MCR200L: QUANTITATION LAB REPORT GROUP 4

I. INTRODUCTION

Microorganisms have structures and functions that can be analyzed through various
techniques including qualitative and quantitative analysis. Evaluating the bacterial growth of a
sample provides the use of both analysis. For qualitative analysis, the growth of the bacteria on
a solid medium to strictly assess whether the bacteria in the sample are living or dead will be
observed. On the other hand, qualitative analysis is the use of the solid growth media to
calculate the actual number of living bacteria on the sample. This laboratory report will focus on
the quantitative analysis of microbial growth specifically, Micrococcus luteus through standard
viable plate count. Objectives of this experiment is to acquire and develop the skills in the
performance of the pour-plate method in the isolation of microorganisms and to determine the
viable cell number in a culture.

II. MATERIALS AND METHODS

The materials used in the experiment are sterile petri dishes, sterile serological pipette,
beaker of disinfectant, 0.1% peptone broth, 0.85% saline (NaCl) solution, Plate Count Agar
(PCA), Nutrient Agar (NA), L-shaped rod, regular sized test tubes, Tryptic Soy Agar (TSA), and
distilled water.

Pour-Plate Method: 6 test tubes of diluent containing 9.0mL each of 0.1% peptone
broth or 0.85%saline (NaCl) solution was prepared and labeled as 10 -1, 10-2, 10-3, 10-4, 10-5 and
10-6. . Likewise, label in small prints 6 sterile plates in duplicate as 10 -4, 10-5 and 10-6. . Then, an
aspirator was used to aseptically pipet 1.0mL from 24 hour old culture broth into the dilution
blank marked as 10-1. The contents of 10-1 was mixed by rotating the tube between the palms of
hand and the pipettes used was dipped in a beaker of 0.5% bleach disinfectant. 1.0mL from the
test tube 10-1 was transferred to 10-2 test tube using a second serological pipet then mixed while
the serological pipet was discarded in the disinfectant. 1.0mL is then transferred from test tube
10-2 to 10-3 using a third serological pipet. The contents of test tube 10 -3 was mixed while the
third pipet was discarded n the disinfectant. Then, 1.0mL from the content of 10 -3 was
transferred to 10-4 test tube using a fourth serological pipet, mixed then discarded in the
disinfectant. The same process was used for test tubes 10 -5 (1.0mL from 10-4), 10-6 (1.0mL from
10-5 and 10-7 (1.0mL from 10-6). 1.0 mL contents from test tubes 10 -5, 10-6, and 10-6 were also
transferred to their corresponding plates.

15-20mL of molten (45-50 C) sterile PCA or NA was aseptically poured into each of the
plates then swirled to spread the agar in the plate. Plates with harden agar was then inverted
and wrapped in newspaper and enclosed plastic to prevent fruit fly infestation and incubated for
2 days at 37 C or room temperature (in our group cabinet). After incubation, the plates with 30-
300 colonies was counted using a permanent marker to prevent counting of colony twice. All
colonies are counted both on the surface and those embedded in the agar medium. Average
number was acquired from duplicated plates to compute for the colony forming units per mL of
the 24 hour old broth.

CFU Average number of colonies x inverse of Dilution Level

=
mL Volume of cell suspension plated

Spread Plate Method: 400mL of Tryptic Soy Agar (TSA) was prepared and sterilized by
autoclaving at 121 for 15-20 minutes and poured individually into 6 sterile plates that solidified.
10-5 was labeled to 2 plates, 2 plates as 10 -2 and so on until 10-6. A 10 fold serial dilution of the
24 hour old broth culture was prepared in the same manner as in the pour plate method. Again
6 test tubes containing 9.0mL of sterile 0.85% saline or 0.1% (0.1g peptone in 100ml distilled
water) peptone was prepared. Then, an aspirator was used to aseptically pipet 1.0mL from 24
hour old culture broth into the dilution blank marked 10 -1. 1.0mL from the test tube 10-1 was
introduced to test tube 10-2 using a serological pipet and then mixed while the pipet was
discarded to the disinfectant and so on until 10-6. The same process as in the pour plate
method was applied except the amount of content transferred to TSA plates was 0.1mL instead
of 1.0mL (for pour plate method). Then, an L-shaped rod was dipped in 95% ethanol and
touched to the walls of the beaker containing the ethanol to remove excess alcohol. The rod is
then used to spread the content on top of the hardened agar. This is done to all 6 plates then
were wrapped inverted in newspaper and enclosed tightly in plastic bags. Plates were incubated
for 2 days in the cabinet. The computations for the CFU/mL is the same as the computation
used in pour plate method.
 III. RESULTS AND DISCUSSION

