Lab 5: Enumeration of Microorganisms
Lab 5: Enumeration of Microorganisms
Introduction
The purpose of this lab will ultimately be to familiarize ourselves with counting microorganism colonies. In the process, we will learn another plating method called spread plating and learn our strengths and weaknesses using these techniques.
The total viable count is based upon the ability of a microorganism placed in a nutrient environment to grow. While individual microorganisms cannot be seen with the naked eye, they can grow in colonies which are visible to any plain Jane. Counting the colonies gives an indication of the number of viable colony forming units (CFU) present in the original sample. However, the appropriate growth medium must be chosen first to get to the counting phase. This part is crucial because the accuracy will be best if the chosen medium is as similar as possible to the sample environment.
Plate counting is the oldest technique in the book, so why teach an old dog new tricks??! The sample is serially diluted, usually ten fold, and portions of each dilution are plated. An end goal of 30-300 colonies in each dish, these plates are used to quantify the number of microorganisms in the original sample. The lower end of the counting range arises from concerns about statistical accuracy, while the upper end is representative of a realistic counting approach and to minimize the possibility of growth overlap.
Two methods of plating are typically used: pour plating and spread plating. Pour plating sees a 1 mL of sample or diluted sample to a plate, followed by a sterile solution of nutrients and melted agar. The sample and solution are mixed and the agar solidifies. It is important to keep in mind that agar, a polymeric extract from marine algae aka the solidifying agent, has a melting point of 40*C. Hence, this method is not to be used for microorganisms that cannot handle that temperature as they will die before they are even incubated. Spread plating, what this lab will consist of, usually gives higher counts with environmental samples and sees 0.1 mL of sample or diluted sample on the surface of a prepared plate (sterile nutrients and agar are already added and solidified). The sample is then spread across the surface with a sterile glass rod. In both methods, plates are counted after an appropriate incubation period for the development of visible colonies.
Two media will be used in this lab: Luria-Bertani (LB) Agar and R2A Agar. LB consists of 10 g tryptone, 5 g yeast extract, 10 g sodium chloride and 15 g agar per liter. LB agar is considered a nutrient-rich medium. R2A consists of 0.5 g each of yeast extract, protease petone, cassation acids, glucose, soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.05 g magnesium sulfate heptahydrate, 0.3 g sodium pyruvate and 15 g agar per liter. R2A media is considered a low-nutrient medium.
Methods and Materials
A sample of water was first chosen as the sample. (Ours was activated sludge, considered 'raw wastewater' for our calculation purposes.) A series of decimal dilutions were first made using a phosphate buffer solution (PBS) as the buffer. (Not DI water as this would lead to the lysing of cells.) 900 µL were pipetted into 5 sterile flasks, along with 100 µL of our solution in the first flask (10^-1 dilution). This first flask was then mixed, and then 100 µL of that solution was placed into the second flask (10^-2). This was repeated until we reached a dilution of 10^-5. We then chose to plate dilutions of 10^-3, 10^-4 and 10^-5 in hopes of plating between 10-1000 (30-300 intended).
All plates were labeled with the medium, dilution, date and sample origin. First plating on LB agar medium, 0.1 mL of the diluted sample was placed onto the plate. A glass 'hockey stick' was then dipped in ethanol and placed in a flame to sterilize. After a few moments of letting this cool down, the diluted sample was then spread on the plate by rotating the plate with the stick gently placed on the surface in a circular motion. This had to be done quickly to prevent contamination. This was repeated for a total of two plated per dilution, with three dilutions and likewise using R2A for the medium. (Total: 12 plates) After all plates were spread, they were inverted and incubated upside down for 1 week.
After 1 week, all 12 plates were counted using a sharpie to mark off the colonies already counted. If some are overgrown, TNTC may be used to indicate too numerous to count.
Results
Table 1. Dilutions, colony counts, original concentration and average concentration for LB Agar.
Table 2. Dilutions, colony counts, original concentration and average concentration for R2A Media.
Sample Calculation: LB Agar, Plate 1, 10^-3 dilution.
Discussion
Cafe Press. Be Nice to Bacteria [Digital image]. Accessed online 11 October 2017 at <http://i3.cpcache.com/product_zoom/98935384/be_nice_to_bacteria_magnet.jpg?height=460&width=460&padToSquare=true>.
Thomson, Ashley. "Lab #5: Enumeration of Microorganisms."
 |
| (Photo: Cafe Press) |
The purpose of this lab will ultimately be to familiarize ourselves with counting microorganism colonies. In the process, we will learn another plating method called spread plating and learn our strengths and weaknesses using these techniques.
The total viable count is based upon the ability of a microorganism placed in a nutrient environment to grow. While individual microorganisms cannot be seen with the naked eye, they can grow in colonies which are visible to any plain Jane. Counting the colonies gives an indication of the number of viable colony forming units (CFU) present in the original sample. However, the appropriate growth medium must be chosen first to get to the counting phase. This part is crucial because the accuracy will be best if the chosen medium is as similar as possible to the sample environment.
