EXERCISE 9

Isolation of Bacteria to Pure Culture

Krisette, David; Fabros, Jamee; Relucio, Mike
Department of Biology, College of Science, University of the Philippines, Baguio City

Abstract
Proper isolation of microbes is important in all microbiological works. It separates a species of
microbes from one another by employing proper techniques. This is performed by a four-
quadrant streak and swabbing in an aseptic environment. At the end of the experiment, colonies
of Bacillus subtilis anf Eschirichia coli were isolated and observed while microbes that were
obtained from swab method were quantitatively and qualitatively observed. It was therefore
concluded that proper separation techniques is important to isolate a specific species of microbes.
KEYWORDS
ISOLATION; Four-quadrant streak; Swab Method
I. Introduction
Microbial populations often occur in aggregates and in order to study their characteristics and
behavior, one must first culture them through contaminating species by purification or isolation
techniques. The aggregates of microorganisms results from one parent cell that is spread through
the medium. Isolating a single cell of microorganism is achieved by streak plating.
The objective of this exercise is to train students how to prepare pure cultures of bacteria by
streak plating and swabbing.
II. Methodology
. A. Streak Plating
The inoculation loop was flame-sterilized by holding it directly above the flame. The
loop was cooled by pressing it to the side of the agar for 5 seconds. The cultures from the
previous experiments were used. A small portion of the colony from the slants was lifted using
the loop and was streaked along one side of the agar in the petri plate. It was repeated until there
were at least 6 streaked lines. The loop was flamed again to kill all residual cells. The plate was
rotated clockwise and then streaking was repeated. It was rotated clockwise so that the next
streak was perpendicular to the first streak. Rotation was repeated again but this time the streak
only touched the previous streak once and the succeeding lines were prevented from touching the
previous streak. On the fourth streak, the plate was rotated again and then the streaks glided over
the middle of the agar. The loop was flamed and cooled every after streaking. Finally, the plates
were incubated inverted inside the autoclavable plastics. The microorganisms were picked up
from isolated colonies for morphological studies.
B. Swab Method
Sterile swabs were dipped into peptone for about twenty minutes. The swabs are then
swabbed to a test area. It is then dipped again to the petone solution. Using a micropipette, 100
microliters of the peptone where the swabs were dipped are transferred in the media and spread
using a hockey stick. The media with the peptone solution then was incubated and were observed
after 24 hours.

III. Results and Discussion
A. Streak Plating
Streak plating is one of the most common and classical method in isolating bacteria into
pure cultures. This is done by using the inoculating loops to be streaked gently over the agar
surface to isolate colonies on at least a portion of the plate. In this exercise, the group practiced
quadrant streak technique. Quadrant streak technique allows sequential dilution of the original
microbial material, Escherichia coli slant (Thiel, 1999). An ideal results is to obtain an isolated
colonies. The area of initial inoculation and first streak yielded heavy growth. The area of the
2nd streak yielded to a less dense growth. The area of the third streak yielded a weak growth.
Lastly, the area of the fourth streak yielded to single colonies. This can be obtained by using a
small amount of bacterial slant on the inoculating loops to be used and by having a dry, free from
water droplets of condensed moisture on the the surface of the plate. E. Coli is a gram negative
rod shaped bacteria mostly studied and well-understood in the field of Microbiology. After the
agar plates had been incubated, it was observed that E. Coli appeared creamy-white in color and
circular in shaped, smooth surface, filamentous in margin, and elevated/ raised.
B. Swab Method
Swab technique can be studied qualitatively and quantitatively. Qualitative study is used
to determine and to identify the microbes present by the distinct morphological features observed
from colony to colony. This aids in identifying what microbes, bacteria to be specific, are
present.
Since swab method produces a semi-quantitative result, quantitative test is conducted.
This is commonly done to test cleaning verification. It is an easy technique in determining and
verifying salinity of objects, whether certain pathogens are present or not. The count of organism
is not relevant. However, an estimate of bacterial count per 100cm2 can be used as an indication
of cleanliness (NSW Government Food Authority, 2003)
In this exercise, not all the class were able to perform this method. Two out of five
groups were able to conduct this exercise. However, all class should be able to observe the step
by step process in doing the swab technique. The results from the two groups were used as the
basis of the result of this report. In qualitative study, as in figure 1 at the appendix, the number of
colonies observed are twenty-two. From that figure, it is showed that the colonies are almost
circular in form and creamy-white in color. One of the colony appeared with little irregularity.
They have an entire type of margin and have raised type of elevation.
In quantitative study, the colony forming units (CFU) per cm2 was computed using the
equation written at the appendix. As observed in figure 2, the number of colonies formed were
too numerous. They reached 1450 in number as computed. Based on the theoretical results, it can
be concluded that the the table top or surface where the sterile swab was slowly rotated is over
contaminated, exceeding the standard value of CFU/ cm2 (<45 CFU/ cm2). *Refer to the table in
the appendix for the comparison of the CFU/ cm2 values.
Therefore, the table surface that was tested has a high probably of contaminating other
objects. If the microbes present are pathogenic than there is a possibility of infecting living
organisms, especially humans, leading to diseases such as diarrhea, gastrointestinal problems,
and so on and so forth.
On the other hand, clean surface can be considered if the surface is visibly clean, dirt and
bacteria are removed with water and detergent and a sanitized surface should be cleaned with a
secondary agent such as sanitizers, (Food Protection Services, 2010).
IV. Conclusion
Proper isolation techniques is important to separate specific colony if microbes. It is done by
distributing he cells in the agar. With the proper isolation technique, one may be able to isolate a
colony. Also, proper swabbing techniques will test if a specified area will test the cleanliness
thru qualitative and quantitative analyses.
V. Answers to Question
ANSWERS TO QUESTIONS
1. What is a pure culture? Why are pure cultures required in the laboratory?
- Pure culture is a culture containing a single kind of microorganisms (Madigan, 2009). Pure
cultures are required in the laboratory because they are used to differentiate species to the other
species.
2. How will you check the purity of an isolate?
-The purity of an isolate can be verified by :
i. Microscopy
ii. Observation of colony characteristics on plates or in shake tubes, and
iii. Tests of culture for growth in other media

