Describes the MTF Method for Bacteriological Analysis

1. Laboratory Testing
Bacteriological Analysis
WATR-082: ADVANCED WASTEWATER TREATMENT
SPRING 2019

2. Bacteriological Testing
Reasons for
conducting
wastewater
effluent
bacteriological
testing
Meeting
requirements
of a
wastewater
discharge
permit
Monitoring
pathogen
impacts of
treated
wastewater
discharge
Assess the level
of contamination
of a public body
of water
Bacteriological
tests involves
detection and
quantification of
one or more of
the following:
Total coliforms
Fecal coliformsE. Coli
Multiple Tube
Fermentation
(MTF) is a widely
used method

3. Multiple Tube Fermentation (MTF)
11 ml
Sample
99 ml
DI water
11 ml
Sample
11 ml
Sample
99 ml
DI water
99 ml
DI water
Original
Sample
1:10
dilution
1:100
dilution
1:1000
dilution
Serial dilutions
may be
required based
upon expected
sample MPN
Test cannot
exactly count
the target
bacteria
Test results
are provided
as Most
Probable
Number –
(MPN)

4. Multiple Tube Fermentation (MTF)
Three stages:
Multiple tubes containing lauryl tryptose broth are inoculated with sample and
incubated for 24 hours
After incubation the tubes are examined for gas formation
Tubes showing gas formation are deemed as “positive”
This stage is to confirm the positives from the presumptive stage
This stage is to reconfirm the Confirmed test result and also serves as a quality
control
PRESUMPTIVE
Stage I
CONFIRMED
Stage II
COMPLETED
Stage III

5. Setup 15 tubes with Lauryl Tryptose Broth and an inverted vial
(Durham tube
5 tubes each for 10 ml, 1 ml and 0.1 ml sample
Incubate 24 hrs
Check tubes for positive results marked by: 1) broth turning cloudy,
and 2) gas collection in the inverted tube. If not postitive, incubate for
another 24 hrs and check if positive
PRESUMPTIVE
The sample in this
example is diluted
serially to a 1:1,000 as
the expected MPN is
between 5,000 –
50,000

6. MTF (Contd.)Setup 15 tubes with Lauryl Tryptose Broth and an inverted vial (Durham tube
5 tubes each for 10 ml, 1 ml and 0.1 ml sample
Incubate 24 hrs
Check tubes for positive results marked by: 1) broth turning cloudy, and 2) gas collection in the
inverted tube. If not postitive, incubate for another 24 hrs and check if positive
Innoculate each positive into bacteria specific broth:
EC Broth for fecal coliform, EC Broth with MUG for E. Coli
and Brilliant Green Bile Broth (BGB) for total coliforms.
Incubate for 24 hours and observe for positive results.
CONFIRMED
Innoculate in: EC Broth for fecal and
EC Broth with MUG for E. Coli
Incubate for 24 hrs
and observe for positive results
Innoculate in: BGB Broth total coliforms
Incubate for 24 hrs
and observe for positive results

7. MTF (Contd.)
CONFIRMED
PRESUMPTIVE
COMPLETED
Count number of confirmed positives for each set of the three
sample volumes
In this Example: 10 ml – 3, 1 ml – 1, 0.1 ml - 1
Establish the MPN using the statistical MPN table
From Table – 14 MPN/100 ml
Given 1:1,000 dilution, MPN=14*1,000 = 14,000 MPN/100 ml
If after 24 hrs, from gram staining of the colonies from
the agar slant, nonsporing gram negative bacteria can
be identified and the lactose broth shows gas formation,
the completed test is deemed positive
Streak agar plate and incubate
Use colonies to inoculate an
agar slant and nutrient broth