Plate counting is the oldest technique in the book, so why teach an old dog new tricks??! The sample is serially diluted, usually ten fold, and portions of each dilution are plated. An end goal of 30-300 colonies in each dish, these plates are used to quantify the number of microorganisms in the original sample. The lower end of the counting range arises from concerns about statistical accuracy, while the upper end is representative of a realistic counting approach and to minimize the possibility of growth overlap.
Two methods of plating are typically used: pour plating and spread plating. Pour plating sees a 1 mL of sample or diluted sample to a plate, followed by a sterile solution of nutrients and melted agar. The sample and solution are mixed and the agar solidifies. It is important to keep in mind that agar, a polymeric extract from marine algae aka the solidifying agent, has a melting point of 40*C. Hence, this method is not to be used for microorganisms that cannot handle that temperature as they will die before they are even incubated. Spread plating, what this lab will consist of, usually gives higher counts with environmental samples and sees 0.1 mL of sample or diluted sample on the surface of a prepared plate (sterile nutrients and agar are already added and solidified). The sample is then spread across the surface with a sterile glass rod. In both methods, plates are counted after an appropriate incubation period for the development of visible colonies.
Two media will be used in this lab: Luria-Bertani (LB) Agar and R2A Agar. LB consists of 10 g tryptone, 5 g yeast extract, 10 g sodium chloride and 15 g agar per liter. LB agar is considered a nutrient-rich medium. R2A consists of 0.5 g each of yeast extract, protease petone, cassation acids, glucose, soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.05 g magnesium sulfate heptahydrate, 0.3 g sodium pyruvate and 15 g agar per liter. R2A media is considered a low-nutrient medium.
 |
| (Photo: Lauren Lukasik) |
 |
| (Photo: Lauren Lukasik) |
Methods and Materials
A sample of water was first chosen as the sample. (Ours was activated sludge, considered 'raw wastewater' for our calculation purposes.) A series of decimal dilutions were first made using a phosphate buffer solution (PBS) as the buffer. (Not DI water as this would lead to the lysing of cells.) 900 µL were pipetted into 5 sterile flasks, along with 100 µL of our solution in the first flask (10^-1 dilution). This first flask was then mixed, and then 100 µL of that solution was placed into the second flask (10^-2). This was repeated until we reached a dilution of 10^-5. We then chose to plate dilutions of 10^-3, 10^-4 and 10^-5 in hopes of plating between 10-1000 (30-300 intended).
All plates were labeled with the medium, dilution, date and sample origin. First plating on LB agar medium, 0.1 mL of the diluted sample was placed onto the plate. A glass 'hockey stick' was then dipped in ethanol and placed in a flame to sterilize. After a few moments of letting this cool down, the diluted sample was then spread on the plate by rotating the plate with the stick gently placed on the surface in a circular motion. This had to be done quickly to prevent contamination. This was repeated for a total of two plated per dilution, with three dilutions and likewise using R2A for the medium. (Total: 12 plates) After all plates were spread, they were inverted and incubated upside down for 1 week.
After 1 week, all 12 plates were counted using a sharpie to mark off the colonies already counted. If some are overgrown, TNTC may be used to indicate too numerous to count.
 |
| (Photo: Amy He) |
Results
Table 1. Dilutions, colony counts, original concentration and average concentration for LB Agar.
Table 2. Dilutions, colony counts, original concentration and average concentration for R2A Media.
Sample Calculation: LB Agar, Plate 1, 10^-3 dilution.
LB Plates
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| (Photo: Lauren Lukasik) |
R2A Plates
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| (Photo: Lauren Lukasik) |
Discussion
In selecting culture media ingredients, some environmental and physiological factors to be considered are whether or not microorganisms in the sample are used to competing with others for resources, cell nutrient demands, component toxicity, waste development of the cells and temperature. All of these factors can either expedite or destroy cell growth. Specifically, when plating bacteria in the lab, one must commonly decide between LB and R2A. LB, a nutrient rich medium, ends up with a less diverse set of colonies because the faster growing bacteria eat up all the readily available nutrients before the slower growing bacteria get a chance to catch on. However, in R2A, a low nutrient agar, there will reflect more differentiation in growth because both the slow & fast growing microorganisms are on a level playing field. In our case of plating activated sludge, the solution plated on R2A was very difficult to count because of its seemingly endless amount of colonies. While this is partly due to our poor dilution practices (discussed later), it was easier to count the colonies on the LB. However, the LB had more overlapping colonies than on the R2A, making us take more time to try to separate the conjoined colonies in our counting. I prefer the R2A because it's more aesthetically pleasing, but the LB was much easier to count.
