Transcript Document

Introduction to Lab Ex. 20: Enumeration of Bacteria Most Probable Number method
Membrane Filter method
Water Quality
• Microbes are filtered from water that percolates into groundwater.
• Some pathogens are transmitted to human in drinking and
recreational water.
• Resistant chemicals may be concentrated in the aquatic food
chain.
• Mercury is metabolized by certain bacteria into a soluble
compound, concentrated in animals
Waterborne Diseases
Table 27.2
Most Probable Number & Membrane Filter methods
These are methods used to enumerate the numbers of bacteria in
water samples.
The Most Probable Number method is used to check potability
(if water is safe enough to be drinking water) of water.
The MPN method looks for the presence of potential pathogenic
bacteria that may be in the water due to fecal contamination
of the water supply.
Water supplies are generally derived from ground sources and have
to be checked for safety levels of bacterial contamination.
MPN method enumerates the enteric bacteria called coliforms,
specifically fecal coliforms (E. coli)
Coliforms are Gram negative bacilli that have the ability to
ferment lactose with the production of acid and gas.
Fecal coliforms are those coliforms that are normally found in the
feces of warm blooded animals (including humans)
MPN method thus enumerates the fecal coliforms in water samples.
E. coli is thus used as an indicator organism.
MPN test includes 3 levels of testing:
Presumptive, Confirmed, Completed.
The presumptive test looks for presence of fecal coliforms in the
water sample by inoculating lactose broths with the water
sample.
Those tubes that show presence of acid and gas are
scored + and those with no acid/gas as -.
Three sets of lactose broths are inoculated with varying
dilutions of the sample:
first set of 3 or 5 tubes inoculated with 10ml of sample;
second set of tubes inoculated with 1ml of sample;
third set of tubes inoculated with 0.1ml of sample.
The combination of positives in the 3 sets is used to figure out
the MPN /100ml of water using the table provided.
The tubes that show positive in the presumptive test should be
confirmed to contain E.coli.
This done in the confirmed test using the selective/differential
medium EMB (that uniquely highlights E.coli growth on it).
The completed test is done only where legal issues are involved
wherein the bacterial culture is then identified by a full
complement of tests including gram stain to show that it is
indeed E.coli
Bacteriological analysis of water:
Most Probable Number (MPN) technique
coliform: acid and gas from lactose <24 hours/370C
indicator organism: E. coli
Water
sample
double strength
single strength
single strength
10 ml sample
1.0 ml sample
0.1 ml sample
*Normally 5 Durham tubes are inoculated but this exercise is modified to three tubes in the interest
of economy.
Table 1.Table of Most Probable Numbers (MPN) Per 100 ML
of Sample using Three Tubes of Each Dilution
Number of positive
tubes in dilutions
10 ml
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1 ml
0
1
0
0
1
1
1
1
2
2
2
2
3
3
3
3
0
0
0
0
1
1
1
1
2
2
2
2
3
3
3
3
0.1 ml
0
0
2
3
0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
3
Number of positive
tubes in dilutions
MPN per
100 ml
3
6
9
3
6.1
9.2
12
6.2
9.3
12
16
9.4
13
16
19
3.6
7.2
11
15
7.3
11
15
19
11
15
20
24
16
20
24
29
10 ml 1 ml
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
0
0
0
0
1
1
1
1
2
2
2
2
3
3
3
3
0
0
0
0
1
1
1
1
2
2
2
2
3
3
3
0.1 ml
0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
MPN per
100 ml
9.1
14
20
26
15
20
27
34
21
28
35
42
29
36
44
53
23
39
64
95
43
75
120
160
93
150
210
290
240
460
1100
Multiple-Tube Method
Figure 6.18a
Multiple-Tube Method
Figure 6.18b
Bacteriological analysis of water: Membrane filter technique