Bacterial Colony Isolation using Serial Dilution Techniques

Al Jay Mejos, John Warner Carag, Jojiemar De Pano, Allison Vincent Labador, Erika Mari Macapagal
Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City

ABSTRACT
A pure culture of an isolated microorganism can be isolated and quantitatively counted using serial dilution techniques. Along with serial dilution,
spread and pour plating methods were done in order to count the number of isolated colonies in a sauce sample. Serial dilution was done by getting
a small amount of the sauce sample and transferring it subsequently to the test tubes. Next spread and pour plating were performed for the last
four dilution of the original sauce sample (10-8, 10-7, 10-6, and 10-5). This was done in order to differentiate the two in terms of procedure and
growth of microorganisms. Lastly, the colony forming units per mL (CFU/mL) was obtained using the average of the cell counts in both plating
techniques. Most of our spread and pour plates gave a spreader result except for one replicate of the 10 -7 spread plate which gave a TNTC.

INTRODUCTION All plates were incubated in an inverted position for 24 hours at

Count of viable cells is important in measuring the amount of 37C, and then stored in the refrigerator. Observations were made
microorganisms in a sample. One of the most common methods of the following week and the colony forming unit per mL (CFU/mL) of
quantifying microorganisms is growing them in a nonselective plate each dilution was calculated.
culture medium with the assumption that a single colony came about
from a single bacterial cell (Madigan & Martinko, 2006). Growing RESULTS
microorganisms directly from a small amount of sample is not Table 1. Number of colonies counted and CFU/mL in spread plate
practical to use in experiments due to the numerous number of Spread plate dilution Trial 1 Trial 2 CFU/mL
microbes that would grow from it. The large number of colonies 10-5 Spreader Spreader NA
overlapping each other when counted gives an inaccurate 10-6 Spreader Spreader NA
10-7 TNTC Spreader NA
representation of the actual number of microorganisms. Serial
10-8 Spreader Spreader NA
dilution, or making ten-fold dilutions of the sample, is implemented
in order to eliminate this problem. By having more diluted assays it Table II. Number of colonies and CFU/mL in pour plate
is relatively easier to produce countable results. Serially diluted
Spread plate dilution Trial 1 Trial 2 CFU/mL
assays can be plated using the spread plate and pour plate methods. 10-5 Spreader Spreader NA
Spread plate technique uses about 0.1 mL of the inoculum; which is 10-6 Spreader Spreader NA
then placed and streaked at the center of the agar plate by a 10-7 Spreader Spreader NA
sterilized hockey stick. The pour plate uses about 1.0 mL of the 10-8 Spreader Spreader NA
inoculum placed in an empty sterile Petri plate with the melted agar
mixture to be poured in only when it is at the right temperature DISCUSSION
(not hot enough to kill the microorganisms present and not cold Colonies grown in Petri dishes by various methods may be used
enough for the agar to solidify prematurely). One disadvantage of to determine the count of viable microorganisms. Plate counts
pour plates is that embedded colonies will be much smaller assume that every colony is founded by a single cell. That cell must
compared to those on the surface; it must be carefully counted so have been alive in order to grow and form a colony. The number of
that none are overlooked. Also, obligate aerobes may grow poorly if colonies obtained in a viable count experiment depends not only on
deeply imbedded in the agar (Fankhauser, 2003). One advantage of the inoculum size and the viability of the culture, but also on the
pour plating is that it is preferred for isolation of microaerophilic sustainability of the culture medium and the incubation conditions.
species since it traps the microorganisms inside the agar where it is (Madigan et al. 2009)
safe from the high concentration of oxygen in the atmosphere. A viable count is a direct counting method in which only viable
cells are counted. It can be accomplished by spread plating or pour
MATERIALS AND METHODS plating and by direct counting the colonies grown on the plates. In
Beef kaldereta sauce was obtained from Mongolian in CASAA in the spread-plate method, only a small volume of the inoculum was
order to be quantitatively tested for microorganism content using spread over the surface of the prepared plate to avoid excess liquid
serial dilution techniques. Culture media preparation was done that may soak in and cause the colonies to coalesce, making them
before the experiment proper. An NA agar solution equivalent to 8 difficult to count. Colonies are expected to grow only on the
NA plates was placed in an Erlenmeyer flask to be used later in pour surface of the medium. In the pour-plate method, a larger volume
plating. On the other hand, eight NA plates were also prepared for of the inoculum may be used since it is mixed with the melted agar
spread plating. medium. However, in this method, the desired organism must be
During serial dilution, eight test tubes containing 4.5 mL of 0.1% able to withstand brief exposure to heat when mixed with the
peptone water were prepared. 0.5 mL of beef kaldereta was melted agar. Colonies from this method are expected to grow not
obtained and transferred into the first test tube labeled 10-1. The only on the surface of the medium but all through out the entire
test tube was mixed. Used pipette tips were discarded into a beaker medium.
containing 70% ethanol. Likewise, similar amounts (0.5 mL) of the In order to be able to provide significant results and execute a
sample coming from the previous test tube were transferred to the reliable colony count, cell suspension containing the desired
next test tube from 10-2 to 10-8. microorganisms must be diluted first to an appropriate
Using the last pipette tip, 1 mL was drawn from the last tube into concentration. Serial dilution will provide dense culture of lesser
two Petri plates while 0.1 mL was transferred into two NA plates. concentration that will provide us an inoculum of much lesser
These four were labeled with 10-8. Similarly, subsequent amounts of amount of organism that will grow colonies on the plate and
diluted samples in order from 10-7 to 10-5 were transferred into the expected
Petri plates and NA plates.
In spread plating, a hockey stick was sterilized in 70% ethanol and REFERENCES
exposed to the alcohol lamp. This was used to spread the sample http://www2.hendrix.edu/biology/CellWeb/Techniques/microspread.html. by
evenly across the agar surface in all NA plates labeled10-8, 10-7, 10-6, Dr. Mark Sutherland. Date accessed: July 19, 2009.
http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Meat_Milk/Pour_Plate.
and 10-5. The agar was left to absorb the sample for 10 minutes. In
htm. by Dr. David D. Fankhauser. Date accessed: July 19, 2009.
pour plating, approximately 15 ml of NA was poured to the plates Madigan MT & Martinko JM. 2006. Brock Biology Of Microorganisms, 11th ed.
containing diluted amounts of 1.0ml of sample culture. The plates USA: Pearson Prentice Hall, Pearson Education, Inc. 140-147.
were then mixed, and afterwards set aside for solidification. http://www.mansfield.ohio-state.edu/~sabedon/biol4038.htm
Figure I. Serial Dilution, Spread Plating and Pour Plating

Figure II. Spreader result in Spread Plate

Figure III. Spreader result in Pour Plate

Figure IV. TNTC result in Spread Plate