title: Detection limit of negative staining electron microscopy for the diagnosis of bioterrorism-related micro-organisms.
abstract: 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.
authors.affiliation: Centre for Biological Safety 4, Robert Koch Institute, Berlin, Germany. michael.laue@med.uni-rostock.de
authors.forename: M
authors.initials: M
authors.lastname: Laue
authors.forename: N
authors.initials: N
authors.lastname: Bannert
mesh_terms: 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
references.citation: J Clin Microbiol. 1981 Aug;14(2):210-21
references.pmid: 6268658
references.citation: JAMA. 2002 May 1;287(17):2236-52
references.pmid: 11980524
references.citation: Lancet. 2003 May 17;361(9370):1730-3
references.pmid: 12767752
references.citation: MMWR Recomm Rep. 2004 Jun 11;53(RR-8):1-27
references.pmid: 15192550
references.citation: J Infect. 2006 Jul;53(1):70-4
references.pmid: 16271397
references.citation: Clin Chem. 2004 Feb;50(2):306-12
references.pmid: 14684621
references.citation: J Dent Res. 2002 Dec;81(12):841-4
references.pmid: 12454099
references.citation: Ultrastruct Pathol. 2003 May-Jun;27(3):133-40
references.pmid: 12775503
references.citation: J Virol Methods. 2006 Jun;134(1-2):92-8
references.pmid: 16417929
references.citation: Arch Exp Veterinarmed. 1978;32(3):427-34
references.pmid: 697528
references.citation: J Ultrastruct Res. 1970 Jul;32(1):107-17
references.pmid: 4193902
references.citation: Emerg Infect Dis. 1999 Jul-Aug;5(4):513-6
references.pmid: 10458955
references.citation: J Clin Virol. 1999 Jun;13(1-2):105-19
references.pmid: 10405897
references.citation: Emerg Infect Dis. 2003 Mar;9(3):294-303
references.pmid: 12643823
references.citation: Nat Med. 2001 Dec;7(12):1271-3
references.pmid: 11726956
references.citation: J Virol Methods. 1987 Jun;16(3):209-16
references.pmid: 3654898
references.citation: J Microbiol Methods. 2007 Jul;70(1):45-54
references.pmid: 17466397
references.citation: Arch Virol. 1980;64(4):311-8
references.pmid: 6772142
references.citation: J Clin Virol. 2007 Feb;38(2):165-8
references.pmid: 17204452
references.citation: J Cell Sci. 1981 Jun;49:383-99
references.pmid: 7309811
references.citation: J Virol Methods. 1984 Nov;9(3):185-92
references.pmid: 6394602
references.citation: J Virol. 1970 May;5(5):598-603
references.pmid: 5419912
references.citation: J Microsc. 1993 Aug;171(Pt 2):125-36
references.pmid: 8411139
references.citation: Biochim Biophys Acta. 1959 Jul;34:103-10
references.pmid: 13804200
references.citation: J Infect. 2007 Mar;54(3):307-8; author reply 308-9
references.pmid: 16716403
references.citation: Biochem J. 1969 Jun;113(1):29-37
references.pmid: 4185146
references.citation: Micron. 2006;37(2):91-106
references.pmid: 16361103
references.citation: Arch Gesamte Virusforsch. 1969;27(2):339-51
references.pmid: 5369726
references.citation: Appl Environ Microbiol. 2008 Jan;74(1):32-7
references.pmid: 17965204
references.citation: J Electron Microsc Tech. 1986;4(3):265-301
references.pmid: 32336873