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
Identification
Typical fecal coliform Escherichia coli
Growth on
mFC agar
Inhibited
by
Rosolic
Acid?
Blue colonies
Blue background
No
Yellow colonies
Yellow background
No
Nonfermentative
Pseudomonas maltophilia
gram-negative bacillus Pseudomonas putrefaciens
Acinetobacter calcoaceticus
Acinetobacter lwoffi
Flavobacterium odoratum
Bordetella bronchiseptica
Yellow colonies
Yellow background
No
Atypical: gramGram-positive streptococcus
positive streptococcus
Blue colonies
Blue background
No
A lactosenonfermenting variety Escherichia fergunsonii
of Escherichia
Standard Membrane Filter
Technique
0.45 mm 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

The Effect of Selected Bacteria on
Agar Surface pH
9
8.5
Final Surface pH
(Initial pH = 7.5)
8
7.5
7
6.5
6
5.5
5
4.5
Escherichia coli
Pseudomonas
maltophila
Pseudomonas
putrefaciens
Acinetobacter lwoffi
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
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Final Surface pH
(Initial pH = 7.5)
Growth of Bacteria in Aerobic and Limited
O2 Conditions: Effect on Agar Surface pH
9
8.5
8
7.5
7
6.5
Aerobic
Limited O2
6
5.5
5
4.5