PREPARATION OF 0.

5 McFARLAND STANDARD

INTRODUCTION
The McFarland Equivalence Standards are intended to be part of a quality control program for
adjusting densities of bacterial suspensions that are used for identification and susceptibility
testing. Each standard is made from different concentrations of latex beads mixed in a buffer
liquid. McFarland Standards are used to standardize the approximate number of bacteria in a
liquid suspension by comparing the turbidity of the test suspension with that of the McFarland
Standard. A McFarland Standard is a chemical solution of barium chloride and sulfuric acid; the
reaction between these two chemicals results in the production of a fine precipitate, barium
sulfate suspension (BSS) [McFarland, 1907].

MATERIALS/ APPARATUS
A. Reagents
1. Sulfuric acid, 1% (vol/vol) (H2SO4)
a. Add approximately 90 mL of deionized water to a 100 mL volumetric flask.
b. Using a 10 mL of concentrated 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 room temperature.

2. Barium chloride, 1.175% (wt/vol) (BaCl2•2H2O).

a. Weigh out 1.175g of BaCl2•2H2O and place in a 100 mL volumetric flask.
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 room temperature.

3. Sterile deionized reagent-grade water.

a. Store in a screw-cap glass bottle for up to1 year at room temperature.

B. Supplies
1. Sterile 1.0-liter and 10.0 mL serological pipettes and pipette bulb.
2. Acid-washed glass screw-cap tubes of a diameter comparable to that used for
inoculum preparation (e.g., 13 by 100 mm).
3. Parafilm.

C. Equipment
1. 100 mL volumetric flasks.
2. 0.5 and 1.0 mL volumetric pipettes for 0.5 standards.
3. Magnetic stirrer and stirring rod.
 4. Vortex.
5. Spectrophotometer.
6. Wickerham card.

PROCEDURE
Preparation of 0.5 McFarland Standard
1. 0.5 McFarland standard is prepared by adding 0.05 mL of a 1.175 (wt/vol) anhydrous barium
chloride (BaCl2•2H2O) solution to 9.95 mL 1% (vol/vol) sulfuric acid (H2SO4). The result is a
fine precipitate of barium sulfate suspension (BaSO4).
2. The turbidity standard is aliquoted into a test tube identical to the test tube used to prepare the
inoculum suspension.
3. The McFarland standard tubes are tightly sealed with parafilm to prevent evaporation and
stored in the dark at room temperature (22° to 25°C) (Cockerill et al., 2012).
The solution should be shaken (agitated) vigorously to mix the fine white precipitate of barium
sulfate in the tube before use and inspected for a uniform appearance (discard if large particles
are present). The suspensions made with latex particles should be gently inverted.

Preparation of Inoculum
1. Each culture to be tested is streaked onto a non-inhibitory agar medium (blood agar, brain
heart infusion agar, or tryptic soy agar) to obtain isolated colonies.
2. After incubation at 35°C overnight, 4 or 5 well-isolated colonies are select with an inoculating
needle (or loop), and the growth is transfered to a tube of sterile saline or non-selective broth
(Mueller-Hinton broth, heart infusion broth, or tryptic soy broth) and mixed thoroughly using a
vortex mixer.
3. The bacterial suspension is compared to the 0.5 McFarland standard.

Comparison of bacterial suspension with 0.5 McFarland Standard

Visual comparison
In visual comparison, the tubes are viewed against a sheet of white paper known as wickerham
card on which sharp black lines are drawn. The turbidity standard should be agitated on a vortex
mixer immediately, prior to examination to sufficiently suspend the barium sulfate particles for
uniform appearance and it is aliquoted into a tube that is the same size and diameter as the test
tube used to prepare the test suspension . If the bacterial suspension does not appear to be the
same density as the 0.5 McFarland standard, the turbidity can be reduced by adding sterile saline
or broth using sterile pipette or increased by adding more bacteria to the test suspension until the
turbidity matches that of the 0.5 McFarland standard.
Bacterial suspensions are standardized if the distortion/attenuation of the black lines is consistent
with the McFarland standard.
Spectrophotometric comparison
A spectrophotometer is an instrument that measures the amount of light absorbed by a sample.
Spectrophotometer techniques are mostly used to measure the concentration of solutes in
solution by measuring the amount of the light that is absorbed by the solution in a cuvette placed
in the spectrophotometer.
The accuracy of the density of a prepared McFarland standard is checked by using a
spectrophotometer with a 1-cm light path.
1. The 0.5 McFarland standard is aliquoted into a selected blank cuvette, the lid is properly
closed and placed in the spectrophotometer.
2. The spectrophotometer is made to read 0.00000 A, by simply clicking on 0 ABS 100%T
button.
3. For the 0.5 McFarland standard, the absorbance is measured using a wavelength of 625 nm ranges
from 0.08 to 0.13.
4. Record the wavelength at the maximum absorbance value. For the 0.5 McFarland standard, the
absorbance ranges from 0.08 to 0.13.
5. The same procedure is repeated to check the absorbance value of the bacterial suspension
using the same wavelength of 625 nm.
If the bacterial suspension does not appear to be the same absorbance as the 0.5 McFarland
standard, the turbidity can be reduced by adding sterile saline or broth using sterile pipette or
increased by adding more bacteria to the test suspension until the turbidity matches that of the
0.5 McFarland standard.

