Characterization of Bacteria Responsible for Background

Anomalies in the Enumeration of Fecal Coliform Bacteria by the

Membrane Filter Technique

Esther P. Tennant-Clegg

Jerald D. Hendrix

Apogee Colony Counting System

Background interference caused by non-fecal coliforms interfered with both automated and manual fecal coliform enumeration.

Inconsistent Colony Counts

Two plates created from identical samples. The plate on the left is overgrown with typical blue

colonies formed by fecal coliforms The plate on the right shows a zero fecal coliform count.

Bacteria Isolated from Water Samples on mFC agar: Typical and Atypical

Group Designation IdentificationGrowth onmFC agar

InhibitedbyRosolicAcid?

Typical fecal coliform Escherichia coli Blue coloniesBlue background

No

A lactose-nonfermenting varietyof Escherichia

Escherichia fergunsonii Yellow coloniesYellow background

No

Nonfermentativegram-negative bacillus

Pseudomonas maltophiliaPseudomonas putrefaciensAcinetobacter calcoaceticusAcinetobacter lwoffiFlavobacterium odoratumBordetella bronchiseptica

Yellow coloniesYellow background

No

Atypical: gram-positive streptococcus

Gram-positive streptococcus Blue coloniesBlue background

No

Standard Membrane Filter Technique

0.45 m nitrocellulose filter mFC Agar Rosolic acid to inhibit background

interference Incubation at 44.5°C ± 0.5°C for 24 hrs in

water bath Plates are incubated in a watertight bag or

box

4.5

5

5.5

6

6.5

7

7.5

8

8.5

9

Escherichia coli Pseudomonasmaltophila

Pseudomonasputrefaciens

Acinetobacter lwoffi

Fina

l Sur

face

pH

(Initi

al p

H =

7.5)

The Effect of Selected Bacteria on Agar Surface pH

Common Features of Atypical Bacteria on mFC

Produce higher pH (up to 8.5) in the mFC Agar

Agar turns transparent yellow Aerobic Non-fermentative Not inhibited by rosolic acid

Methods Used to Identify Bacteria

Gram Stain Wet mount slide to observe motility Catalase and Oxidase Test SIMS Agar to discover Indole reactions, and

Oxygen requirements Fermentation Tubes (Sucrose, Lactose, Glucose) Oxiferm and Enterotube rapid biochemical test

(Beckton Dickinson)

Limited Oxygen Incubation Method

Agar is poured close to the top lip of plate limiting the area between bacterial growth and lid.

Each plate is individually wrapped with parafilm prior to incubation.

All other aspects of the Standard Method of Membrane Filter Technique remain unchanged.

Effect Of Limited 02 on Background and Growth of E. coli

Atmospheric O2 Levels Limited O2 Levels

Effect Of Limited 02 on Background and Growth of Atypical Isolates

Atmospheric O2 Levels Limited O2 Levels

Growth of Bacteria in Aerobic and Limited O2 Conditions: Effect on Agar Surface pH

4.5

5

5.5

6

6.5

7

7.5

8

8.5

9

Fin

al S

urf

ac

e p

H(I

nit

ial p

H =

7.5

)

Aerobic

Limited O2