An experiment using the aseptic technique to cultivate Staphylococcus aureus (S. aureus) on an agar plate to observe the isolation of its respective colonies 4,8/5(53) 4 rows · · The milk agar was incubated at 37 degrees Celsius for 48 hours. After observation there was a clear · Columbia CNA Agar (or C-CNA for short) is a classic example of a selective media enriched with two antibiotics, colistin and nalidixic acid, and a defibrinated sheep’s blood (5%) · Bacillus subtilis Staphylcoccus epidermidis Enterococcus faecalis Methyl Red (Positive) Bacillus cereus Milk Agar (positive) Glycerol Test (positive) Maltose Test (positive) · For example, there are particular bacterial species that have the capacity of degrade red blood cells. This process of hemolysis is caused by exotoxins that are produced ... read more
Naveena Varghese. Introduction Microorganism is an organism that is microscopic or submicroscopic, which is too small to be seen under naked eyes. However, the numbers of microorganisms in a given sample are required to know in certain aspect such as dairy industries, diseases investigation, and so on. Because of this, a variety of methods have been developed for the enumeration of microorganisms like direct microscopic counts, filtration and viable plate counts Jacquelyn G. Black Among the methods of enumeration, viable plate counts are being used most frequently to measure bacterial populations. In viable plate counts, we are measuring the number of viable cells, unlike the microscopic counts which cannot distinguish live from dead cells. However, it takes some time for the visible colonies to grow Gerard J.
Tortora, Before doing plate counts, serial dilutions are required. This is because it is hard to count more than colonies on an agar plate if we inoculated directly from the original bacterial suspension or sample without serial dilutions Kathleen Talaro, To complete plate counts, we could either use pour plate method or spread plate method and each of them have their advantages and limitations. All the visible colonies are calculated and represented as colony forming units CFU. Then, the CFU is multiplied with the corresponding dilution factor.
As a result, the population of original sample is known. The objectives of this experiment are to learn the process of enumeration to determine the number of microorganisms in a sample and utilize two methods in enumeration which are pour plate method and spread plate method. Pour Plate Method The pour plate, like other viable plate count methods, involves adding a sample to a solid medium that will support microbial growth incubating the plates so that each bacterial cell multiplies to form a colony, and counting the number of colonies that develop. Generally we have no idea of the number of bacteria in a sample, so it is almost always necessary to prepare a dilution series to ensure that you will obtain a dilution containing a reasonable number of bacteria to count.
Six nutrient agar pours are melted in a boiling water bath. After they liquefy, they are mixed and placed in a 50°C water bath until they are ready to use. Three 99 mL dilution blanks are labeled as 10 -2, , and respectively. Six Petri plates are labeled through The unknown sample is shaken to ensure an even distribution of microorganisms generally shaking side to side for 25 times. The dilution blank is shaken vigorously to distribute the bacteria evenly. Using a new sterile pipette, 0. With the same pipette, an additional 1. The original 1 mL of sample has now been diluted 1 part in a total of 10, parts.
The shaking procedure is repeated for the 10 -4 blank and 0. This same procedure is repeated to form a 10 -6 dilution blank from which you will establish 10 -7 0. A tube of melted agar 50°C is poured aseptically into each Petri plate to which already added a dilution of the sample. The plate is swirled to mix the sample with the agar. The agar is made sure does not run over the edges of the plate. The lid is replaced. The agar is allowed to cool and solidity. The inverted plates are incubated at 30°C. After incubation, the colonies are counted on each plate. Both the colonies on the agar surface and the colonies growing within the agar must be counted. The colonies are counted by marking their position on the back of the Petri plates with a marking pen.
This aid in keeping track of those colonies previously counted and avoids recounts. Counts are recorded. If a plate has more than colonies record it as TNTC too numerous to count. From the plate-count data, the concentration of bacteria in the original sample is calculated. For statistical reasons only plates with between 30 and colonies are used in this calculation. Each colony forming unit CFU represents the progeny of a single cell. Therefore, the number of bacterial cells in the original sample is determined by multiplying the number of colonies on a dilution plate by the corresponding dilution factor.
Generally replicates of each dilution are plated, and the mean count recorded. Spread Plate Method Another technique for performing a standard plate count is the spread plate method. As the name implies, serial dilutions of a sample are spread onto the surfaces of agar plates. Three 9 mL dilution water blanks are labeled as 10 -1, , and respectively. Four nutrient agar plates are labeled through The dilution tube is mixed thoroughly by vortex or vigorous shaking. Using a new pipette, 1 mL from the 10 -1 dilution tube is transferred aseptically to the dilution tube. The sample is vortexed. Using this same procedure, 1 mL from the 10 -2 dilution tube is transferred to the dilution. Because only 0.