Due to plate quantity limitations, duplicate plates were created at each dilution to minimize error in both plating technique and counting. By having two plates at each dilution, we had two different opportunities to perfect our spread plating technique (which we were pretty successful with) and two different opportunities to compare colony counts. A third plated dilution would have been even better. Also, the solution was plated at different dilution due to the range of initial bacteria concentration. The intention was that at least one of our dilutions would fit into the 30-300 colony window and we would also see a ten fold transition between the dilutions. The minimum 30 is to ensure there is statistical accuracy and the 300 maximum exists because colonies start to grow on top of each other and over 300 is unreasonable to keep track of on a small plate. This however did not happen consistently, most likely due to our poor dilution techniques. All 6 plates of the LB exist within the 30-300 range, but they should not according to our dilutions. Only one dilution should be correct while the others should increase or decrease ten fold. Our R2A plates are a hot mess--too many colonies all around. We should definitely have diluted more. Our spread plate technique was good as there seems to be colonies in all aspects of the plate, but the amount is what's troubling across the board leading me to believe our dilution were not well mixed.
Only 0.1 mL is plated with the spread plate technique to ensure we are not inundating the plate with solution. Otherwise, the solution will just remain as liquid on the media surface. Also, it is important to note that we diluted with a phosphate buffer solution (PBS) and not DI water so as to prevent the lysing of the cells. The DI water, void of any nutrients, would naturally be drawn to the nutrient rich insides of the cell and water would rush into the cell through the cell membrane and ultimately cause the cell to burst.
The plates are incubated in an inverted position to minimize the contamination from the condensation on the lid of the plates. The condensation will therefore not make our solution runny again and ruin our spreading nor interfere with the plating of a specific solution.
The spread plate technique normally yields higher counts from environmental samples than the pour plate technique because in the pour plating, the agar must be at its 40*C melting point to liquify to allow pouring. This extreme temperature can be detrimental to all kinds of microorganisms, killing them before they even begin to grow in the media. Imagine someone pouring very hot water on you and then expecting you to thrive...it would probably be difficult.
Overall, our spread plate technique was pretty good, but our dilution skills could use some work. It's also worthy to note that we did use activated sludge, but we based our calculations off of raw wastewater. This would lead to me to think that we would be end up with even less counted colonies as raw wastewater is probably originally more concentrated than activated sludge, but this is not the case. Good thing we are learning now instead of on the job!!!!!
ReferencesDue to plate quantity limitations, duplicate plates were created at each dilution to minimize error in both plating technique and counting. By having two plates at each dilution, we had two different opportunities to perfect our spread plating technique (which we were pretty successful with) and two different opportunities to compare colony counts. A third plated dilution would have been even better. Also, the solution was plated at different dilution due to the range of initial bacteria concentration. The intention was that at least one of our dilutions would fit into the 30-300 colony window and we would also see a ten fold transition between the dilutions. The minimum 30 is to ensure there is statistical accuracy and the 300 maximum exists because colonies start to grow on top of each other and over 300 is unreasonable to keep track of on a small plate. This however did not happen consistently, most likely due to our poor dilution techniques. All 6 plates of the LB exist within the 30-300 range, but they should not according to our dilutions. Only one dilution should be correct while the others should increase or decrease ten fold. Our R2A plates are a hot mess--too many colonies all around. We should definitely have diluted more. Our spread plate technique was good as there seems to be colonies in all aspects of the plate, but the amount is what's troubling across the board leading me to believe our dilution were not well mixed.
Only 0.1 mL is plated with the spread plate technique to ensure we are not inundating the plate with solution. Otherwise, the solution will just remain as liquid on the media surface. Also, it is important to note that we diluted with a phosphate buffer solution (PBS) and not DI water so as to prevent the lysing of the cells. The DI water, void of any nutrients, would naturally be drawn to the nutrient rich insides of the cell and water would rush into the cell through the cell membrane and ultimately cause the cell to burst.
The plates are incubated in an inverted position to minimize the contamination from the condensation on the lid of the plates. The condensation will therefore not make our solution runny again and ruin our spreading nor interfere with the plating of a specific solution.
The spread plate technique normally yields higher counts from environmental samples than the pour plate technique because in the pour plating, the agar must be at its 40*C melting point to liquify to allow pouring. This extreme temperature can be detrimental to all kinds of microorganisms, killing them before they even begin to grow in the media. Imagine someone pouring very hot water on you and then expecting you to thrive...it would probably be difficult.
Overall, our spread plate technique was pretty good, but our dilution skills could use some work. It's also worthy to note that we did use activated sludge, but we based our calculations off of raw wastewater. This would lead to me to think that we would be end up with even less counted colonies as raw wastewater is probably originally more concentrated than activated sludge, but this is not the case. Good thing we are learning now instead of on the job!!!!!
Cafe Press. Be Nice to Bacteria [Digital image]. Accessed online 11 October 2017 at <http://i3.cpcache.com/product_zoom/98935384/be_nice_to_bacteria_magnet.jpg?height=460&width=460&padToSquare=true>.
Thomson, Ashley. "Lab #5: Enumeration of Microorganisms."
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