966.24 NMP

 

17.2.02

Table Ta ble 966.24B.

AOAC Offi Official Method 966.24 Coliform Group and Esch Esche erichia coli 

Indole

Micro Mi crobi bio olog logiical (MPN) Method First Action Action 1966 Final Fi nal Action Action 1971

Seed 3-tube most mo st prob a ble num ber (MPN) se series ries into lauryl sulfate sulfate tryptose broth, 966.23A 966.23A((b)  see 17.2.01),  (see 17.2.01), using 1 mL inocula of 1:10,

Streak gas-pos itive tubes on Levine’s Levine’s eosin methy methylene blue agar   plates, 966.23A  plates,  966.23A((d) ( see 17.2.01),  see 17.2.01), and incu incubate plates 24  2 h at 35°C. Pick 2 or more well-isolated typi typ ical colo colonies from Levine’s eosin methy meth ylene blue agar plates and transfer trans fer to agar slants pre pared from agar medium, medium, 940.36A 940.36A((g) ( see 17.1.02).  see 17.1.02). Incu Incu bate 18–24 h at 35°C. If typi typical colo colonies are not present, present, pick 2 or more col onies most likely to be E. be  E. coli. Pick coli. Pick 2 from every every plate.

Table 966.24A.

a

MR

VP

Citrate

Type

+

+

–

–

Typical E. coli 

 –

+

–

–

Atypiical E. coli  Atyp

+

+

–

+

Typical inter interme medi diate ate

 –

+

–

+

Atypiical inter Atyp interme medi diate ate

 –

–

+

+

Typiical Enterobacter aerogenes Typ

–

+

+

Atypical Enterobacter aerogenes

+

1:100, and[halves 1:1000 and di lutions, dilu tions, triplicate tripli cate each dination dilu lution. tion. (For nut meats largerwith pieces], be begin gintubes MPNatdeter de termi mina tion with 0  –1 10  di  dilu lution; tion; for nut meal, m eal, be gin with 10 di dilu lution.) tion.) Incu Incu bate 48  2 h at 35°C for gas forma formation tion as evi evidenced by dis place  placement ment of liquid liquid in insert in sert tube or by vigor vigorous ous effer efferves vescence cence when tubes are shaken gently shaken gently.. Exam Ex amine ine tubes tub es for gas for ma mation tion at 24 and 48 h inter intervals. Trans Transfer, fer, using us ing 3 mm loop, from gassing gassing tubes to BGLB, 966.23A 966.23A((c) ( see  see 17.2.01), (omit this transfer transfer for tree nuts), and EC broth, 966.23A 966.23A((g) ( see 17.2.01),  see 17.2.01), at time gas forma for mation tion is noted. In cu cu ba  ba te BG LB br ot h 48    2 h at 35°C. Using MPN Ta ble 966.24A, com pute MPN MPN on ba basis sis of num ber of tubes of BGLB  broth produc producing ing gas by end of incu in cu ba  bation tion period. period. Re port as MPN of  coliform bacte bacteria/g. ria/g. Incu In cu bate EC broth 48  2 h at 45.5  0.02°C in i n covered water bath. Submerge Sub merge broth tubes in bath so that water level is above highest highest level of medium. medium. Exam Examine ine tubes for gas forma for mation tion at 24 and 48 h inter intervals.

Classi Clas sifi fica cation tion of biochem biochemiical types

a

Other groupings groupings may appear; appear; in such cases, cultures cul tures are usually usually mixed. Restreak to deter determine mine their purity. purity. Compute Compute MPN of E. coli /g, /g, consid consider ering ing Gram-negative, nonspore-forming rods produc producing ing gas in lac tose and produc pro ducing ing + + – – or – + – – IMViC patterns patterns as E. coli.

Transfer growth from plate c ount agar slants into fol lowing Transfer lowing broths for identi identifi fica cation tion by biochem biochemiical tests: (a) Tryptophane broth.— Incu Incu bate broth broth,, 966.23A 966.23A(( j) ( see 17.2.01),  see 17.2.01), 24  2 h at 35°C and test for indole by adding adding 0.2–0.3 mL Kovacs reagent, re agent, 967.25B 967.25B((a) ( see  see 17.9.01),  17.9.01), to 24 h cul ture. Test is posi pos itive if  up per layer layer turns turns red. red. ( b)  MR- VP me medium.—  dium.— Incu In cu bat e me medium, dium, 966.23A 966.23A((k ) ( see 17.2.01), 48  2 h at 35°C. Aseptically Aseptically transfer transfer 1 mL culture cul ture to 13  100 mm test tube to test for acetylmethylcar acetylmethylcarbinol. binol. Add 0.6 mL 5% alco al coholic holic -naphthol solu solution tion (w/v), 0.2 mL KOH solu solution tion (4 + 10), and few crystals crystals of creatine. Shake and let stand 2 h. Test is posi positive if eosin pink devel develops. ops. Alter Alterna natively, tively,  see 967.27D 967.27D((c)( )(1 1) ( see 17.9.03). Incu In cubate remain mainder der of MR-VP medium medium for addi additional tional 48 h and test for methy l red re action action by adding adding 5 drops methyl red solu solu tion tion to culture. cul ture. Test is posi positive if culture culture turns red; nega negative, if yellow. (Pre pare methyl red solu lution tion by dissolv dissolving ing 0.1 g methyl red in 300 mL 90% alco cohol hol and dilut diluting to 500 mL with H2O.)

Most probable numbers (MPN) (MPN) per 1 g test portion, us using ing 3 tubes with each of 0.1, 0.01, and 0.001 g portions Posiitive tubes Pos

0.1

0.01

0.001

0

0

0

0

0

1

MPN

0.1

0.01

0.001