Universiti Tunku Abdul Rahman (Kampar Campus)

Faculty of Science, Engineering, and Technology

Bachelor of Science (Hons) Biotechnology

Year 2 Semester 1

UESB 2142 Laboratory 2A

(III) Microbiology

Lecturer: Dr. Teh Yok Lan

Student’s Name: Cheah Hong Leong

Student’s ID: 08AIB03788

Experiment No.

1: The culture of Microorganisms

4: Determination of Microbial Numbers

Date: 22 June 2009

Title: The Culture of Microorganisms and Determination of Microbial Numbers

Objectives:

- To learn the techniques and precaution steps in culturing microorganisms in Broth

culture, streak plate culture, pour plate culture, and spread plate culture.

- To determine the colony-forming units per ml through spread plate culture by

counting the numbers of colonies formed.

Result:

A. Broth culture

Table: Description on Broth Cultures Appearances after Inoculation

Broth Cultures Observations

Control Clear solution without any changes of color and turbidity of

solution.

Escherichia coli The solution had becomes more turbid and cloudy than control

solution. The color of solution also turned slightly yellowish.

Micrococcus luteus The color of solution turned yellowish. Pellicles were observed

appeared in the solution.

B. Streak plate

C. Pour plate (Loop dilutions)

Table: Numbers of Colonies Formed on Agar Plate with First, Second, and Third Loop

Dilution

Loop dilution Numbers of colonies formed

First 4

Second 0

Third 0

Confluent growth, no isolated colony was observed at the beginning of the streak. Isolated colonies were observed to appear at the end of the streak.

No isolated colony, only confluent growth was observed at the beginning part of the streak. Only few isolated colonies were observed at the ending part of the streak.

D. Spread plate

Table: Numbers of Colonies Formed on Agar Plate

Dilution Numbers of colonies Total *cfu/ml

Yellow Red

*Water solution (undiluted) 150 11 161 1610

First dilution (10-1) 29 4 33 3300

Second dilution (10-2) 3 0 3 3000

Third dilution (10-3) 0 0 0 0

*cfu/ml: colony-forming units per ml of the original water sample

* Some colonies were found to be overlapping each other

Discussion:

The observations on broth cultures can be explained by the arrangement and mobility

characteristics of the two different bacterial species. Escherichia coli usually exist as

single rod shape form and they are mobile by the presence of flagella. Therefore, the

Broth culture tends to appear turbid in showing the growth of Escherichia coli

population. Micrococcus luteus usually exist in clusters and they are immobile. Hence,

the Broth culture tends to form pellicles. Sediment might also form in the culture with

Micrococcus luteus but it was not observed to appear in the tube. One possible reason is

the sediment was already swirling up before the observation was made.

The main purpose of both techniques for streak plate method and loop dilution pour plate

method is to form well-isolated colonies so that pure culture can be obtained. For both the

techniques of streak plate method, isolated colonies were obtained at the end of streak.

Loop dilution pour plate method also showed isolated colonies but only four colonies

were formed on the plate of first dilution and colony was absent for the rest of the plates.

One possible reason is the water sample was not mix well before inoculation;

microorganisms might not concentrate in the surface area of the water sample where the

inoculation loop was immersed.

The cfu/ml obtained from the undiluted water sample was largely different from the

cfu/ml obtained from first and second dilutions. In agar plate of undiluted water sample,

some colonies were observed to overlap each other and some of the possible tiny colonies

were missed to be counted. Not all the inoculated microorganisms would form colonies at

the same rate; invisible tiny colonies formed due to short incubation period might be

missed when counting. The cfu/ml of undiluted water sample should be higher than that

of obtained (1610 cfu/ml).

The numbers of viable cells in the original water sample per ml should be higher than the

cfu/ml calculated through the spread plate method. There are many possible errors that

occurred in performing this method to estimate the microbial numbers per ml in the

original water sample.

Beside the difference in colony forming rate between each microorganisms, cfu/ml

cannot representing the numbers of viable cells per ml as a colony formed may derived

from originally one or more viable cells. For the water sample that containing more than

one microbial species, they might require different conditions for population growth into

colonies, the culture agar that was used might not suitable for some of the

microorganisms in the water sample and they were therefore not grown into colony. The

cfu/ml value might just underestimate the actual numbers of viable cells per ml in the

original sample.

Errors made by students also responsible for the underestimation of the actual numbers of

viable cells per ml of original water sample. One possible error made was the

inhomogeneity of the water sample and diluted samples when it was pipette to the agar

plate. The water sample and dilution samples were also might not spread evenly on the

agar plate, leading to the overlapping of colonies or clusters of cells developed into one

single colony. Technical error when using the micropipette might also affect the result.

The inhomogeneity problem can be solved by mix well the water sample and dilution

samples before transfers. The spreading procedure should be performed carefully to make

sure that the samples were spread evenly. Micropipette should be used properly so that

the volumes of samples transferred (0.1 ml) were consistent for each transfer.

References:

Campbell, N. A., Reece, J. B. (2005). Biology, 7th ed., San Francisco, CA: Pearson

Benjamin Cummings.

Madigan, M. T., Martinko, J. M., Dunlap, P. V., & Clark, D. P. (2009). Brock Biology of

Microorganisms, 12th ed., San Francisco, CA: Pearson Benjamin Cummings.

Attachment: Calculation of colony-forming units per ml of the original water sample for

each dilution.

Colony-forming units per ml, cfu/ml of original sample

= [(Numbers of colony formed)/0.1 ml]/Dilution

For undiluted water sample, cfu/ml of original sample = [161 colonies/0.1 ml]/1

= 1610 cfu/ml

For sample of first dilution, cfu/ml of original sample = [33 colonies/0.1 ml]/10-1

= 3300 cfu/ml

For sample of second dilution, cfu/ml of original sample = [3 colonies/0.1 ml]/10-2

= 3000 cfu/ml

For sample of third dilution, cfu/ml of original sample = [0 colony/0.1 ml]/10-3

= 0 cfu/ml