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Journal of Microbiological Methods 47 2001.



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
Department of Pharmacology, Microbiology and Food Hygiene, Norwegian School of Veterinary Science,
P.O. Box 8146 Dep, N-0033, Oslo, Norway

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


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

Keywords: Bacteria; Gram staining; Magnetic beads

As a basic method, Gram staining of bacteria wall staining properties of Gram-positive and Gram-
segregates bacteria into two categories based on cell negative bacteria. It remains today as an almost
wall composition. Gram-negative bacteria possess a fundamental step in bacterial classification. The reac-
bilayered outer membrane, a thin peptidoglycan layer, tion is based on the retention of a blue-violet colour
and a bilayered plasma membrane Beveridge and within the cell wall of Gram-positive organisms
Graham, 1991.. The cell envelope of Gram-positive crystal violet complexed with iodine. following an
bacteria consists of a cytoplasmic membrane, many alcohol wash. Those bacteria that do not retain the
polymer layers of peptidoglycan connected by amino blue-violet stain Gram-negative. are counterstained
acid bridges, and a variable outer layer called the with carbolfuchsin or safranin, and are therefore pink
capsule Jawets et al., 1987.. in colour.
The Gram stain procedure developed by Hans Alternative bacterial staining methods have been
Christian Gram in 1884 relies on the differential cell 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
) technique for unfixed organisms in suspension has
Corresponding author. Tel.: q47-22-96-49-95; fax: q47-22-
been developed Mason et al., 1998.. The technique
E-mail address: siamak.p.yazdankhah@veths.no employs two fluorescent nucleic acid binding dyes,
S.P. Yazdankhah.. 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- cal binding of aminogroups carrying aminogroups
ditional Gram staining and require expensive instru- such as proteins, peptides, and glycoproteins Anon-
ments such as epifluorescence microscopes or flow ymous, 1994..
cytometers. One colony from fresh culture of each bacterial
In this study, we have developed a method using species was inoculated into Luria Bertani LB. or
tosylactivated monodisperse magnetic beads, which Heart Infusion HI. broth Difco, Detroit, MI., and
bind to the bacteria in a culture suspension. The incubated at 37 8C overnight. Fifty microliters 10
bacteria concentrating on the surface of magnetic mgrml. tosylactivated monodisperse magnetic parti-
beads were stained as with traditional Gram staining cles Dynabeads M280, Dynal, Oslo, Norway. was
and could easily be visualised Fig. 1.. added to 1.25 ml bacterial culture, in a 1.5 ml
The method for production of monodisperse mag- eppendorf microtube, and incubated at room temper-
netic beads has been described elsewhere Ugelstad ature 20 8C. for 10 min with end-over-end mixing.
et al., 1980.. Generally, magnetic beads are coated Uninoculated LB or HI medium was used as a
by polymeric compounds with affinity to biological negative control. After incubation, the beads were
molecules. The large surface area of magnetic beads washed once with 1 ml distilled water. The beads
increases their binding capacity. Magnetic beads used were resuspended in 200 ml of 500 mgrl methyl
in this study are polysterene beads activated by p- violet Sigma, MO. and incubated at room tempera-
toluenesulfonyl chloride treatment to enable chemi- 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
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
Fig. 1. Simple diagram of the magnetic bead-based Gram staining ATCC 25923, S. intermedius, Streptococcus dys-
of bacteria. galactiae, S. zooepidemicus, Escherichia coli, Pas-
S.P. Yazdankhah et al.r Journal of Microbiological Methods 47 (2001) 369371 371

teurella multocida, Pseudomonas aeruginosa, Bacil- Acknowledgements

lus subtilis, and Proteus mirabilis.
In this study, the bacteria are separated from the This work was supported by Oslo Research Park,
bacterial culture and concentrated from a large vol- Procaryo AS and VESO National Centre for Veteri-
ume 1.25 ml. to a volume suitable for downstream nary Contract Research and Commercial Services.,
application. The sample volume may be increased, Oslo, Norway.
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 References
bacteria on beads may also be enriched in a nutrient
and selective broth, depending on the bacterial strain, Allman, R.R., Manchee, R., Lloyd, D., 1993. Flowcytometric
before Gram staining. analysis of heterogeneous bacterial populations. In: Lloyd, D.
For further identification of bacteria, the bacteria Ed.., Flowcytometry in Microbiology. Springer Verlag, Lon-
concentrated on beads may be used to perform rapid don, England, pp. 2747.
biochemical analysis, e.g. oxidase, catalase, etc. Anonymous, 1994. Dynabeads w M-280 tosylactivated. Product
description. Dynal, Oslo Norway
In traditional Gram staining on glass slide, smears Beveridge, T.J., Graham, A.A., 1991. Surface layers of bacteria.
may not be properly fixed by heating and tend to be Microbiol. Rev. 55, 684705.
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