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)。

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.

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.

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.

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

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.

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

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

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

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)

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)

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

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

LST Tryptose …...........................................20.0 g Lactose ................................................ 5.0 g Dipotassium Phosphate ....................... 2.75 g Monopotassium Phosphate .................. 2.75 g Sodium Chloride .................................... 5.0 g Sodium Lauryl Sulfate ........................... 0.1 g

BGLB Peptone…….............................. 10.0 g Oxgall ........................................ 20.0 g Lactose ..................................... 10.0 g Brilliant Green ............................ 13.3 mg

Difco™ EC Medium with MUG Tryptose ................................................ 20.0 g Lactose .................................................. 5.0 g Bile Salts No. 3 ...................................... 1.5 g Dipotassium Phosphate ......................... 4.0 g Monopotassium Phosphate ................... 1.5 g Sodium Chloride ..................................... 5.0 g MUG (4-methylumbelliferyl-β-D-glucuronide) 0.05

EMBA Pancreatic Digest of Gelatin ..................... 10.0 g Lactose ...................................................... 5.0 g Sucrose ...................................................... 5.0 g Dipotassium Phosphate ............................. 2.0 g Eosin Y ...................................................... 0.4 g Methylene Blue ..................................... 65.0 mg Agar ........................................................ 13.5 g

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.

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

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

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)

LST EMBA BGLB EC-MUG

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

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)

example Case 1: no dilution, positive number 330, MPN 240, possible range: 36-1300. Result: 240 MPN/100 mL Case 2: dilute to 10-2, positive number 330, MPN 240, Result: 240 x 102 = 24000 = 2.4 x 104 MPN/100mL

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.