CVEEN 3615 Environmental Engineering Laboratory

Lab # 6 Coliforms Lab

Objective
To understand the concepts and procedure to computing the total coliforms in sample solutions
by demonstrating the two methods, the Multiple Fermentation Tube and Membrane Filtration
technique.

Method
During this lab experiment, there are two methods that are put to the test, the multiple-tube
fermentation technique and the membrane filter technique (MF Test). The first portion of this
lab, the multiple tube was conducted and this required 48 hours of incubating. There are five or
ten test tubes that are used to conduct this test. If the sample solution is a finished water
sample, this is no need for dilution, but if the sample solution is not a finished water sample,
dilution is needed. So for these dilutions, two of our water samples (Millcreek and Jordan River)
the dilutions were according; 0, 10X (0.1), 102X (0.01), 103X (0.001), and 104X (0.0001). For the
third non finished water (Activated Sludge), the dilutions were 10 3X (0.001), 104X (0.0001), 105X
(0.00001), 106X (0.000001), and 107X(0.0000001). Now that we have all our samples ready, they
are added to the Laural tryptose broth which are inside the test tubes and the tubes are
incubated for 24 hours at 35oC. If any of the tubes from Day 1 are cloudy or turbid on Day 2, we
will assume “presumptive positive”, then we move these solutions and transfer them to the
green bile broth and incubate for another 24 hours at 35oC. By Day 3, if the tubes that have a
cloudy feature and also have a gas bubble, they are counted as a positive. Here we then count
the number of positive tubes per set and since knowing the sample volume, we can refer to the
Most Probable Number (MPN) Index table to find the MPN per 100 mL.
The second portion of this lab experiment is the membrane filter test, which we have a certain
amount of sample filtered through a sterile membrane filter. Once the sample solution has
been filtered through, we can then remove the filter and placed on the absorbent pad that
contains m-Endo broth. Then this filter, can be incubated for 24 hours at 35 oC. After incubation,
recording the colonies with a green metallic shine to them, these are considered coliform
bacteria. Once counting the colonies, these are the results as number of colonies per 100 mL.
 Sample Dilution Volumes filtered, mL

Millcreek Water None 100

10 to 1 10
100 to 1 1
10^3 0.001
Activated Sludge 10^4 0.0001
10^5 0.00001
CVWRF Effluent None 100
10 to 1 10
Jordan River Water None 100
10 to 1 10
100 to 1 1
Table 1. Samples with Dilution and Volumes filtered, for the Membrane Filter Test

Sample Dilution mL of raw sample used # of tubes

Millcreek Water 0 to 10^4 1, 0.1, 0.01, 0.001, 0.0001 5

Activated Sludge 10^3 to 10^7 0.001 to 0.0000001 5
CVWRF Effluent none 1 10
Jordan River Water 0 to 10^4 1, 0.1, 0.01, 0.001, 0.0001 5
Table 2. Samples with Dilution, mL of sample used, and tubes used for the Multiple Tube Test
Results
No. of Colonies Observed
Sample Dilution Volumes filtered, mL
24 hr 24 hr
Millcreek Water None 100 0 0
10 to 1 10 0 0
100 to 1 1 0 0
Activated Sludge 10^3 0.001 10 19
10^4 0.0001 0 0
10^5 0.00001 0 0
CVWRF Effluent None 100 0 3
10 to 1 10 0 0
Jordan River Water None 100 45 51
10 to 1 10 0 0
100 to 1 1 0 0
Table 3. Summary of the number of colonies observed in the Membrane Filtration

# of Tubes positive
Sample Dilution mL of raw sample used
tubes Presumptive Confirmed
Millcreek 1, 0.1, 0.01, 0.001,
0 to 10^4 5 5, 3, 2, 1, 0 3, 2, 0, x, x
Water 0.0001
Activated
10^3 to 10^7 0.001 to 0.0000001 5 5, 3, 1, 0, 0 5, 3, 1, x, x
Sludge
CVWRF
none 1 10 3 2
effluent
Jordan River 1, 0.1, 0.01, 0.001,
0 to 10^4 5 5, 4, 4, 1, 1 2, 0, 1, x, x
water 0.0001
Table 4. Summary of the number of tubes positive in the Multiple-Tube Fermentation test

Calculating the Most Probable Number (MPN) in each test, using this equation and referring to
tables that are provided by instructor and TA, we will be able to obtain the MPN/100 mL.

Table MPN
MPN
=
( 100 mL )
∗10

100 mL V
MULTIPLE TUBE TEST CALCULATION TO FIND MPN

Figure 1. Table used for Multiple Tube Test, for 10 test tubes
Since we only have one sample that uses 10 tubes which is the CVWRF Effluent, this is how we
calculate the MPN/100 mL, I used the confirmed positives.