Table 1. showing the McFarland Standard in comparison to Bacterial suspension

 Catalogue McFarland 1% BaCl2 1% Approximate Bacterial
number Standard mL) (H2SO4) suspension/ mL
TM50 0.5 0.05 9.95 1.5 x 108
TM51 1.0 0.10 9.90 3.0 × 108
TM52 2.0 0.20 9.80 6.0 × 108
TM53 3.0 0.30 9.70 9.0 × 108
TM54 4.0 0.40 9.60 1.2 × 108
Source: McFarland (1907)

Precaution
1. The tubes are closed tightly at all times and kept in the dark, because McFarland
standard are sensitive to air and light.
2. The level of McFarland standards are checked occasionally to ensure that
evaporation does not occur.
3. The McFarland Standards is vigorously agitated on a mechanical vortex before each
use and inspected for a uniform turbid appearance.
4. Products beyond the expiration date are not to be used.
5. Directions are carefully read prior to use.
6. The same size tubes are used in comparing bacterial suspensions to the 0.5
McFarland Standard.
Advantages of McFarland Standard
1. It is used in the antimicrobial susceptibility testing procedure where the bacterial
suspension is compared to Standard McFarland, prior to swab on MHA media.
2. It is a part of quality control to check and adjust the densities of bacterial suspension that
can be used for identification and susceptibility procedure.
3. It helps to maintain and/or ensure that the number of bacteria will be within a given range
to standardize microbial testing.

Disadvantages of McFarland Standard

1. In the case of the colored media, it may not provide proper contrast with McFarland
Equivalent Standards.
2. The older cultures (>24 hours) of bacterial suspensions may not compare to expected
bacterial counts.
3. The McFarland standards have been adjusted by a spectrophotometer analysis, and use of
any other instrumentation may not give reliable results during the testing procedure.
4. During the storage time, exposure of the McFarland standard to the light can affect the
turbidity measurement.
Appendix
 Source: Bioanalytic GmbH (2021)
Plate 1. McFarland Standards.

Source: Bioanalytic GmbH (2021)

Plate 2. McFarland Standard set and Wickerham Card
Source: Wheal (2012)
Plate 3. Spectrophotometer.
 REFERENCES
Cockerill, Franklin, R. (2012). Methods for Dilution Antimicrobial Susceptibility Test for
Bacteria That Grow Aerobically; Approved Standard. 9th edition. p.12
Dalynn Biologicals (http://www.dalynn.com/dyn/ck_assets/files/tech/TM53.pdf).
Himedia (http://himedialabs.com/TD/R092.pdf)
http://www.wheal-jane-laboratory.co.uk/spectrophotometer-%E2%80%93-dr-lang-xion-500

http://www.wheal-jane-laboratory.co.uk/wp-content/uploads/2012/01/xion-resized.jpg

McFarland, J., Amer, J. (1907). Document, Performance Standards for Antimicrobial. Medical
Association.14:1176-1178.
NCCLS .(1990). Document, Performance Standards for Antimicrobial Disk Susceptibility Tests.
4th edition. 10:7.
NCCLS. (1996). Quality assurance of commercially prepared microbiological culture media.
2nd edition.
Pro-Lab Diagnostic (http://www.pro-lab.com/wp-content/uploads/2017/01/SD2300-
SD2350_en.pdf)
Washington, J.A., Warren, E., and Karlson, A.G. (1972). Stability of barium sulfate turbidity standards.
Applied Microbiology. 24:1013.