Laboratory Procedures

Risk assessment

B. anthracis is a Hazard Group 3 pathogen, and should thus be covered by existing risk assessments for handling such organisms in diagnostic microbiological laboratories. Note that blood samples from anthrax patients for clinical chemistry and haematology pose no special risk and can be handled according to normal procedures.

Receipt of samples

Samples should have been labelled as 'High risk' by the submitting staff, and should be handled according to local protocols for such samples. All laboratory procedures should be performed, by experienced scientists, in a Containment Level 3 facility using a Class 1 protective safety cabinet. Chain-of-evidence documentation should accompany specimens. In larger incidents, this would only be required for several of the initial cases.

Protection of laboratory staff

All laboratory procedures should be performed in a Containment Level 3 facility using a Class 1 biological safety cabinet. Under these circumstances there is no indication for antibiotic prophylaxis for laboratory staff unless there is an inoculation injury or a spillage releasing aerosols containing spores. Anthrax vaccine is only indicated for laboratory staff who routinely work with the organism.

Any member of laboratory staff, working with specimens or cultures of anthrax, who develops a febrile/respiratory illness should seek urgent medical attention.

Isolation and identification

Two smears should be made on microscope slides and fixed by immersion in absolute ethanol for 1 minute. Slide 1 should be stained with Giemsa or Gram's stain, and the typical capsulated short chains of "box-car" bacilli looked for under oil immersion. Their presence is highly suggestive of anthrax. If numerous bacilli in short chains are visible, dispatch the second slide to a reference laboratory for confirmation. The specimens should also be cultured on to blood agar for incubation at 37°C in air/CO2. Antimicrobial susceptibility tests must be set up as soon as possible.

Culture

B. anthracis is a non-motile, Gram-positive, aerobic bacillus 1.2 to 10µm in length, capable of forming central and terminal spores. Cultures should be inoculated onto an agar slope in a bijoux bottle and incubated overnight. After incubation, the typical white, non-haemolytic colonies, with bees-eye appearance (that is, oval, slightly granular but not dry, about 2-5mm diameter) and characteristically tacky on teasing with a loop, will be apparent in large numbers. (Click here for laboratory images ).

These can be subcultured to a slope in a Class 1 protective cabinet within a Containment Level 3 facility that can then sent to the Reference Laboratory for confirmation.

Follow this link for information on the laboratory investigation of possible anthrax in drug users.

Antibiotic sensitivity

Organisms should be tested for sensitivity to antibiotics including ciprofloxacin, penicillin, doxycycline and gentamicin.

Confirmation

Clinical microbiology laboratories should take care not to regard all isolates of Bacillus species as contaminants, especially if isolated from sterile sites (blood, cerebrospinal fluid) and/or multiple cultures are positive from the same patient.

All sterile site Bacillus isolates should be further evaluated, and if non-motile or non-haemolytic (particularly if they form short chains), and/or if the clinical syndrome is suggestive of anthrax, the isolates should be immediately referred to reference laboratory.

Waste disposal

In the laboratory, hypochlorite (10,000ppm) disinfection is necessary for decontaminating surfaces that may have been exposed to B. anthracis spores. All other waste containers should be autoclaved.

Reference laboratory

All positive isolates and cultures should be sent to the reference laboratory for confirmation. In addition, samples may be sent there directly if local laboratories lack the facilities for dealing with them. All samples and cultures must be packaged appropriately, taking care to observe the procedures outlined in transport of clinical specimens. The Reference Laboratory should be telephoned prior to sending to expect the sample. Samples should be forwarded urgently to:

HPA Centre for Emergency Preparedness and Response
Special Pathogens Reference Unit
Porton Down, Salisbury
Wiltshire, SP4 0JG

Tel: ( 44) 01980 612100 (24hours)

Last reviewed: 24 December 2010