2. 2
• The most famous family member of
Enterobacteriaceae.
• It known to be a part of normal
intestinal flora but can also be the
cause of intestinal and extraintestinal
illness in humans.
• Some versions of E. coli make you sick
by making a toxins, this toxins can
damage the lining of your intestine
One especially bad strain, O157:H7
Escherichia coli
6. 6
Escherichia coli
E. coli Types :
•Enteropathogenic
E. coli
•(EPEC)
•Enterotoxigenic
E. coli
•(ETEC)
•Enteroinvasive
E. coli
•(EIEC)
•Shiga-Toxin
Producing E. coli
(STEC)
7. Virulence factors of E. coli :
7
Escherichia coli
• Endotoxins
• Exotoxins
• Phili
• Capsule
9. Horizontal method for the detection and enumeration of ß-
glucuronidase-positive Escherichia coli
Colony-count technique at 44 °C using 5-bromo-4chloro-3-indolyl
ß-glucuronide
9
ISO 16649-2
Escherichia coli
10. 10
• Scope
o Applicable to products intended for human and the
feeding of animals at 44 °C using solid medium
containing a chromogenic ingredient for detection of
the enzyme ß-glucuronidase.
• Terms and definitions
ß-glucuronidase-positive Escherichia coli :
o Bacteria which form typical blue colony on TBX at 44 °C
Escherichia coli
11. 11
• Culture medium
Tryptone-bile-glucuronic medium (TPX)
Enzymatic Digest of
Casein
Provides amino acids, nitrogen, carbon, vitamins
and minerals for organisms growth.
Bile Salts selective agent inhibiting most Gram-positive
bacteria.
X-Glucuronide the chromogenic substrate cleaved by the
enzyme β-glucuronidase of E. coli. Agar is the
solidifying agent.
Agar The solidifying agent.
Escherichia coli
12. 12
• Culture medium
Tryptone-bile-glucuronic medium (TPX)
o Chromogenic selective medium
o Adjust the pH after sterilization at 7.2 ± 0,2 at 25 °C
o Sterilize at 121 °C for 15 min in an autoclave
o Cool the medium in the water bath at 44 °C to 47 °C .
Escherichia coli
13. 13
• Procedure
o Homogenize the test representative sample with
Saline Peptone Water (9:1)
o Add 1ml of the appropriate dilution to petri dish
(Inoculate two plates per dilution)
o Repeat with further dilution, using a new sterile
pipette
o Pour 15ml of the TBX medium into each petri dish
Escherichia coli
14. 14
Incubation :
o Incubate at 44 °C for 18h to 24h
o The total incubation time shall not longer than 24h
o Invert the inoculated dished
The time elapses between the distribution of the inoculum in the dishes
and pouring the medium shall not exceed 15min
If the presence of stressed cells is suspected, incubate for initial period
of 4h at 37 C and then raise the incubation temp. to 44 for 18h to 24h.
Escherichia coli
16. 16
• Counting the colony-forming unit
Escherichia coli
o Count the typical CFU in each dish containing less
than 150 typical CFU and less than 300 total
(typical and non-typical) CFU
o The CFU at least on one dish containing as a
minimum 15 blue CFU
o 𝑁 =
𝞢𝑎
𝑉 𝑛1+0.1𝑛2 𝑑
17. 17
Special cases :
o When the number of the blue colonies is higher than 150 for the
two dishes from the first dilution 𝑑1 , and the number of the blue
colonies is less than 15 for the subsequent dilution 𝑑2 :
- If the number of blue CFU for the two dishes from 𝑑1 is
within the range 150 to 167 use the calculation method :
𝑁 =
𝞢𝑎
𝑉 𝑛1+0.1𝑛2 𝑑
- If the number of blue CFU for the two dishes from 𝑑1 is higher
than 167, only take into account the low number count, of 𝑑2 :
𝑁 =
𝞢𝑐
𝑉 . 𝑛 . 𝑑