Therefore, the greatest dilution plate is Starting with the greatest dilution, the tube is vortexed and 0. Because starting with the greatest dilution, same pipette can be used for each of the tubes. The glass spreader is sterilized by dipping it in alcohol and flaming it in a Bunsen burner flame. Be careful because alcohol fires can easily result. After the glass rod has cooled, the sample is spread over the surface of the plate by touching the rod to the agar and rotating the plate. This spreading procedure is repeated for each of the plates, making sure to sterilize and cool the glass rod before spreading each sample. Inverted plates are incubated at 30°C for hours.
After incubation, the colonies are counted. As in the pour plate method, count plates with a range of colonies are required. The concentration of bacteria in the original sample is calculated. Results A. Viable Plate Count-A. Why are counts above and below 30 statistically unreliable? In statistics, the sample size is inversely proportional to the margin of error. Thus, below 30 colonies per plate has a larger error in the results. If there are too many colonies in an agar plate, the closer colonies may form a single colony, or only one colony will grow as competing for nutrients with each other.
Thus, above colonies per plate is statistically unreliable too. Did you enumerate all the bacteria using this method? No, using pour plate method to enumerate heat-sensitive bacteria are not suitable as it may damage or kill them. What factors make the pour plate method selective? How can you reduce the selectivity of this procedure? Pour plate method might kill the hear-sensitive microbe with hot agar. To reduce the selectivity of this procedure, the agar is left to be cool but still molten before poured into the Petri dish. We can add 0. What does the term colony forming unit mean and how does it differ from a colony? Colony forming unit CFU is the smallest functional unit of colony formation to predict or estimate the number of viable cells in a sample.
What types of errors would be introduced by using a single pipette for all operations in this procedures? Systematic error. What are the advantages and limitations of the spread plate method compared to other enumeration procedures? Relative heat-sensitive microorganisms are not damaged or killed by the spread plate method. The colonies are grow only on surface of medium, the obligate anaerobes will not be able to grow. How could you modify the spread plate procedure to enumerate obligate anaerobes? After inoculation, using anaerobic jar to create an anaerobic environment so that anaerobes are able to grow well on the agar.
Discussion Normally it is very difficult to determine the actual number of microorganisms in a population. Thus, we calculate the number of microorganisms by enumeration from the sample of population. Viable plate count is the method being used most frequently in enumeration of bacterial population. We can either use pour plate method or spread plate method for viable plate count. Colony forming unit CFU is introduced in viable plate count for the enumeration. Viable plate count has it's limitations such as CFU only accounts the visible colony in enumeration, take some time for the visible colonies to grow and too many colonies could cause error in the count.
To overcome the overcrowded problem, dilution is required. There are multiple choices for the dilution. Although dilution is made to reduce the sample size for ease in enumeration, sometimes the growth of microorganisms is very fast until there are too many visible colonies. Since the margin of error is dependent on the sample size, which means the larger the sample size, the smaller the error would be. However, the result is more difficult to handle and calculate if the sample size is too large. Thus, only CFU within 30 to are used to calculate the concentration of bacteria. In pour plate method, the bacterial suspension is introduced into a Petri dish either in 1. Even though pour plate method is easy to conduct, we cannot use pour plate method only in enumeration because of the limitations of pour plate method which is damaging or killing heat-sensitive bacteria.
As a result, no visible colonies could be found. The aerobes also trapped inside the agar, no oxygen can penetrate or diffuse into the agar, causing aerobes fail to survive. Therefore, pour plate method is considered as selective method. The spread plate method allows the bacteria grow on the surface. The bacterial suspension is pour on the agar, and then spread evenly over the surface with a sterile glass rod. By using spread plate method, we can avoid the damage or dead of heat-sensitive bacteria, however, we are exposing the obligate anaerobes to the oxygen from the atmosphere which may kill them.
To enumerate obligate anaerobes, we could modify the spread plate method by using anaerobic jar after inoculation. The anaerobic jar will create an anaerobic environment for the obligate anaerobes. This is because there are more microorganisms in the dilution of , overcrowded occurred, thus, less visible colonies can be grown on the agar plate due to competition among each other. Although we still can found 3 visible colonies in the dilution of , it is not suitable to used in the calculation of the concentration of bacteria as only in the range of 30 to are statistically suitable. Since the warm, molten agar is poured onto the bacterial suspension, the temperature could damage or kill the heat- sensitive bacteria. Thus, pour plate method is considered as selective. To prevent this, either the agar is left to be cooled and still molten before pour onto the bacterial suspension, or instead of using pour plate method, spread plate method is used.
