| Limulus Amoebocyte Lysate (LAL) test for Endotoxin | - LAL: extract of horseshoe crab's blood
- detection of gram neg. bacteria
- Lipopolysaccharide (LPS) layer of g- bacteria is called endotoxin, is pyrogenic (causes fever), causes clotting of LAL |
| Limulus Amoebocyte Lysate (LAL) test for Endotoxin: pros and cons | advantages:
- sensitive test
- small quantities of endotoxin and low numbers of gram neg bacteria detected
- rapid evaluation of foods spoiled by gram neg bacteria
disadvantages:
- detects viable and non-viable gram neg bacteria
- gram positive bacteria NOT detected |
| Thermostable Nuclease | - detects pathogenic Staphylococcus aureus
- thermostable nuclease has a high correlation with enterotoxin producing S. aureus
- can withstand boiling for 15 min
- degrades DNA |
| Thermostable Nuclease testing procedure | 1. Boil sample for 15 min, then cool to room temperature
2. cut wells in Thermonuclease agar
3. Fill well with sample
4. Incubate (37 C) agar plate for 2-4 hours
5. S. aureus positive wells will form a pink ring in agar |
| Thermostable Nuclease: advantages | advantages:
- rapid
- more sensitive than enterotoxin detection test
- thermostable nuclease withstands heat treatment as do S. aureus enterotoxin |
| Fluoro- and Chromogenic Substrates | Enzymatic degradation of substrates gives fluoro- or chromogenic products
- MUG (4- methylumbelliferyl-β-D-glucuronide)
- ONPG (o-nitrophenyl β-D-galactopyranoside) |
| MUG (4- methylumbelliferyl-β-D-glucuronide) | - β-D-glucuronidase: enzyme produced by 95% of E. coli strains
- β-D-glucuronidase cleaves MUG to release 4- methylumbelliferyl, which fluoresces under long wave UV light |
| ONPG (o-nitrophenyl β-D-galactopyranoside) | - ONPG is degraded by E. coli β-galactosidase to o-nitrophenyl β-D-galactopyranoside
- Nitrophenyl moiety is released and gives a yellow color measured at 420 nm |
| Adenosine Triphosphate (ATP) Measurement | - ATP present in all living cells at 0.5 fg ATP/bacterial cell
- ATP lost rapidly after cell death
- utilize Luciferin-luciferase system from Fireflys (use ATP to produce light)
- detects amount of viable cells by measuring cellular ATP, light intensity is proportional to numbers of viable cells
- luminometer used to measure emitted ligth |
| Adenosine Triphosphate (ATP) Testing Procedure | 1. Swab food contact surface
2. use ATP test kit for measuring ATP
3. Add sterile cotton swabs to buffer mixture containing luciferin and luciferase
4. Use luminometer to measure light emission |
| Adenosine Triphosphate (ATP) Testing: pros and cons | advantages:
- rapid estimation of microbes in foods
- automation possible
disadvantages/limitations:
- ATP can vary with physiological state of cell
- yeast cells contain 100X more ATP than bacteria
- non-microbial sources of ATP must be removed
- natural pigments can interfere with results
- less effective with low pH foods
- ATPases from somatic cells in milk |
| Radiometry | - place radioactive substrate in growth medium (14C-glucose, 14C-glutamate, or 14C-formate)
- add food sample to growth medium in capped serum vial
- organisms metabolize substrate and release 14CO2
- measure amount of 14CO2 released in a given time OR time before a specific amount of 14CO2 is released |
| Radiometry: pros and cons | - advantage: very sensitive method
- disadvantage: radioactive isotopes not popular in the food industry |
| LUX Gene background | - luminescence in marine bacteria caused by luxA and luxB genes that code for luciferase (enzyme with alpha and beta subunit) |
| Lux Gene Luminescence | - lux genes inserted in bacteriophage specific for the target microbe
- bacteriophages inject DNA into the target microbes and more lux genes are produced
- target microbes fluoresce and are detected |
