Journal of Microbiological Methods 47 2001.

369371
www.elsevier.comrlocaterjmicmeth

Note

Use of magnetic beads for Gram staining of bacteria

in aqueous suspension
Siamak P. Yazdankhah a,) , Henning Srum a , Hans J.S. Larsen a , Geir Gogstad b
a
Department of Pharmacology, Microbiology and Food Hygiene, Norwegian School of Veterinary Science,
P.O. Box 8146 Dep, N-0033, Oslo, Norway
b

Procaryo AS, Kjelsaseien 172, 0884, Oslo, Norway
Received 18 July 2001; received in revised form 30 August 2001; accepted 18 September 2001

Abstract

A Gram staining technique was developed using monodisperse magnetic beads in concentrating bacteria in suspension for
downstream application. The technique does not require heat fixation of organisms, electrical power, or a microscope.
Gram-negative and Gram-positive bacteria were identified macroscopically based on the colour of the suspension. The
bacteria concentrated on magnetic beads may also be identified microscopically. q 2001 Elsevier Science B.V. All rights
reserved.

Keywords: Bacteria; Gram staining; Magnetic beads

As a basic method, Gram staining of bacteria
segregates bacteria into two categories based on cell
wall composition. Gram-negative bacteria possess a
bilayered outer membrane, a thin peptidoglycan layer,
and a bilayered plasma membrane Beveridge and
Graham, 1991.. The cell envelope of Gram-positive
bacteria consists of a cytoplasmic membrane, many
polymer layers of peptidoglycan connected by amino
acid bridges, and a variable outer layer called the
capsule Jawets et al., 1987..
The Gram stain procedure developed by Hans
Christian Gram in 1884 relies on the differential cell
) technique for unfixed organisms in suspension has
Corresponding author. Tel.: q47-22-96-49-95; fax: q47-22-
96-48-18.
E-mail address: siamak.p.yazdankhah@veths.no
S.P. Yazdankhah..
wall staining properties of Gram-positive and Gram-
negative bacteria. It remains today as an almost
fundamental step in bacterial classification. The reac-
tion is based on the retention of a blue-violet colour
within the cell wall of Gram-positive organisms
crystal violet complexed with iodine. following an
alcohol wash. Those bacteria that do not retain the
blue-violet stain Gram-negative. are counterstained
with carbolfuchsin or safranin, and are therefore pink
in colour.
Alternative bacterial staining methods have been
developed using fluorescence-labeled wheat germ
agglutinin Sizemore et al., 1990. and rhodamine
123, a lipophilic cationic dye Allman et al., 1993;
Shapiro, 1995; Davey and Kell, 1996.. A staining
been developed Mason et al., 1998.. The technique
employs two fluorescent nucleic acid binding dyes,
hexidium iodide and SYTO 13. All of these alter-

