EXPERIMENTAL WORK IV

“ANALYSIS OF YEAST AND MOLD IN TOMATO SAUCE

SAMPLE”

Group-1
Manisha Subedi
Namrata Khanal
Priyanka Gharti

Date of the Experiment: 10/7/2023

Date of the Accomplishment: 10/11/2023
Summary
The main objective of the experiment was to analyze the presence of yeast and molds in a tomato sauce
sample. For the experiment, series of decimal dilution were carried out from the initially prepared
suspension (25 gm tomato sauce and 225 ml of sterilized water) using the stomacher. DG18(Dicloran 18%
Glycerol Agar) was used as the culture medium as recommended by the ISO 6887-1 Standard and is rich
in nutrients for the growth of yeasts and molds coagulase. For the experiment, series of decimal dilution
was carried out for 1 mL of mother solution sample (10−1 ) using ringer solution. For the inoculation of the
medium, o.1mL from each dilution tube, in our case from sample and dilution factor 10−2 , 10−4 and 10−5
(Group 1) were inoculated by spreading technique in Yeast agar medium at 25°C for 7 days.

Yeasts and molds were obtained and the concentration of yeasts-molds in the sample was found to be
𝐶𝐹𝑈
2.8 × 103 𝑔
.

Experimental Observations and Results

The obtained values of CFU count for each petri plates of each group are tabulated below in Table 1:
Table 1: Values of CFU count obtained for each decimal dilution obtained in petri plates incubated at 25°C
for 7 days.

Group 10−1 10−2 10−3 10−4 10−5 10−6

G1 - 2 0 0 0 -

G2 18m 2 0 0 - -

G3 19m 1 - 0 0 -
1y
G4 43m - 1 0 0 -
1y
G5 - 2 1 - 0 0

G6 25m 9 0 - - 0
1y

From the above table, i.e., Table 1, The CFU count for 10−1 dilution performed by G2, G3, G4, and G6
were found to be 18 (m), 20 (19 m + 1 y), 44 (43 m + 1 y), and 26 (25 m + 1y) respectively. The CFU value
i.e., 44 obtained by G4 is much higher compared to other groups. There could be two possibilities either
G4 agitated mother solution much better or inoculated volume higher than 0.1 mL. Similarly, the CFU value
for 10−2 performed by G1, G2, G3, G5 and G6 were found to be 2, 2, 1, 2, and 9. The value 9 obtained by
G6 is much higher than it was supposed to be (3/4). The error in pipetting inoculated volume could have
led them to the greater CFU value. The CFU value for 10−3 obtained by G1, G2, G4, G5 and G6 were
found to be 0, 0, 1, 1 and 0 which is in accordance with the decimal dilution principle. Likewise, 0 colonies
were found for all the samples of decimal dilution 10−4 , 10−5 𝑎𝑛𝑑 10−6 .

Calculations
The value of the most precise value was taken (i.e., the value that satisfied the counting criterion). In this
case, the dilution 10−1 is the lowest decimal that obeys the counting criterion, CFU< 150 was taken and
the concentration of the yeasts and molds in tomato sauce sample was calculated using the equation below;

𝐶𝐹𝑈 ∑𝐶
N( ) = ……. (Equation 1)
𝑔 (𝑛1+ 0.1𝑛2 )𝑑×𝑉(𝑚𝐿)

Where,
N – number of coliforms per mL sample (CFU/mL)
∑𝐶 – sum of the characteristic’s colonies counted on the plates considered valid
𝑛1 - number of plates where counting was made in the lowest valid dilution
𝑛2 - number of plates where counting was made in the second dilution
d- value of the lowest dilution where the counting was made
V- inoculated volume of sample or decimal dilution, mL
From Table1
The mean of the counted colonies in the plates,
∑𝐶 = 18 + 20 + 44 + 26 + 2 + 2 + 1 + 2 + 9 = 125 CFU ……... (Equation 2)
𝑛1 =4
𝑛2 = 5
d= 10−1
V= 0.1 mL
Now,
The number of coliforms per mL of water sample (CFU/ mL);
𝐶𝐹𝑈 125
N( ) = ………. (Equation 3)
𝑔 (4+0.1×5)0.1×0.1

= 2.8 × 103
𝐶𝐹𝑈
Therefore, the concentration of yeasts-molds in tomato sauce sample was found to be 2.8 × 103 .
𝑔
Images
The typical colonies of yeasts are white and that of molds are fuzzy. The images showing the inoculation at
four different dilutions 10−1 , 10−2, 10−3 and 10−5 are below:

Figure 1: Yeasts and molds colonies in 10−1(G4), 10−2 , 10−3 and 10−5(G1).
10−1: 43m + 1 y (G4)

10−2: 2 y + 0 m (G1)

10−3: 0 y + 0 m (G1)

10−5: oy + 0 m (G1)
Conclusion
The tomato sauce sample was contaminated with yeast molds. The concentration of yeast molds in the
𝐶𝐹𝑈
tomato sauce sample was 2.8 × 103 .The presence of yeast molds can cause severe health problems to
𝑔
both humans and animals. So, they should be stored in the refrigerator and used within a few days and
separate tomatoes from raw meat, poultry, and seafood because they could be contaminated from these
sources too.

Reference
Iso Standards