Diagnostic Test for Bacillus: Ascoli's Thermo Precipitation Test
GROUP No.: | DATE: 02/14/13 | | NAME: | | EXPERIMENT No.25BACILLUS: Diagnostic Tests | |
ASCOLI’S THERMOPRECIPITIN TEST
Purpose: used to identify anthrax bacilli in animal hides and meat.
Principle:
This test was designed to detect B. anthracis antigens in the tissues of animals being utilized in animal by-products and thereby to reveal when these products contained ingredients originating from animals that had died of anthrax. The thermostable antigens involved are common to other Bacillus species so the test depends on the fact that the only Bacillus likely to have proliferated within and throughout an animal depositing extensive precipitating antigens in the tissues is B. anthracis.
Procedure:
1. Chop or slice the specimen into fine pieces or strips. 2. Boil approximately 2g of the specimen for 5 minutes in 5mL saline containing 1:100 (final concentration) acetic acid. Alternately soak in saline containing 0.5% phenol for 24-48 hours in a refrigerator. 3. After cooling, filter through filter paper until completely clear. 4. Insert a few drops of antiserum in bottom of a small test tube and carefully add some of the filtrate down the side of the tube to form a layer of antigen above antiserum. 5. Include appropriate positive and negative specimen controls.
Reagents used: * Acetic acid * Saline containing 0.5% phenol * Anthrax antiserum:
Antiserum is prepared in rabbits by the subcutaneous Inoculation of Sterne anthrax vaccine on days 1 and 14. On days 28 and 35, the rabbits receive 0.5 mL of a mixture of several strains of virulent B. anthracis not exceeding 105 colony-forming units (CFU)/ml suspended in saline. Alternatively, the live virulent bacteria can be inactivated by prolonged suspension in 0.2% formalized saline, but the antigen mass needs to be increased to 108 –109 CFU/ml. The suspension should be checked for inactivation of the B. anthracis before animal inoculation by culture of 0.1 ml into 100 ml
ASCOLI’S THERMOPRECIPITIN TEST
Purpose: used to identify anthrax bacilli in animal hides and meat.
Principle:
This test was designed to detect B. anthracis antigens in the tissues of animals being utilized in animal by-products and thereby to reveal when these products contained ingredients originating from animals that had died of anthrax. The thermostable antigens involved are common to other Bacillus species so the test depends on the fact that the only Bacillus likely to have proliferated within and throughout an animal depositing extensive precipitating antigens in the tissues is B. anthracis.
Procedure:
1. Chop or slice the specimen into fine pieces or strips. 2. Boil approximately 2g of the specimen for 5 minutes in 5mL saline containing 1:100 (final concentration) acetic acid. Alternately soak in saline containing 0.5% phenol for 24-48 hours in a refrigerator. 3. After cooling, filter through filter paper until completely clear. 4. Insert a few drops of antiserum in bottom of a small test tube and carefully add some of the filtrate down the side of the tube to form a layer of antigen above antiserum. 5. Include appropriate positive and negative specimen controls.
Reagents used: * Acetic acid * Saline containing 0.5% phenol * Anthrax antiserum:
Antiserum is prepared in rabbits by the subcutaneous Inoculation of Sterne anthrax vaccine on days 1 and 14. On days 28 and 35, the rabbits receive 0.5 mL of a mixture of several strains of virulent B. anthracis not exceeding 105 colony-forming units (CFU)/ml suspended in saline. Alternatively, the live virulent bacteria can be inactivated by prolonged suspension in 0.2% formalized saline, but the antigen mass needs to be increased to 108 –109 CFU/ml. The suspension should be checked for inactivation of the B. anthracis before animal inoculation by culture of 0.1 ml into 100 ml
References: Forbes, B., Sahm, D., &Weissfeld, A.(2007).Bailey & Scott’s Diagnostic Microbiology 12thed.Philadelphia, USA: Mosby Incorporation http://www.oie.int/fileadmin/home/eng/health_standards/tahm/2.01.01_anthrax.pdf http://ahd.kerala.gov.in/index.php/livestockdiseases?start=3 http://www.who.int/csr/resources/publications/anthrax/whoemczdi986text.pdf http://books.google.com.ph/books?id=EKYihvnaA7oC&pg=PA136&lpg=PA136&dq=ascoli%E2%80%99s+thermo+precipitation+test+procedure&source=bl&ots=EghgCCoqbO&sig=t0pQ_LpGC0HLOqsod228WGWYP5k&hl=en&sa=X&ei=tF4bUZ7xIsftrAf584CQBw&ved=0CGIQ6AEwCA#v=onepage&q&f=false http://www.austincc.edu/microbugz/starch_hydrolysis.php http://www.microbelibrary.org/index.php?option=com_resource&controller=article&article=3172&Itemid=0&category_id=1 http://www.mesacc.edu/~johnson/labtools/Dbiochem/nit.html