The method of most probable number MPN MPN

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 ECMUG NA or TSA (general medium) IMVi. C test Biochemical, immunological, DNA tests

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

MPN (most probable number) �Medium �LST (2 X and 1 X) � 2 x LST use 71. 2 g for one liter, � 1 X LST use 35. 6 g for one liter �BGLB �EC �Sample (100 m. L) �Reverse osmosis (RO) water (negative control) �RO water inoculated with 1 m. L Escherichia coli and Enterobacter aerogenes (inoculated water) �Water or beverage samples (supplied by students)

samples 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. inoculated water -8 (add one m. L) inoculated water -7 (add one m. L) Water samples -6 (add one m. L)

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

�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

Eosin methyl blue agar (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. coli O 157: H 7 in contrast to most other E. coli. �β-D-glucuronidase cleaves 4 -methylumbelliferyl-β-Dglucuronide to 4 -methylumbelliferone and glucuronide. The fluorogen 4 -methylumbelliferone can be detected under a long wavelength (366 nm) UV lamp.

schedule �Monday (9/26) �MPN �Inoculated 10 m. L of water sample into the first dilution of LST medium �inoculate 1 m. L of water sample into the second dilution �Inoculate 0. 1 m. L of water sample into the third dilution �Incubate at 37 C for 48 h

�Wednesday (9/28) �Check LST medium �Transfer positive tube (turbid and with gas inside the fermentation tube) into BGLB medium and EC medium �Incubate BGLB (37 C) and EC (44. 5 C) �Thursday (9/29) �Check results of BGLB and EC tubes

�Monday (10/3) �MPN calculation �Transfer 0. 1 m. L of EC positive sample into EC-MUG tubes �Streak EC positive samples onto EMBA plate �Incubate at 37 C for 24 h �Observe on Tuesday (10/4)

LST EMBA BGLB EC-MUG

MPN calculation �查表 (10/4) �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 m. L ，若有皆為負，則表示 3/1的100 m. L 的樣品 無細菌，<3 MPN/100 m. L � 3 -tube (33. 3 m. L) and 5 -tube (55. 5 m. L)

example �Case 1: no dilution, positive number 330, MPN 240, possible range: 36 -1300. �Result: 240 MPN/100 m. L �Case 2: dilute to 10 -2, positive number 330, MPN 240, �Result: 240 x 102 = 24000 = 2. 4 x 104 MPN/100 m. L

�Usually, if a sample needs to be diluted, pour or spread 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.