Diagnostic Test for Bacillus: Ascoli's Thermo Precipitation Test

Topics: Bacillus, Bacteria, Bacilli Pages: 6 (1533 words) Published: February 24, 2013
GROUP No.: | DATE: 02/14/13| |
NAME: | |
EXPERIMENT No.25BACILLUS: Diagnostic Tests| |

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 of nutrient broth containing 0.1% histidine and, after incubation at 37°C for 7 days, subculture on to blood or nutrient agar. The dose regimen for the formalized suspension after initial vaccination on days 1 and 14 is increasing doses of 0.1, 0.5, 1, and 2 ml given intravenously at intervals of 4–5 days. Expected results:

* Positive: Whitish ring appear at the junction of the two fluids * Negative: No whitish ring appear at the junction of the two fluids Consideration:
The test is not suitable for detection of B. anthracis in environmental specimens; numerous other Bacillus species can be expected to occur in these. GELATIN LIQUEFACTION/GELATIN HYDROLYSIS
Purpose: used to identify Bacillus anthracis and Bacillus cereus which yield positive result in this test. Principle:
This test is used to determine the ability of an organism to produce proteolytic enzymes (gelatinase) that liquefy gelatin. Procedure:
1. Inoculate gelatin deep with 4 to 5 drops of a 24-hour broth culture. 2. Incubate at 35⁰C in ambient air for up to 14 days. Note: incubate the medium at 25⁰C if the organism grows better at 25⁰C than at 35⁰C. 3. Alternatively, inoculate the gelatin deep from a 24-hour-old colony by stabling 4 to 5 times ½ inch into the medium. 4. Remove the gelatin tube daily from the incubator and place at 4⁰C to check for liquefaction. Do not invert or tip the tube, because sometimes the only discernible liquefaction will occur at the top of deep where inoculation occurred. 5. Refrigerate an uninoculated control along with the inoculated tube. Liquefaction is determined only after the control has hardened (gelled). Expected results:

* Positive: partial or total liquefaction of the inoculated tube at 4⁰C within 14 days. * Negative: complete solidification of tube at 4⁰C
Quality control:
* Positive: Proteus vulgaris
* Negative: Enterobacteraerogenes
Purpose: used to identify Bacillus anthracis which yield positive result...

References: Forbes, B., Sahm, D., &Weissfeld, A.(2007).Bailey & Scott’s Diagnostic Microbiology
12thed.Philadelphia, USA: Mosby Incorporation
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