Comparing both pour plate method and spread plate method results, we found that spread plate method result shows more CFU than pour plate method result. Unknown streak A showed evidence of contamination during the gram staining process. A gram negative tube was given by the instructor and another gram stain had been done. Unknown streak A was red and had rods, therefore it was a gram negative. These tests were done and followed by the help of the McDonald lab manual. Unknown A or also known as Proteus vulgaris was difficult to find. To start this process an isolation streak had to be done. Once the isolation streak had been done, several gram stains had been attempted, but since there were some contaminations in this process it made the bacteria a bit more difficult to find.
After many contaminated gram stains the gram negative was given by the instructor in a test tube. Finally a clear gram negative with rods has appeared on the slide. Using a sterile inoculating loop a few drops of Proteus vulgaris was taken placed in an Urea broth tube as well as a Mannitol broth tube. These tubes were incubated at 37 degrees for two days. After the incubation was completed the tubes were thoroughly examined. The mannitol test came out negative, because it failed in producing acid. The outcome of the Urea test was positive, because the color of the broth changed to pink.
This is considered a positive reaction. Unknown B or also known as Enterococcus faecalis was much easier to find than Unknown A. As the McDonald lab says the first step is an isolation streak. Once the isolation streak had been completed the next step would be a gram stain. The first try at this gram stain went perfectly, but just to be safe another gram stain was performed. The second gram stain came out much more clear and had better result. The results that were seen on the slide were a clear purple positive with circles as well as noticeable colonies.
The next step was to use a sterilized inoculating loop that took a bit of Enterococcus faecalis and was dipped into a Urea broth tube, Mannitol broth tube and as well as a Nitrate broth tube. These test tubes were then placed in the incubator at 37 degrees for two days. The Urea test came back negative with no color change to the broth. The Mannitiol test had produced acid , which is considered a positive reaction. When the reagent A and B were placed into the Nitrate tube there was no change. Once a pinch zinc was added to the tube it immediately turned red which means it produced acid and that is a negative reaction.
All techniques and instructions done as laid out in the LAB MANUAL BIO by MCDONLD. Proteus vulgaris is found in human intestinal tract, soil, water and plants. This bacterium has been connected with wound infections due to direct contact. This bacterium has also been linked to food spoilages, such as fresh meat, poultry, and seafood. This bacterium is actively motile and swarming behavior. In order to treat this bacterium you will need ampicillin and aminoglycosides. Tortora, Gerard J. pg Microbiology An Introduction eleventh edition.
By CPR Louisville at June 19, pm Print. In this paper I will discuss the processes of how I came to find my two unknown bacteria. This will be a vital task to take with me into my profession for many reasons. In the medical field bacteria and infections of different kinds are the core of the practice. These bacteria must be able to be identified in order to treat patients properly, efficiently and safely. Materials and Methods:. The unknown number handed out by the Professor on March 20, contained both a gram positive bacteria and a gram negative bacteria. At this point everything that had been learned in microbiology lab and that had been explained in our lab manual 1 was put into action.
The first step to figuring out the unknowns, was to separate the two bacteria. In order to do this, a nutrient agar plate was used. The streak method was used to spread the bacteria across the nutrient agar in hopes of isolating a pure culture of one of the bacteria. In order to do the streak method, an inoculating loop was sterilized with a Bunsen burner and put into the unknown specimen. After removal with bacteria on the loop, the quadrant streak method was used. The streak plate was then incubated at 37 degrees Celsius for 48 hours. Upon returning and observing the streak plate, there was an abundance of green across the plate.
There was only one colony that was apparent. After observation, a sample was taken from the isolated colony on the streak plate and another streak plate was done with that, trying to further isolate the colonies. As well as using the quadrant method to further isolate the colonies, a sample was taken from the best colony on the original streak and gram stained. The gram stain procedure was performed as directed in the lab manual 1. The gram stain showed a result of red, gram negative rods. To decipher between which biochemical tests to perform, the gram positive and negative tables handed out by the Professor, were referred to. From previous biochemical tests done in the semester, Pseudomonas aeruginosa was already suspected because of the green pigment of the original streak plate.
