1. 利用最大可能數(most probable number, MPN)測定水樣品大腸菌群數
最常使用的MPN是水品質測定，測定水中的大腸菌桿群(coliform)， 糞便大腸菌桿群 (fecal coliform)，與大腸桿菌 (E. coli)。

2. The method of most probable number (MPN)
MPN is a statistical method to determine the bacterial population and usually is used for water or liquid samples.
MPN usually is used for the samples with lower bacterial populations.
The most common use of MPN is to determine the populations of coliform, fecal coliform, and Escherichia coli.

3. Indicator microorganism
Definition: indicator microorganism which can be used as an indication to monitor contamination situation of target foods.
Laboratory can not detect every pathogen but use level of indicator to monitor microbial quality of foods.
Indicator microorganisms should not be pathogens but closely related to pathogen. Thus, occurrence of indicator can be associated with pathogens.

4. Coliform, fecal coliforms
The most commonly used indicators are total and fecal coliforms.
Reasons:
Closed related to feces.
Exists in feces at high numbers.
Strongly survival ability in natural environment.
Fecal-oral transmission rout.

5. Coliform (大腸桿菌群): microorganisms mainly live in intestinal tracks and sometimes live in natural environment. Positive results of coliform indicate possible contamination from animal feces. The 6 genera in the family of Enterobacteriaceae
Escherichia, Enterobacter, Klebsiella, Serratia, Edwardsiella, and Citrobacter.
Non-spore forming, motile, facultative aerobic, ferment lactose to produce gas

6. Fecal coliforms (糞便大腸桿菌群):
Fecal coliforms are capable of growth in the presence of bile salts or similar surface agents, oxidase negative, and produce acid and gas from lactose within 48 hours at 44 ± 0.5ºC
Microorganism only live in animal intestinal tracks. Positive results of fecal coliform mean contamination of animal feces.
Fecal coliforms include the genera that originate in feces; Escherichia as well as genera that are not of fecal origin; Enterobacter, Klebsiella, and Citrobacter.

7. MPN Statistical estimate 3-tube or 5-tube
More sensitive than plate count
Key procedure
LST initial checking
BGLB confirmation for coliform
EC confirmation for fecal coliform
EMBA and EC-MUG confirmation for E. coli

8. MPN (most probable number) for total and fecal coliform
Presumptive test
in LST
Confirmation test in BGLB for total
coliform at 37℃
Confirmation test in EC for fecal
coliform at 44.5℃
EMBA (selective agar) or EC-MUG
NA or TSA (general medium)
IMViC test
Biochemical, immunological, DNA tests

9. LST: presumptive medium
BGLB: positive coliform
EC: positive fecal coliform
EC-MUG and EMBA: positive E. coli

10. MPN (most probable number)
Medium
LST (2X and 1X)
2x LST use 71.2 g for one liter,
1X LST use 35.6 g for one liter
BGLB EC
Sample
Reverse osmosis (RO) water (negative control)
RO water inoculated with Escherichia coli and Enterobacter aerogenes (inoculated water)
Water or beverage samples (supplied by students)

11. samples inoculated water -9 (add one mL)
Water samples -8 (add one mL)
Water samples -7 (add one mL)
Water sampels -7 (add one mL)

12. Dilutions For LST The fist dilution (the first three tubes) use 2X LST
Remaining LST use 1 X LST
Three dilutions per sample
Total 9 tubes per group

13. Use fermentation tube (small tube)
BGLB EC
Three dilutions, 9 tubes
Use fermentation tube (small tube)
Each dilution tube should has one small tube (fermentation tube) inside

14. LST
Tryptose … g
Lactose g
Dipotassium Phosphate g
Monopotassium Phosphate g
Sodium Chloride g
Sodium Lauryl Sulfate g

15. BGLB
Peptone…… g
Oxgall g
Lactose g
Brilliant Green mg

16. Difco™ EC Medium with MUG
Tryptose g
Lactose g
Bile Salts No g
Dipotassium Phosphate g
Monopotassium Phosphate g
Sodium Chloride g
MUG (4-methylumbelliferyl-β-D-glucuronide) 0.05

17. EMBA Pancreatic Digest of Gelatin ..................... 10.0 g
Lactose g
Sucrose g
Dipotassium Phosphate g
Eosin Y g
Methylene Blue mg
Agar g

18. The absence of β-D-glucuronidase activity in E
The absence of β-D-glucuronidase activity in E. coli O157:H7 in contrast to most other E. coli.
β-D-glucuronidase cleaves 4-methylumbelliferyl-β-D-glucuronide to 4-methylumbelliferone and glucuronide. The fluorogen 4-methylumbelliferone can be detected under a long wavelength (366 nm) UV lamp.

19. schedule Monday (10/6) MPN
Inoculated 10 mL of water sample into the first dilution of LST medium
inoculate 1 mL of water sample into the second dilution
Inoculate 0.1 mL of water sample into the third dilution
Incubate at 37C for 48 h

20. Wednesday (10/8) Thursday (10/9) Check LST medium
Transfer positive tube (turbid and with gas inside the fermentation tube) into BGLB medium and EC medium
Incubate BGLB (37C) and EC (44.5C)
Thursday (10/9)
Check results of BGLB and EC tubes

21. Monday (10/13) MPN calculation
Transfer 0.1 mL of EC positive sample into EC-MUG tubes
Streak EC positive samples onto EMBA plate
Incubate at 37C for 24 h
Observe on Tuesday (10/14)

23. LST
EMBA
BGLB
EC-MUG

24. MPN calculation
查表 (10/1)
BGLB for coliform, EC for fecal coliform, EC-MUG and EMBA for E. coli.

27. MPN table Most Probable Number (最大可能數) 單位為 MPN，而非CFU Probability (機率)
測低數目菌量
取100 mL ，若有皆為負，則表示3/1的100 mL 的樣品無細菌，<3 MPN/100 mL
3-tube (33.3 mL) and 5-tube (55.5 mL)

28. example
Case 1: no dilution, positive number 330, MPN 240, possible range:
Result: 240 MPN/100 mL
Case 2: dilute to 10-2, positive number 330, MPN 240,
Result: 240 x 102 = = 2.4 x 104 MPN/100mL

29. Usually, if a sample needs to be diluted, pour or streak plating is more accurate than MPN.
MPN method can be used for water and solid samples.
MPN method is usually used for water (liquid) samples and for Vibrio species and Staphylococcus aureus.