Christian C.

Banania

BSFT 2B

Submitted on Dec, 23 2021

Laboratory Activity # 2 and # 3

Basic Microbiology Calculation

Serial Dilutions and Plating: Microbial Enumeration

Questions and answers

1. What is the importance of serial dilution and plating techniques in food microbiology
laboratory?

In a food microbiology lab, serial dilution and plating technique are critical. The successive
dilutions aid in the isolation of germs from a Winogradsky column. It also aids in reducing the
concentration of bacteria by re-suspension in predetermined liquid volumes of diluent and is
used to organize the reduction of an organism's growth by a multiple of 10. The enumeration of
an organism or bacteria is aided by serial dilution. Because the quantity of colony in a liquid
dilution is unknown, plating procedures come in helpful because they aid in the isolation and
enumeration of the sample.

2. Which plating technique should be used if the aim is to observe colony morphology?
What about if the purpose is to estimate the number of colony forming units per millilitre
in a given suspension?

The streak plating technique is used to observe colony morphology during serial dilution.
As indicated in the video clip, the streak plating technique aids in the differentiation of
two separate bacterial species. After serial dilution, the spread plating technique will be
used to estimate the number of colony forming units per millilitre (CFU/mL). A spread
plate is prepared for each dilution to identify the numbers of each colony.

3. What is the time and temperature/pressure usually employed in the sterilization of media
and diluent? Why?
The time and temperature for sterilizing media and diluent is 15 to 20 minutes at 121
degrees Celsius. The reason for this is that before working with the agar media, it must be
melted without being burned before being transferred to separate containers.

4. . Why plates containing colonies fewer than 30 or more than 300 are eliminated from
counting?
A colony multiplies in the agar plate after serial dilution. There are plates with more than 300
and plates with less than 30. For calculating the dilution factor and CFU/mL, only the plate with
colonies between 300 and 30 is used. Because there may be flaws in the final product or data,
colony counts larger than 300 and less than 30 are deleted. More over 300 colonies can lead to
data underestimate, whereas less than 30 colonies can lead to more inaccuracies.

Problem
 Supposed the spore suspension has a titer of 6.5 x 106 spore per ml. Dilute the spore
suspension to obtain a quantifiable counts when plated in nutrient media. First, transfer volume
(TV1) from the spore flask into the 10.0 ml DV1, then transfer TV2 from the 10.0 ml DV1 into the
100.0 ml DV2, and lastly plate PV. For TV1, TV2, and PV there are two values, 0.1 and 1.0 ml.
Calculate dilution factors and spores per millilitre.
A. Illustrate and describe the dilution scheme.
B. Calculate colony-forming units (CFU) per plate for each of the options below. (Use
spores per ml instead of CFU per ml).

Table 1. Dilution Factor and Colony-Forming Units (CFU/ml)

Item No. Dilution Factor Spores/ml

1 9.0009 x 10-4 7.2 x 109

2 9.0009 x 10-5 7.22 x 1010

3 9.803 x 10-5 6.63 x 1010

4 9.803 x 10-6 6.63 x 1011

C. Show your solutions here.

k
 D. Conclusion
Each number has a titer of 6.5 x 106 per spores/ml, regardless of TV1, TV2, DV1, DV2, or
PV. The Dilution Factor totals 9.0009 x 10-4, but the spores/ml is 7.2 x 109, when TV1 and TV2
are equal to 1.0 ml, DV1= 10.0 ml, DV2= 100.0 ml, and PV is equal to 1.0 ml. The spores/mL is
7.22 x 1010 when the dilution factor is 9.0 x x 10-5. The DF result will be 9.803 x 10-5 and its
spore value will be 6.63 x 1010 if the TV1=0.1 ml and TV=1.0 ml while the DV1 and 2 remain in
contact. The TV1 and 2 and Dv1 and 2 do not change for the fourth number, but the PV
increased to 0.1ml, implying that the DF is 9.803 x 10-6 while the spores/ml is 6.63 x 1011.