0167-7012r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 7 - 7 0 1 2 0 1 . 0 0 3 5 0 - 5
370 S.P. Yazdankhah et al.r Journal of Microbiological Methods 47 (2001) 369371
native-staining methods are more expensive than tra-
ditional Gram staining and require expensive instru-
ments such as epifluorescence microscopes or flow
cytometers.
In this study, we have developed a method using
tosylactivated monodisperse magnetic beads, which
bind to the bacteria in a culture suspension. The
bacteria concentrating on the surface of magnetic
beads were stained as with traditional Gram staining
and could easily be visualised Fig. 1..
The method for production of monodisperse mag-
netic beads has been described elsewhere Ugelstad
et al., 1980.. Generally, magnetic beads are coated
by polymeric compounds with affinity to biological
molecules. The large surface area of magnetic beads
increases their binding capacity. Magnetic beads used
in this study are polysterene beads activated by p-
toluenesulfonyl chloride treatment to enable chemi-
Fig. 1. Simple diagram of the magnetic bead-based Gram staining
of bacteria.
cal binding of aminogroups carrying aminogroups
such as proteins, peptides, and glycoproteins Anon-
ymous, 1994..
One colony from fresh culture of each bacterial
species was inoculated into Luria Bertani LB. or
Heart Infusion HI. broth Difco, Detroit, MI., and
incubated at 37 8C overnight. Fifty microliters 10
mgrml. tosylactivated monodisperse magnetic parti-
cles Dynabeads M280, Dynal, Oslo, Norway. was
added to 1.25 ml bacterial culture, in a 1.5 ml
eppendorf microtube, and incubated at room temper-
ature 20 8C. for 10 min with end-over-end mixing.
Uninoculated LB or HI medium was used as a
negative control. After incubation, the beads were
washed once with 1 ml distilled water. The beads
were resuspended in 200 ml of 500 mgrl methyl
violet Sigma, MO. and incubated at room tempera-
ture for 1 min. After incubation, the beads were
washed with 1 ml distilled water. The beads were
resuspended in 200 ml of 2500 mgrl iodine Sigma.
and incubated at room temperature for 30 s, and
subsequently washed with 200 ml ethanol 96%.
followed by washing with distilled water. Concen-
trated beads were resuspended in 100 ml of 1000
mgrl carbolfuchsin Sigma. and incubated at room
temperature for 30 s, and then washed in 1 ml
distilled water. Washing steps consisted of concen-
trating the beads for 30 s in a Magnetic Particle
Concentrator for microtubes MPC w -M, Dynal., fol-
lowed by aspiration of the supernatant. Finally, the
beads were resuspended in 100 ml distilled water,
and then concentrated on the wall of the microtube
by using MPC for microtubes. Bacterial cultures,
which retained the violet colour in supernatant, were
termed Gram-positive, those staining pink from the
carbolfuchsin counterstain were termed Gram-nega-
tive. Supernatant of negative controls appeared
colourless.
For microscopic examination, a 10 ml magnetic
beadsbacteria suspension was transferred to a glass
slide, covered by a cover glass and added with one
drop of immersion oil before viewing =100..
The technique was applied to a range of bacterial
suspensions and correctly identified the Gram stain
of overnight cultured suspensions of the following
clinically relevant bacteria: Staphylococcus aureus
ATCC 25923, S. intermedius, Streptococcus dys-
galactiae, S. zooepidemicus, Escherichia coli, Pas-
 S.P. Yazdankhah et al.r Journal of Microbiological Methods 47 (2001) 369371
teurella multocida, Pseudomonas aeruginosa, Bacil-
lus subtilis, and Proteus mirabilis.
In this study, the bacteria are separated from the
bacterial culture and concentrated from a large vol-
ume 1.25 ml. to a volume suitable for downstream
application. The sample volume may be increased,
aiming to increase the sensitivity of the technique.
This item may be used in samples with low concen-
tration of bacteria in suspension. The concentrated
bacteria on beads may also be enriched in a nutrient
and selective broth, depending on the bacterial strain,
before Gram staining.
For further identification of bacteria, the bacteria
concentrated on beads may be used to perform rapid
biochemical analysis, e.g. oxidase, catalase, etc.
In traditional Gram staining on glass slide, smears
may not be properly fixed by heating and tend to be
washed away during staining and washing. Bead-
based staining of bacteria does not require heat
fixation. Tosylactivated magnetic beads strongly bind
to bacteria, avoiding losses during staining and wash-
ing. The colourless result of negative controls con-
firmed that the nonspecific binding between beads
and the chemicals involved in Gram staining is
negligible.
Other commercially available particles may also
be used in this study. Although magnetic bead parti-
cles add a certain advantage related to the smooth
conduction of the procedure, bead-based Gram stain-
ing of bacteria in aqueous suspension has the poten-
tial to be applied in clinical and environmental
microbiology, and in field studies with limited labo-
ratory facilities.
371
Acknowledgements
This work was supported by Oslo Research Park,
Procaryo AS and VESO National Centre for Veteri-
nary Contract Research and Commercial Services.,
Oslo, Norway.
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