General Microbiology lab | Date 06.04.
2024 | Report 04
MICROBIAL ENUMERATION: MOST PROBABLE
NUMBER METHOD
Student Name Dinh Hang Giang
Student ID 20210286
Class ID 743706
I. PURPOSE
In this experiment, the purpose is to quantify the amount of bacteria that can
ferment lactose to produce gas in drinking water, using the MPN method but
only the presumptive test. After incubating at 35 oC for 48 ± 2 hours, the result
is observed and we can determine the MPN of these bacteria in the sample
using the statistical table.
II. MATERIALS & METHODS
1. Materials & Equipment
The water sample our group used in the experiment is drinking water in plastic
bottles, manufactured by Lavie. The culture medium is Lactose broth at single
strength (60 mL) and double strength (30 mL). The ingredients used for
preparing the medium is peptone, lactose, beef extract and water.
The required equipments are micropipettes (5 mL, 1 mL, 100 µL) and their
corresponding tip boxes; 9 test tubes with inverted Durham tubes; 2 beakers.
The medium is prepared in test tubes with inverted Durham tubes inside to
capture gas produced by lactose fermenting bacteria.
2. Procedure
Medium Preparation
The Lactose Broth medium contains peptone, lactose, beef extract and water.
All ingredients were measured in two beakers follows the Table II.1 below, then
mixed until dissoved completely. After autoclaving, the mixture is distributed
100 mL for each of three groups.
Double Strength Single strength
Peptone 3g 1.5 g
Lactose 3g 1.5 g
Beef Extract 1.8 g 0.9 g
Water 300 mL 300 mL
Table II.1. Lactose Broth medium ingredients
After that, the lactose broth is dispensed into test tubes containing inverted
Durham tubes following the instructions in Table II.2 below.
Sets of Medium Strength Dispensed volume Number of
tubes tubes
F1 Double 10 mL 3
F2 Single 10 mL 3
F3 Single 10 mL 3
Table II.2. Distribution of lactose broth into test tubes
In this experiment, we used Lactose broth, a liquid and general-purpose
medium, because the purpose of this experiment is to determine the density of
microorganisms in water sample. These microbes have the ability able to
ferment lactose and produce gas (mostly H 2). The MPN method is suitable for
this experiment because the sample, which is drinking water, has low cell
density.
Sample collection
Around 50 mL of sample is transferred from a plastic Lavie bottle to a sealed
flask.
Inoculation
Firstly, we inoculated each of three tubes of double strength lactose broth
(containing 10 mL broth) with 10 mL sample using a micropipette. Secondly,
we inoculated each of three tubes of single strength lactose broth (containing 9
mL broth) with 1 mL sample using a micropipette. Finally, each of three tubes
of single strength lactose broth (containing 9.9 mL broth) was inoculated with
0.1 mL sample using a micropipette.
As it can be seen from the data, the amount of sample inoculated was higher
than other experiments, with the highest amount being 10 mL. This proves the
another advantage of MPN method, it allows a large amount of sample with low
cell level.
Incubation and observation
The 9 tubes containing lactose broth culture were incubated at 35 oC for 48 ± 2
hours. After 48 ± 2 hours, students came to the laboratory to observe the gas
accumulation in the Durham tubes. The tubes shows significant gas growth are
considered positive. Finally, we determine the MPN of lactose fermenting, gas
producing bacteria in the sample by refering to the statistical table 4.3 (Lab
Manual – page 16) [1].
III. RESULTS & DISCUSSION
1. Result
These observations was made on 08/04/2024, around 11:30 AM.
Figure III.1. Set of tubes F1
Figure III.1 shows the set of tubes F1. There observed no formation of gas -
indication for gas (Durham tubes) shows negative results. However, it is able to
see the spores forming, and turbidity in the growth medium. Its color change
from transparent to slightly murky.
Figure III.2. Set of tubes F2
Figure III.2 shows the set of tubes F1. There observed no formation of gas -
indication for gas (Durham tubes) shows negative results. However, it is able to
see the a small amount of spores forming, and turbidity in the growth medium.
Figure III.3. Set of tubes F3
Figure III.3 shows the set of tubes F1. There observed no formation of gas -
indication for gas (Durham tubes) shows negative results. However, it is able to
see the a small amount of spores forming, and turbidity in the growth medium.
2. Discussion
The result is 000.
All three sets of tubes are considered negative result since no gas emission
was detected: neither did the Durham tubes rise up from the bottom of the
tubes nor the gas bubble form inside the tubes. However, there appears
formation of white and opaque cells in all sets of tubes. Out of three set of
tubes, the set F1 with doubled medium strength shows the most significant
formation of spores: the medium color has the most noticable change
compared to other sets. From the FDA’s Bacterial Analytical Manual [2], the
result with no positives tubes specifies that MPN per 100 mL is lower than 3.0
and MPN limit at 95% confidence level ranges from 0 to 9.5. This result is
reasonable because the sample chosen is drinking water, which should remain
at the least possible of fecal contamination. It can be concluded that the
amount of coliforms in the sample from Lavie bottled product is not
considerate and product is safe to drink.
The MPN method comprise three distinct steps, in which the presumptive test –
the one being conducted in the experiment - is only the first one. The test is
presumptive only because under these conditions several other types of
bacteria can produce similar results. To obtain an accurate estimation of
coliform concentration in water samples, a confirmary test and completed test
should also be conducted. However, there are disadvantages of the MPN
method. The accuracy and precision is indeed questionable because of its
inherent variability, whose obtained result come with a relatively large margin
of error. The probability of obtaining false positives is another limitation,
because the test is evaluated by human eye. [3]
IV. Appendix
[1] Microbiology Lab Manual – 2023.2 version.
[2] The Most Probable Number Method and Its Uses in Enumeration,
Qualification, and Validation | Scott Sutton
[3] Most Probable Number (MPN) Test - Principle, Procedure, Results |
microbiologynote
