RESULT

Violet Red Bile agar (VRBA), Pour plate technique:

Microorganism: Escherichia coli

Dilution No. of Colonies
factor 100 10-1 10-2 10-3 10-4 10-5 10-6
Plate 1 TNTC LA LA 122 TSTC LA TSTC
Plate 2 TNTC LA LA 15 TSTC LA TSTC
Average TNTC LA LA 69 TSTC LA TSTC
Table 1: The Total number of colonies for coliform bacteria that represent in each dilution
factor observed after 18 - 24 hour hours incubation at 350C.

Calculation:

Dilution factor of plate counted: 10-3

𝟏
𝟔𝟗 ×
𝟏𝟎−𝟑 = 6.90 x 104 CFUs per mL
𝟏 𝒎𝑳
Number of CFUs sample: 𝟔. 𝟗𝟎 𝐱 𝟏𝟎𝟒 𝐂𝐅𝐔𝐬 𝐩𝐞𝐫 𝐦𝐋

Brilliant Green Lactose Bile (BGLB), Most Probable Number (MPN) technique:

Microorganism: Escherichia coli

Dilution Positive/negative (+/-) of gas production
factor 100 10-1 10-2 10-3 10-4 10-5 10-6
Tube 1 + + + + + + +
Tube 2 + + + + + + -
Tube 3 + + - + + + +
Number of 3 3 2 3 3 3 2
positive
tube(s)

Table 2: The total numbers of positive tubes for each tube in each serial dilution factor
observed after 24 and 48 hours of gas production.

Based on the 3-tubes MPN table, MPN in the inoculum of the middle set of tubes is 11 x 105
CFUs per mL
DISCUSSION

Coliform bacteria are defined as rod-shaped Gram-negative organisms which ferment

lactose with the production of acid and gas when incubated at 350C. Coliforms are abundant
in the feces of warm-blooded animals, but can also be found in the aquatic environment, in
soil and on vegetation. In most instances, coliforms themselves are not the cause of sickness,
but they are easy to culture and their presence is used to indicate that other pathogenic
organisms of fecal origin may be present like Escherichia coli (E. coli) bacteria used in this
experiment. Based on these two methods result, it is clearly that coliforms were found in
taken water sample.

There two types of culture media that being used in these experiment to determine the
coliform bacteria that is Escherichia coli. The sample that is being used in this experiment
was taken at a water fountain. The first method is by using Violet Red Bile Agar (VRBA) and
the second method is Brilliant Green Lactose Bile (BGLB). VRBA method refers to a
medium for the enumeration of coliform organisms in food and dairy products.

The selectivity of the medium was due to the presence of bile salts and crystal violet.
Lactose fermenters produce red/purple colonies often surrounded by a halo of the same color.
Non lactose fermenters produce pale colonies. Selectivity can be increased by incubation at
42-440C. Meanwhile for the BGLB method, the objective was same like VRBA. But the
difference was their developing the coliform bacteria by water bath for 24 hours to develop
gas production in a test tube containing Durham tubes as a result of lactose fermentation.

The VRBA technique were related to the pour plating method where the sample water
were taken under serial dilution (100 – 10-6) in order to obtain certain range of total bacteria
count in each plate. The plates inverted and incubated at 350C for 24 – 48 hours under aerobic
condition for detection of colony formation. Escherichia coli were isolated and cultured in
VRBA medium by using pour plating method.

For the BGLB technique, the sample was diluted by using the same serial dilution as
in VRBA technique manner up to 10-6 dilution factor. Then it was transferred to each tube of
BGLB broth that comes with inverted Durham tube and was incubated at 350C. Gas
production was then observed after 24 to 48 hours incubation.

Based on the results from the experiment above, there are two types of method in
enumeration and determine coliform bacteria. The result must be observed 24 hours after the
experiment for VRBA medium. Based on the result that we observed for VRBA pour plate
method in the petri dish, there’s a bacterial contamination observed and can be countable is
serial dilution factor 10-3 which gave result 6.90 x 104 CFUs per mL of sample.

Inoculated tubes were incubated at 350C for 48 hours. Positive tubes (gas collected in
Durham’s tube) were recorded and count was calculated using MPN tables. For MPN
method, the result after being observed 48 hours almost all tube contains gas production.
These positive tubes give result11 x 103 CFUs per mL .

The methods were Most Probable Number or MPN and pour plating method. From
the results obtained as above, plate count method by using VRBA has the lower number of
total bacteria plate count which is 6.90 x 104 CFUs per mL than the MPN method which
is 11 x 105 CFUs per mL.

Relatively, MPN methods give higher values for coliforms population than the plate
counts methods due to the greater sensitivity of liquid media in MPN methods rather than
with solid plating media. The high level of contamination of water sample is due to the water
sources that are drain from the lab.
CONCLUSION

As a conclusion, these experiments are about to familiarize with the enumeration techniques
employed for coliform bacteria. It is also to determine coliform bacteria form by using Most
Probable Number (MPN) method. Based on the result shows by using the violet red bile agar
(VRBA), the calculation shows that the coliform form is lower, 6.90 x 104 CFUs per mL than
the brilliant green lactose bile (BGLB), 11 x 105 CFUs per mL. It is because the BGLB only
shows the amount of bubble in the tube while for the VRBA shows the colony of the coliform
on the plate of agar. Besides, sensitivity to detect the microorganism is more likelihood liquid
media rather than solid plating media. Nevertheless, the presence and growth of coliform
microorganisms may lead to hazard against public health. Therefore, it is highly
recommended that hygienic practices and regulations should be applied for consumer and
water treatment as water is a major source for human need.