Other advanced tools in verifying pure isolate is by laser tweezers. It is an in light inverted
microscope equipped with a strongly focused infrared laser and micromanipulation (Madigan,
2009).
3. Cite a practical application of the quantitative swab method.
-Quantitative swab method is practically used in environmental sampling and sanitary purposes.
For instance, food premises swabbing is often used in the investigation of food borne illness and
the verification of cleaning and sanitation in surfaces and utensils (Environmental Swabbing,
2003). This is commonly used for inspection and cleaning validation in putting up food
businesses. This is also used in testing non-arterial chronic wounds and other open wounds.
V. References
Clark, David P.; Dunlap, Paul V.; Martinko, J ohn M.; Madigan, Michael T. 2009. Brock
Biology of Microorganisms.12th ed., Pearson education, Inc.
Environmental Hygiene Monitoring. A guide for Environmental Health Officers. Version
3. 2010. Provincial Health Services Authority. Vancouver, BC V5Z 4R4. Ph:
604.707.2458. Retrieved last October 31, 2014 from
http://www.bccdc.ca/NR/rdonlyres/EF1461BE-0301-4A59-8843-420072412721/0/E
nvMonitoringHygieneGuideforEHOs.pdf
Environmental Swabbing. A guide to method selection and consistent technique. 2003. NSW
Government. Food Authority. Retrieved last October 31, 2014 from
http://www.foodauthority.nsw.gov.au/_Documents/science/environmental_swabbing.
pdf
Thiel, Teresa. 1999. Streaking Microbial Cultures on Agar Plates. Department of Biology,
University of Missouri-St.Louis. Science in the Real World: Microbes in Action.
Retrieved last October 31, 2014 from
http://www.umsl.edu/~microbes/streakplates.pdf

Appendix

APPENDIX
B. SWAB METHOD

Figure 1. Plate showing colonies produced in the qualitative swab method



Figure 2. Plate showing colonies produced in quantitative swab method





Figure 3. This figure shows the colonies number counted by marking each colony with different
colors. Each set of colors are composed of 50 colonies.
(29 sets of colors x 50 =1450)

COMPUTATIONS

CFU / cm
2
=Total count per plate x DF
2.5 cm
2

Where, DF = Dilution = 10
Volume plated 0.1
CFU / cm
2
=1450 x 100
2.5 cm
2

=58, 000


STANDARD VALUE OF CFU AND ITS INTERPRETATION

*This table was obtained from the pamphlet Environmental Hygiene Monitoring. A guide
for Environmental Health Officers. 2010.
Website: http://www.bccdc.ca/NR/rdonlyres/EF1461BE-0301-4A59-8843-
420072412721/0/E nvMonitoringHygieneGuideforEHOs.pdf

*This
table was obtained from the pamphlet Environmental Hygiene Monitoring. A guide
for Environmental Health Officers. 2010. Originally from a finish publication, Orion
Diagnosticsa, A Guide to Monitoring Surface Hygiene
Website: http://www.bccdc.ca/NR/rdonlyres/EF1461BE-0301-4A59-8843-
420072412721/0/E nvMonitoringHygieneGuideforEHOs.pdf