APPENDIX 6

(a) McFARLAND NEPHELOMETER BARIUM SULFATE STANDARDS (FROM

LENETTE ET AL., 1974)

1. Prepare 1% aqueous barium chloride and 1% aqueous sulfuric

acid solutions.

2. Add the amounts indicated in Table A6.1 to clean dry

ampoules. Ampoules should have the same diameter as the

test tube to be used in the subsequent density

determinations.

3. Seal the ampoules and label them.

Table A.3. Preparation of McFarland nephelometer barium sulfate

standards.
Barium Sulfuric Corresponding approx.
chloride acid density of bacteria
Tube 1% (ml) 1% (ml) (million/ml)
1 0.1 9.9 300
2 0.2 9.8 600
3 0.3 9.7 900
4 0.4 9.6 1,200
5 0.5 9.5 1,500
6 0.6 9.4 1,800
7 0.7 9.3 2,100
8 0.8 9.2 2,400
9 0.9 9.1 2,700
10 1.0 9.0 3,000
(b) TURBIDITY ADJUSTMENT OF THE BACTERIAL SUSPENSION

For bacterial agglutinations, the cell suspension is usually

adjusted to approximately 1x109 cells/ml. In the McFarland

standards, tubes 3 and 4 will have approximately 9.0x108 (1x109)

and 1.2x109 cells/ml respectively. The arbitrary selection of

these two densities will yield satisfactory results for many

systems.

With dust-free saline in a tube (blank) similar in diameter to

the standards, set the nephelometer to a low nephelometric

unitage. Read the corresponding unitage on tubes 3 or 4.

With approximately 8 ml of saline in another clean tube, add the

turbid washed suspension of rhizobial cells dropwise with a

Pasteur pipette until a turbidity is reached which is slightly

lower than the corresponding standard chosen. Place the tube in

the nephelometer and adjust the turbidity to the required unitage

by further additions of the turbid rhizobial suspension.

If a nephelometer is not available, the turbidity is adjusted to

fall between tubes 3 and 4 by visual comparison.