into each glass screw) cap tube.

&. raw a line to mar0 the meniscus on each tube. This mar0 can be used as a

guide to chec0 for evaporation at a later time.!.Seal the tubes with para-n or +ara,lm.



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Baltimore, MD USA

!!.?epeat the procedure to ma0e additional standards using volumes indicated

in (ppendix (.!2.Store the prepared standards in the dar0 at room temperature for * months

or longer as per 9% acceptability.

!":

!. (t time of preparation chec0 the optical density 46>7 of the McFarland

standard at a wavelength of :2'nm and record results. The acceptable range

for a McFarland .' standard is .3 to .!.6>. For standards

other than .' establish acceptable ranges in)house.2. 1isually chec0 standards with each use for evidence of clumping and@or

evaporation. >iscard the standard if either is apparent*. %hec0 the line drawn to indicate the position of the meniscus during the

preparation. ;f there has been signi,cant evaporation discard the standard.. %hec0 the optical density of a representative standard 4that has not been in

use7 at three months to determine if the optical density is still within limits. ;fin control" the shelf life can be extended for another month. ?epeat chec0

monthly for up to a year.

#se of McFarland Standards:

!. Mix standard and test suspension using a vortex" prior to examination.2. Aith good lighting" visually compare the turbidity of the test suspension to

the McFarland standard.*. ;f the suspension is too dense in comparison to the standard dilute the

suspension until it is comparable to the McFarland standard.. ;f the suspension is too dilute in comparison to the standard" inoculate it with

additional organism until the concentration matches that of the standard" orprepare a new suspension.

'. (ll adjustments to the bacterial suspension should be performed using sterile

technique.:. ( Aic0erham %ard 4see (ppendix $7 can also be used as an additional guide

when adjusting a bacterial suspension to match the appropriate standard.

References:

!. (SM Manual of %linical Microbiology 2&.2. B.%. %hapin and T. .%.

$ppendices

!. (ppendix (C Guide for the +reparation of McFarland Standards2. (ppendix $C Aic0erham %ard ;nformation



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Appendix A: Guide for the Preparation of McFarland Standards

%olume in m&Standard 15 a)(2 15 2S8 Number of acteria

m(: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

/0 96/0 12

= =/0 9=/0 1=

6 6/0 9/0 1;

7 7/0 93/0 21

; ;/0 92/0 2

9 9/0 91/0 2710 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 aMcFarland Standard. A handy visual aid to be used for the preparation of inocula forthe 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

http://www.hardydiagnostics.com/http://www.carolina.com/http://www.hardydiagnostics.com/http://www.carolina.com/

Documents

MacFarland Standard