Experiments 5-8 Flashcards
Listeria blackening of medium
Hydrolysis of esculin
Product (esculetin) reacts with ferric iron
Creates black/brown complex
Listeria acidification
Fermentation of mannitol changes color from red to yellow
PALCAM
Selectivity PALCAM
LiCl
Polymyxin B Sulfate
Acriflavine
Ceftazidime
Differentiation PALCAM
Listeria
Esculin
Mannitol
Phenol Red
EB selective (Listeria)
Nalidixic acid
Acriflavin
Cycloheximide
Catalase reaction
2 H2O2 –> 2 H2O + O2
Alpha hemolysis
Cloudy zone under colony
Beta hemolysis
Clear zone around colony
Gamma hemolysis
No hemolysis
TASYE purpose
Further purification
Listeria Characteristics
Small
Catalase +
Gram +
Non spore forming
Rods
Issue with antibiotic residues
Inhibition of growth of lactic acid bacteria
Mastitis
Bacterial infection of mammary glands
Beta lactam antibiotics
Bind to peptidoglycan and inhibit cell wall synthesis
Penicillin
Ampicillin
Amoxicillin
Cephalosporin
Antibiotic regulations
Cow cannot be supplemented with antibiotics for 48 hours prior to milking
All milk that is shipped from farm to factory to be tested for antibiotics
Antibiotic tests
Disc assay
Delvo P
Delvo P test
If antibiotic is not present: media changes from purple to yellow (shows growth via acid production)
BSDA:
Negative for inhibitory substances
No zone around disk containing untreated milk
BSDA:
Positive for β-lactam residue
A zone around disk containing untreated milk but no zone around disk containing penicillinase-treated milk
BSDA:
Presence of inhibitors other than β-lactam residues
Clear zone of equal size around both disks
BSDA:
Presence of β-lactam residues as well as another inhibitor(s)
Clear zone of 4 mm around penicillinase-treated milk smaller than that around untreated milk disk
Penicillin positive control
Clear, well defined zones
16-20 mm
Antibiotic assay controls
PM
PM + A
PM + A + B
Vehicle of transmissions for listeriosis
Consumption of contaminated food products
Listeria plating order
Enrichment broth (selective)
PALCAM (black halo)
TASYE (purify)
Listeria Blood Agar results (if pathogenic)
Narrow zone of clearing around colonies
Hemolytic
Antibiotics select for
Bacteria that have acquired mutations/genes that confer antibiotic resistance
Beta lactamase is
An enzyme that some bacteria produce that renders them resistant to beta lactam antibiotics
Sulfonamides
A group of antibiotics
Lactic ring has
1 x N and 3 x C
Organisms used as standard test for antibiotic residues
Bacillus stearothermophilus
Lactococcus lactis
Delvo P
Result: purple medium
An antibiotic is present, no growth of B. stearothermophilus will occur
LST MUG Assay
Based on presence of GUD
GUD cleave MUG
Creates MU (fluorescent)
Identify coliforms by gas production from lactose
Methyl Red
1-2 drops of methyl red
MR VP broth
Methyl Red: Red color
Mixed acid fermentation
pH < 4.4
Methyl Red: Yellow color
Butanediol fermentation
pH > 6.2
Methyl Red: Orange color
equivocal reaction
pH 4.4 - 6.2
VP Test
Detect presence of acetoin
α-naphthol reagent
KOH
MR VP broth
VP: red color
Positive reaction
Presence of acetoin (neutral end product)
Indole test
Kovac’s reagent
Tryptone broth
Determine the ability of an organism to split tryptophan to form indole