Table 1.1
Spread Plate Method Pour Plate Method
Plate 1 Plate 2 Plate 1 Plate 2
10-5 105 95 TNTC TNTC
10-6 20 13 60 152
10-7 3 2 4 9

Figure 1.1

SPREAD PLATE
METHOD. Results show

that there is a decreasing trend in the number of colonies formed as the level of
dilution increases. Plates 1 and 2 with a dilution level of 10 -5 formed 105 and 95
colonies respectively. With a dilution level of 10 -6, the colonies formed were
significantly reduced to just 20 for plate 1, and 13 for plate 2. Finally for plates with
a dilution level of 10-7, plate 1 only had 3 colonies formed while plate 2 only had 2
colonies.

Figure 1.2. Left most picture shows the plates with dilution level of 10 -5. Middle picture shows the
plates of dilution level 10-6, and last picture shows plates with dilution level of 10 -7.
POUR PLATE METHOD. Results also share the same trend of decreasing
colonies present as the levels of dilution increases. Both plates with a dilution level
of 10-5 had colonies counting over 300. Only about one quadrant has been marked
and the colony count has already reached over 250, thus both plates were labelled
as TNTC, or too numerous to count. As for the plates with dilution levels of 10-6,
plate 1 had 60 colonies while plate 2 had 152 colonies formed. Plates with dilution
levels of 10-7 had the least number of colonies formed, with only 4 and 9 colonies
for plates 1 and 2 respectively.

Figure 1.3. Left most picture shows the plates with dilution level of 10 -5. Middle picture shows the
plates of dilution level 10-6, and last picture shows plates with dilution level of 10 -7.

As seen with the tables and figures, both the spread plate and pour plate
method follow the same sequence of decreasing colonies present in plates with
higher dilution levels. With the initial step of placing 1.0ml of the previous test tube
into a new one containing 9ml of NaCl, this allows the next test tube to be more
diluted than the previous one. Thus, the ratio of the number of microorganisms
present to the volume is decreased. Therefore when a certain volume of the diluted
solution is plated (0.1ml for spread plates and 1ml for pour plates), the number of
microorganisms that will be plated will be related to that of the said ratio. With this,
having a more diluted solution means having a smaller number of microorganisms
present per ml of the solution. This is evident as plates with a dilution level of 10-5,
which is the least diluted solution among the three dilution levels plated, had the
most number of colonies formed, both for the pour and spread plates. As the
dilution levels increase, the colonies present are significantly reduced in each plate,
again for both pour and spread plates.

In addition, it can also be inferred that among the two methods, the pour
plates generally have more colonies compared to those of the spread plates. This
may also be in relation to the volume used in transferring the diluted sample to the
plate. The volume used for spread plates is 0.1ml, while 1ml was used for pour
plates. This may have been the reason as to the difference in the number of
colonies present, since increasing the volume may also increase the amount of
microorganisms present.

Moreover, the pour plates may also have had higher numbers of colonies
present since this allows the colonies to form both in the surface and the
subsurface, since the sample was introduced in the plate first followed by the
pouring of the media. By increasing the area and/or surface area that is allotted for
the microorganisms to grow on, it may translate into having more colony formation
and growth. As for the spread plates, the microorganisms can only grow on the
surface of the media since the sample was only introduced in the plate after the
media had been placed and had been dried already. This may limit the number and
growth of the colonies.

In terms of the media used, specifically Tryptic Soy Agar (TSA) and Plate
Count Agar (PCA), these cannot be used as a basis for the difference and
comparison of the number of microorganisms present since neither of the two
medium are considered as selective media. TSA is a general medium, which is
usually suitable for enumeration and counting of bacteria. PCA is also a general
medium that is commonly used for quantifying microorganisms, or rather bacteria,
present.

IV. CONCLUSION
V. REFERENCES (APA FORMAT)