Gram positive spore forming bacilli o Remove daily and place

at 4 deg. C
Bacillus species Positive result: partial or complete
Specimen: Bacillus anthracis liquefaction
Skin lesion Liquefaction is confirmed of the
Blood control tube has solidified at 4
Lymph node aspirates deg. C within 14 days
Specimen: Bacillus cereus Positive organism- Bacillus cereus
Stool ( complete liquefaction)
Gram stain reaction: bacillus anthracis Positive- Bacillus anthracis
(Serpentine) ( inverted fir tree appearance due
to slow liquefaction)
Culture media 2. String of pearls test
5% sheep blood agar: bacillus Purpose: determine the
anthracis organisms susceptibilty to
Medium-large penicillin
Gray flat Procedure:
Medusa head o MHA
Non-hemolytic o 10 units of penicillin
5% sheep blood agar: bacillus
cereus o Inoculate MHA
Large o Apply a cover slip
Feathery o Incubate at 35-37 deg.
Spreading C for 3-6 hours
Beta-hemolytic o Focus under the
Chocolate agar microscope
Phenylethyl alcohol agar Positive result: morphology
Selective for B. Anthracis : resembles a string of pearls (cell
Polymixin lyzozyme EDTA wall disappears)
thallous acetate 3. Ascoli test
o Polymixin inhibits g+ Serological test
bacteria Uses heat extracted antigen from
o Lyzozymes inhibit the organism
vegetative cells Procedure:
o Thallous acetate inhibit o Tissue of infected animal is
both gram + and boiled in NSS for 5-10 min
bacteria then filter
Contains: o Heat extracted antigen
o 1 polymixins o Filtrate(source of the
o 2 lysozymes antigen): overlay with
o 3 ethylene dioxide anthrax antiserum
Plet AGAR Positive result: white precipitin
Bicarbonate agar: whitish colonies line at the junction
4. Gamma phage lysis test
that are mucoid
Lysis of the organism
Biochemical test Susceptible: positive result
1. Gelatin hydrolysis (Bacillus anthracis)
Purpose: determine the ability 5. To differentiate B. cereus
of organism to produce Motile
gelatinase Beat hemolytic
Gelatin ---> polypeptides----> Grows at 45 deg. C
amino acids (through action Growth in PEA
of gelatinase) Positive gelatin hydrolysis
Procedure Salicin fermenter
o Inoculate the gelatin Negative phage lysis
o Incubate at 35-37 deg.
C
 Gray to grayish yellow
Circular, glossy
Double zone of beta hemolysis
Anaerobic blood agar: C. tetani
Gray
Matte surface
Rhizoid margin
Narrow beta hemolysis
Swarming
Anaerobic blood agar : C.
Botulinum
Clostridium group Gray- white
Circular to irregular
Clostridium perfringes Beta- hemolytic
Isolated from: Anaerobic blood agar: C. defficile
Stool Large white, glossy matte
Wound material Circular, convex
Clostridium tetani: Horse-stable odor
Difficult to isolate Gamma hemolysis
Clostridium botulinum Anaerobic PEA
Cannot be isolated Egg yolk agar
Clostridium difficile Thioglycollate broth
Stool (Spore formers gram positive bacilli:
diagram in bailey and scott)
Gram stain
C. perfringes: G (+) Selective media: CCFA (cycloserine
C. tetani: tennis racket cefoxitin fructose agar) for C. Difficile
C. botulinum:
C. defficile: subterminal spores Biochemical test
Naglers test
Culture media
Anaerobic blood agar: C.
perfringes