Journal of Magnetism and Magnetic Materials 122 (1993) 371-373

North-Holland A4"

A magnetic concentration method using hydrosol of ferric particles

for diagnosing tuberculosis
M.A. Vladimirsky ~, A.A. K u z n e t s o v b and V,I. P h i l i p p o v b
" Moscow Tubercuh)sis, Research Institute, Moscow, Russia
h hzstitute o f Chemical Physics, Russian Academy of Sciences, Moscow 117977, Russia

A method has been developed for rapid identification of M. tuberculosis. A suspension of finely dispersed ferromagnetic
particles links with the bacterial cells and makes possible a higher sensitivity of bacteria identification compared to the
standard techniques.

Identification of Mycobacterium tuberculosis real preparations, having good adhesion to M.

in the diagnostic specimens (sputum, urine) is the tuberculosis. We have not studied details of the
most reliable evidence of the active tuberculosis mechanism of this interaction, but it is interesting
process. The available techniques for identifica- to mention that our measurements of magnetic
tion of M. tuberculosis in smears (direct micro- susceptibility of M. tuberculosis have shown that
scopic observation with staining of acid-fast bac- susceptibility of Mycobacteria has an anomalous
teria or fluorescent microscopy using staining with shift to paramagnetism.
auramine and rhodamine) are not sensitive
enough while the more sensitive techniques, Methodology
namely, cultures, take too long, up to 4-8 weeks
[1]. The rapid and sensitive technique of multipli-
Finely dispersed iron powder was prepared by
cation of the DNA fragments (polymerase chain
the plasmocheomical method [6]. The particle size
reaction) developed in the last two years involves
was 100-500 A. The suspension of ferromagnetic
complicated analytical procedures [2].
particles in distilled water at a concentration of 3
Several reports have described the use of fer-
m g / m l was subjected to 22 kHz ultrasound treat-
romagnetic particles for separation and accumu-
ment for five minutes at a power of 150 W / c m 2.
lation of bacterial and cell suspension [3,4]. In
The sputum specimens were homogenized, lique-
ref. [5] it has been demonstrated with models of
fied and decontaminated with a 10% solution of
cell suspensions that ferromagnetic particles can
trisodium phosphate in the ratio 1"1 (v/v) and
be used for concentrating the M. tuberculosis
then neutralized by 0.1 N HCI. The hydrosol of
cells thus enhancing the sensitivity of fluorescent
ferromagnetic particles was added to the tested
microscopic bacterioscopy.
material in the ratio of 1:10 (v/v), then the
We have studied the use of finely dispersed
specimens were incubated at the room tempera-
ferromagnetic particles for concentrating M. tu-
ture for one hour and continuously stirred avoid-
berculosis from sputum specimens for subsequent
ing extreme agitation. The bacterial cells linked
identification of them by fluorescent microscopic
with ferromagnetic particles were concentrated
examination of auramine-rhodamine-stained
either by centrifugation at 1000 g for 15 minutes
smears.
or by a special magnetic concentration device
We have made a broad screening of highly
with a non-uniform magnetic field during 1
dispersed ferromagnetic particles and found opti-
minute. The sediment was placed on slides (slides
Correspondence to: Dr. A.A. Kuznetsov, Institute of Chemical
pre-treated with egg protein, are recommended),
Physics, Russian Academy of Sciences, Kosygin st. 4, Moscow which then were dried and fixed by heating at
117977, Russia. 70°C during 1 hour. The slides then were stained

(1304-8853/93/$06.00 © 1993 - Elsevier Science Publishers B.V. All rights reserved

