Detection limit of negative staining electron microscopy for the diagnosis of bioterrorism-related micro-organisms.
OBJECTIVE
To determine the detection limit of diagnostic negative staining electron microscopy for the diagnosis of pathogens that could be used for bioterrorism.


RESULTS
Suspensions of vaccinia poxvirus and endospores of Bacillus subtilis were used at defined concentrations as a model for poxviruses and spores of anthrax (Bacillus anthracis), both of which are pathogens that could be used for bioterrorist attacks. Negative staining electron microscopy was performed directly or after sedimentation of these suspensions on to the sample supports using airfuge ultracentrifugation. For both virus and spores, the detection limit using direct adsorption of a 10-μl sample volume onto the sample support was 10(6) particles per ml. Using airfuge ultracentrifugation with a sample volume of 80 μl, the detection limit could be reduced to 10(5) particles per ml for spores and to 5 × 10(4) particles per ml for poxviruses. The influence on particle detection of incubation time, washing and adsorption procedures was investigated.


CONCLUSIONS
The reproducibility and sensitivity of the method were acceptable, particularly considering the small sample volume and low particle number applied onto the sample support.


CONCLUSIONS
Diagnostic negative staining electron microscopy is used for the diagnosis of pathogens in emergency situations because it allows a rapid examination of all particulate matter down to the nanometre scale. This study provides precise detection limit for the method, an important factor for the validation and improvement of the technique.
Laue
M
M
Centre for Biological Safety 4, Robert Koch Institute, Berlin, Germany. michael.laue@med.uni-rostock.de
Bannert
N
N
D001412:Bacillus subtilis / Q000648:ultrastructure; D023482:Bioterrorism; D057230:Limit of Detection; D008854:Microscopy, Electron / Q000379:methods*; D016624:Negative Staining; D011212:Poxviridae / Q000648:ultrastructure; D013171:Spores, Bacterial / Q000648:ultrastructure; D014771:Virion / Q000648:ultrastructure
J Clin Microbiol. 1981 Aug;14(2):210-21
6268658
JAMA. 2002 May 1;287(17):2236-52
11980524
Lancet. 2003 May 17;361(9370):1730-3
12767752
MMWR Recomm Rep. 2004 Jun 11;53(RR-8):1-27
15192550
J Infect. 2006 Jul;53(1):70-4
16271397
Clin Chem. 2004 Feb;50(2):306-12
14684621
J Dent Res. 2002 Dec;81(12):841-4
12454099
Ultrastruct Pathol. 2003 May-Jun;27(3):133-40
12775503
J Virol Methods. 2006 Jun;134(1-2):92-8
16417929
Arch Exp Veterinarmed. 1978;32(3):427-34
697528
J Ultrastruct Res. 1970 Jul;32(1):107-17
4193902
Emerg Infect Dis. 1999 Jul-Aug;5(4):513-6
10458955
J Clin Virol. 1999 Jun;13(1-2):105-19
10405897
Emerg Infect Dis. 2003 Mar;9(3):294-303
12643823
Nat Med. 2001 Dec;7(12):1271-3
11726956
J Virol Methods. 1987 Jun;16(3):209-16
3654898
J Microbiol Methods. 2007 Jul;70(1):45-54
17466397
Arch Virol. 1980;64(4):311-8
6772142
J Clin Virol. 2007 Feb;38(2):165-8
17204452
J Cell Sci. 1981 Jun;49:383-99
7309811
J Virol Methods. 1984 Nov;9(3):185-92
6394602
J Virol. 1970 May;5(5):598-603
5419912
J Microsc. 1993 Aug;171(Pt 2):125-36
8411139
Biochim Biophys Acta. 1959 Jul;34:103-10
13804200
J Infect. 2007 Mar;54(3):307-8; author reply 308-9
16716403
Biochem J. 1969 Jun;113(1):29-37
4185146
Micron. 2006;37(2):91-106
16361103
Arch Gesamte Virusforsch. 1969;27(2):339-51
5369726
Appl Environ Microbiol. 2008 Jan;74(1):32-7
17965204
J Electron Microsc Tech. 1986;4(3):265-301
32336873