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 Count of viable cells is important in measuring the amount of microorganisms in a sample. One of the most common methods of quantifying microorganisms is growing them in a nonselective plate culture medium with the assumption that a single colony came about from a single bacterial cell (Madigan & Martinko, 2006). Growing microorganisms directly from a small amount of sample is not practical to use in experiments due to the numerous number of microbes that would grow from it. The large number of colonies overlapping each other when counted gives an inaccurate representation of the actual number of microorganisms. Serial dilution, or making ten-fold dilutions of the sample, is implemented in order to eliminate this problem. By having more diluted assays it is relatively easier to produce countable results. Serially diluted assays can be plated using the spread plate and pour plate methods. Spread plate technique uses about 0.1 mL of the inoculum; which is then placed and streaked at the center of the agar plate by a sterilized hockey stick. The pour plate uses about 1.0 mL of the 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 (not hot enough to kill the microorganisms present and not cold enough for the agar to solidify prematurely). One disadvantage of pour plates is that embedded colonies will be much smaller compared to those on the surface; it must be carefully counted so that none are overlooked. Also, obligate aerobes may grow poorly if deeply imbedded in the agar (Fankhauser, 2003). One advantage of pour plating is that it is preferred for isolation of microaerophilic species since it traps the microorganisms inside the agar where it is safe from the high concentration of oxygen in the atmosphere. MATERIALS AND METHODS Beef kaldereta sauce was obtained from Mongolian in CASAA in order to be quantitatively tested for microorganism content using serial dilution techniques. Culture media preparation was done before the experiment proper. An NA agar solution equivalent to 8 NA plates was placed in an Erlenmeyer flask to be used later in pour plating. On the other hand, eight NA plates were also prepared for spread plating. During serial dilution, eight test tubes containing 4.5 mL of 0.1% peptone water were prepared. 0.5 mL of beef kaldereta was obtained and transferred into the first test tube labeled 10-1. The test tube was mixed. Used pipette tips were discarded into a beaker containing 70% ethanol. Likewise, similar amounts (0.5 mL) of the sample coming from the previous test tube were transferred to the next test tube from 10-2 to 10-8. Using the last pipette tip, 1 mL was drawn from the last tube into two Petri plates while 0.1 mL was transferred into two NA plates. These four were labeled with 10-8. Similarly, subsequent amounts of diluted samples in order from 10-7 to 10-5 were transferred into the Petri plates and NA plates. In spread plating, a hockey stick was sterilized in 70% ethanol and exposed to the alcohol lamp. This was used to spread the sample evenly across the agar surface in all NA plates labeled10-8, 10-7, 10-6, and 10-5. The agar was left to absorb the sample for 10 minutes. In pour plating, approximately 15 ml of NA was poured to the plates containing diluted amounts of 1.0ml of sample culture. The plates were then mixed, and afterwards set aside for solidification.
All plates were incubated in an inverted position for 24 hours at 37C, and then stored in the refrigerator. Observations were made the following week and the colony forming unit per mL (CFU/mL) of each dilution was calculated. RESULTS Table 1. Number of colonies counted and CFU/mL in spread plate Spread plate dilution 10-5 10-6 10-7 10-8
Trial 1 Spreader Spreader TNTC Spreader
Trial 2 Spreader Spreader Spreader Spreader
CFU/mL NA NA NA NA
Table II. Number of colonies and CFU/mL in pour plate Spread plate dilution 10-5 10-6 10-7 10-8
Trial 1 Spreader Spreader Spreader Spreader
Trial 2 Spreader Spreader Spreader Spreader
CFU/mL NA NA NA NA
DISCUSSION Colonies grown in Petri dishes by various methods may be used to determine the count of viable microorganisms. Plate counts assume that every colony is founded by a single cell. That cell must have been alive in order to grow and form a colony. The number of colonies obtained in a viable count experiment depends not only on the inoculum size and the viability of the culture, but also on the sustainability of the culture medium and the incubation conditions. (Madigan et al. 2009) A viable count is a direct counting method in which only viable cells are counted. It can be accomplished by spread plating or pour plating and by direct counting the colonies grown on the plates. In the spread-plate method, only a small volume of the inoculum was spread over the surface of the prepared plate to avoid excess liquid that may soak in and cause the colonies to coalesce, making them difficult to count. Colonies are expected to grow only on the surface of the medium. In the pour-plate method, a larger volume of the inoculum may be used since it is mixed with the melted agar medium. However, in this method, the desired organism must be able to withstand brief exposure to heat when mixed with the melted agar. Colonies from this method are expected to grow not only on the surface of the medium but all through out the entire medium. In order to be able to provide significant results and execute a reliable colony count, cell suspension containing the desired microorganisms must be diluted first to an appropriate concentration. Serial dilution will provide dense culture of lesser concentration that will provide us an inoculum of much lesser amount of organism that will grow colonies on the plate and expected REFERENCES http://www2.hendrix.edu/biology/CellWeb/Techniques/microspread.html. by Dr. Mark Sutherland. Date accessed: July 19, 2009. http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Meat_Milk/Pour_Plate. htm. by Dr. David D. Fankhauser. Date accessed: July 19, 2009. Madigan MT & Martinko JM. 2006. Brock Biology Of Microorganisms, 11th ed. USA: Pearson Prentice Hall, Pearson Education, Inc. 140-147. 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