372 M.A. Vladimir~'ky et al. / 14vdros'ol oj'.&rric partich,s .[~>rdiagnosing tuberculosis

by the fluorescent stain auramine-rhodaminc C, Table l

followed by decolorization by 3% hydrochloric
acid alcohol and counterstained by 0.025% mety-
lone blue. According to literature data [1] speci-
ficity of staining is determined by presence of
mycol acid on the cell surface. Concentration of
bacteria cells was determined by fluorescence mi-
croscopy scanning of this smears at 400 × magni-
fication for orange fluorescent Mycobacteria
against a dark background. Each slide was
scanned in 100 fields. The result was considered
as positive, if more than 3 MbT were observed at
the slide.
Experiments on M. tuberculosis determination
in model systems were performed with non-
bacterial sputum with addition of a known con-
centration of MbT by means of different tech-
niques: standard technique without use of the
ferromagnetic hydrosole; application of the hy-
drosole with concentrating by centrifugation or by
magnetic concentration by a special magnetic de-
vice. Further treatment of smears was as de-
scribed above, and the results of this experiments
are given in table 1. The results demonstrate the
effectiveness of the concentration technique with
the use of ferromagnetic hydrysol. The difference
between centrifugation and magnetic concentra-
tion is rather small, and therefore, during clinical
trials we used a method of centrifugation more
available in clinical laboratories.
Identification results for M. tuberculosis in model systems
obtained with the use of the standard smear technique and
the magnetic concentration technique
M. tuberculosis M. tuberculosis cells in the smear
cells in 1 cm ~
Standard Application of
of specimen technique ferromagnetic particles
Centrifugation Magnetic method
10 a 2 92 86
5 × 102 0 50 44
102 0 25 21
We havc examined sputum specimens taken
from 140 patients with various type of pulmonary
tuberculosis at various treatment stages. The
specimens were analyzed simultaneously with
three methods: cultures on a Lowenstein solid
medium, fluorescent microscopy of auramine-
stained smears, and a similar examination of
smears after aggregation of M. tuberculosis with
ferromagnetic particles.
The examination results are presented in fig.
1. The upper unshaded rectangle represents the
number of positive identifications of M. tubercu-
losis (68) for the Lowenstein culture technique.
The shaded rectangle below represents the num-
ber of positive results (48) obtained with tradi-
tional fluorescent microscopy. It can be seen that
the culture technique identified 26 cases of tuber-

i
I

!
, I
i I
t

q2 25"

Fig. 1. Number of tuberculosis positive cases identified with different techniques. 1 culture, I1 traditional fluorescent
microscopy, l l I - fluorescent microscopy with the use of ferromagnetic particles.
 M.A. Vladimirsky et al. / Hydrosol of ferric particles .for diagnosing tuberculosis
culosis infection which fluorescent microscopy
failed to identify. But the microscopic tests iden-
tified 6 positive cases which the culture technique
could not reveal. These cases, apparently, in-
volved unviable bacterial cells. The lower hori-
zontally shaded rectangle represents the cases
(88) identified by fluorescent microscopy of the
smears with the ferromagnetic hydrosol. Not only
almost all cases identified with the culture tech-
nique (with the exception of one) were identified
with the magnetic technique but also 21 more
positive cases were identified.
The use of ferromagnetic hydrosol for concen-
tration of M. tuberculosis from the sputum speci-
mens improved the sensitivity of rapid bacteria
identification with fluorescent microscopy in au-
ramine-stained smears by 83.3% in comparison
with the standard technique.
The identification efficiency of this method
373
was better by 29.4% than that of the long-term
culture technique.
The magnetic method in future will be useful
for identification tests employing other tech-
niques, including the polymerase chain reaction.
References
[1] T.N. Yashchenko and I.S. Mecheva, Manual of Labora-
tory Tests for Tuberculosis, Moscow, 1973 (in Russian).
[2] J. Sjobring et al., J. Chim. Microbiol. 28 (1990) 2200-2204.
[3] P. Kronic and R.W. Gilpin, Biochem. Biophys. Meth. 12
(1986) 56-58.
[4] L. Tor, Rapp Ingenjorsvetenkapsakad 355 (1988) 56-58.
[5] A.A. Rukhlina, V.I. Philippov, M.A. Vladimirskii, A.A.
Kuznetsov et al., Proc. 3rd Conf. on Application of Mag-
netic Liquids in Biology and Medicine (SukhumL 1989)
112-113.
[6] S.I. Malashin, E.K. Dobrinskii, N.P. Glukhoedov, V.G.
Gerlivanov et al., ibid., 69-70.