 2 Confirmed Positive Tubes

 Volume = V = 1 mL that was added to the 10 mL media
Refer to Figure 1, for two positive tubes, we get a MPN Index/100 mL = 2.2. Now we use the

equation; MPN
=
( Table MPN
100 mL )
∗10

100 mL V
2.2
MPN
=
( 100 mL )
∗10
=.22
MPN
100 mL 1 100 mL
Figure 2. Table used for 5 Tubes in the Multiple Tube Test
Now we will do one calculation for the 5 tubes in the Multiple tube test. We will do the
Activated Sludge sample and using values from the confirmed positive column.

 Confirmed Positive – 5, 3, 1 , x, x
 Volume = 103X (0.001), 104X (0.0001), 105X (0.00001), 106X (0.000001), and
107X(0.0000001)
 Now we can refer to Figure 2, using the Confirmed positive of 5-3-1 which will give us a MPN
Index/100 mL value of 110. Then using the same equation:

Table MPN
MPN
=
( 100 mL )
∗10

100 mL V
Looking at the value of 5-3-1-x-x , we will corresponding with the volume of 103X (0.001), 104X
(0.0001), 105X (0.00001), 106X (0.000001), and 107X(0.0000001). We will use the lowest volume
value which is our 1, being 105 (0.00001)

110
MPN
=
( 100 mL )
∗10
=1100000
MPN
100 mL 0.00001 100 mL

Tubes positive MPN

mL of raw sample # of MPN/100
Sample Dilution Index/
used tubes Presumptive Confirmed mL
100 mL
1, 0.1, 0.01, 0.001,
Millcreek Water 0 to 10^4 5 5,3,2,1,0 3,2,0,x,x 14 140
0.0001
10^3 to 0.001 to
Activated Sludge 5 5,3,1,0,0 5,3,1,x,x 110 1100000
10^7 0.0000001
CVWRF Effluent none 1 10 3 2 2.2 0.22
Jordan River 1, 0.1, 0.01, 0.001,
0 to 10^4 5 5,4,4,1,1 2,0,1,x,x 6.8 68
Water 0.0001
Table 5. Results of the MPN/100 mL for each sample solution using the Multiple Tube Test

MEMBRANE FILTRATION CALCULATION TO FIND MPN

For this section, there is not a specified calculation to finding the MPN, but these results are
number of colonies/100 mL. I provided images that were taken as record data, as we will be
counting the number of green shiny metallic coliforms thus these will be known as colonies.
This membrane filtration method isn’t very reliable for water samples that are very turbid or
cloudy.
Figure 3. Membrane Filtration Test for Activated Sludge (Top “Replicate 1”, Bottom “Replicate
2”)
 Figure 4. Membrane Filtration Test for CVWRF Effluent (Top “Replicate 1”,Bottom “Replicate
2”)

Figure 5. Membrane Filtration for Jordan River (Top “Replicate 1”,Bottom “Replicate 2”)

Replicate 1 Replicate 2 Number of Colonies/100 mL

Volumes
Sample Dilution No. of Colonies Observed Replicate Replicat
filtered, mL Average
24 hr 24 hr 1 e2
none 100 0 0 0 0 0
Millcreek
10 to 1 10 0 0 0 0 0
Water
100 to 1 1 0 0 0 0 0
10^3 0.001 10 19 10 19 14.5
Activated
10^4 0.0001 0 0 0 0 0
Sludge
10^5 0.00001 0 0 0 0 0
CVWRF none 100 0 3 0 3 1.5
Effluent 10 to 1 10 0 0 0 0 0
Jordan River none 100 45 51 45 51 48
Water 10 to 1 10 0 0 0 0 0
 100 to 1 1 0 0 0 0 0
Table 6. Results for Number of Colonies/100 mL of all samples for Membrane Filtration Test
During this lab session, we tested the two methods, membrane filtration technique and
multiple tube test technique. The results are as followed of the multiple tube test on Table 5,
these showed the MPN/100 mL for the four samples, Millcreek water, Activated Sludge, CVWRF
Effluent, and Jordan River water. We expected very few coliforms to form in the Disinfected
Effluent and expected many coliforms to show for the other three sample solutions. For the
membrane filtration results are in Table 6, for the same four samples. These techniques main
goal is to investigate possible sources of bacterial contamination.

Discussion
There are a few differences between the two techniques, such as membrane filtration methods
isn’t very reliable when it comes to water sample solutions that are very turbid or cloudy, which
means that high concentration of bacteria in water solutions, you shouldn’t be using the
membrane filtration method. Also, during this experiment of the multiple tube test, a 10 4X
showed up negations and a 105X sample showed up presumptive positive. This is assumed to be
a false positive as the coliform may have derived from the researcher or the pipette. I believe
that the multiple tube test was most likely the more accurate results due to the number of test
tubes used for one sample.

Questions
- Rank the samples in order of expected number of fecal coliform bacteria. Did the laboratory
results match the expected order? If not, what are some possible reasons why this occurred?
1. Central Valley WRF activated sludge (Highest)
2. Central Valley WRF Effluent
3. Millcreek
4. Jordan River Water
The lab results didn’t match my expected order, the one out of place is the CVWRF Effluent.
This should have been number 4 on my list because it has gone through the disinfection process
of the treatment plant, thus meaning lab shows that it has very little fecal coliform bacteria.