Upon reviewing the identification tables, the deciding biochemical test was the Casein test which tests for the production of the enzyme casease to break down the milk protein casein. The milk agar was incubated at 37 degrees Celsius for 48 hours. After observation there was a clear positive result, which showed the bacteria produced casease. After confirming the gram negative bacteria, the process of isolating the gram positive bacteria began. Returning to the original unknown stock , a quadrant streak plate was done with a sterilized inoculating look on a mannitol salt agar, which inhibits the growth of gram negative bacteria. This MSA plate was incubated at 37 degrees Celsius for 48 hours. Upon return and observation, the MSA did not yield a good isolated colony.
Professor Snaric advised to do another MSA agar from the original stock, but this time with a sterile swab instead of an inoculating loop. This test was also incubated at 37 degrees Celsius for 48 hours and returned a good isolated colony. A sample was taken from this colony and transferred to a nutrient broth agar to further isolate it. After incubation, this nutrient agar had great results with many isolated colonies. A sample was taken from the isolated nutrient agar and a gram stain was done as directed by the lab manual 1. The gram stain showed clear purple gram positive cocci. After getting a good gram stain the identification tables were referred to in order to choose between appropriate biochemical tests.
A nitrate test was performed in order to detect if the bacteria was able to reduce nitrate into nitrate or some further reduced form. Then a urea test was performed to check for the production of urease. All of the biochemical tests performed were explained, in the lab manual provided by Professor 1 and were practiced earlier in the semester. Tables 1 and 2 lists the tests, purposes, reagents used and the results of each test. The first test performed on the gram negative bacteria, was a Casein Test. This test gave a positive result turning a brown color, meaning the gram negative bacteria produced the enzyme casease in order to break down the milk protein casein. This was the only test necessary to determine the unknown gram negative bacteria in unknown stock white opaque Milk Agar changed color where bacteria was smeared, turning a brown color Positive, the bacteria produced casease.
The first test performed on the gram positive bacteria was the Nitrate Test which turned red after adding reagents giving a positive result meaning the bacteria reduced nitrate into nitrite or something further. Following the nitrate test was the Urea Test to determine if the bacteria produced urease. This gave a positive result showing a hot pink broth, meaning the bacteria did produce urease. The unknown contained two different specimen of bacteria, one being a gram positive bacteria and one being a gram negative bacteria. The first unknown in found to be a gram negative bacteria, was identified as Pseudomonas aeruginosa. This identification was reached by only one biochemical test and close observation.
A gram stain was done originally and found red rods identifying the bacteria as gram negative. The streak plate that was originally done, showed a heavy green pigment, which is a main characteristic of Pseudomonas auruginosa. The next test performed was a Casein Test which showed a clear positive result. The only gram negative bacteria that shows a positive result on the Casein Test is Pseudomonas aeruginosa , therefore correctly confirming the unknown gram negative bacteria. The second unknown bacteria was identified as a gram positive bacteria with a coccus shape. This immediately ruled two of the gram positive bacteria Bacillus cerus and Bacillus subtilis. This left three bacteria that the second unknown could be Staphylococcus aureus , Staphylococcus epidermidis, or Enterococcus faecalis.
The next test performed was a Nitrate Test which gave a positive result. The two bacteria that give a positive result for this test are Staphylococcus aureus and Staphylococcus epidermidis. The second test performed was a Urea Test which also gave a clear positive result confirming that unknown gram positive bacteria as Staphylococcus epidermidis because Staphylococcus aureus gives a negative result. Consultation with the Professor, confirmed the two unknown bacteria correctly as Pseudomonas aeruginosa and Staphylococcus epidermidis.
The only problem that was encountered appeared when trying to isolate the gram positive bacteria. The gram negative bacteria in unknown is a very aggressive bacteria that makes the growth of a gram positive bacteria difficult. Even on an MSA agar, it took a few tries and several isolations to get the gram positive to successfully grow. After successful isolation of both bacteria, there were no more issues encountered in identifying either. Pseudomonas aeruginosa is a gram negative rod shaped bacteria that was first discovered in by a pharmacists named Carle Gessard 2.
His study picked up on the unique blue-green pigmentation of P. This bacteria can catalyze in many environments which makes it very common and found almost everywhere such as soil, water, humans, plants, sewage and hospitals. aeruginosa is what is called an opportunistic human pathogen, because it rarely affects a healthy individual. This bacteria more so affects individuals with compromised immune systems the most common being those with cystic fibrosis, cancer, or AIDS. It builds resistance against a lot of antibiotics and even chemotherapeutic agents. aeruginosa is a facultative aerobe; its preferred metabolism is respiration. aeruginosa are those in hospital settings, especially ones on breathing machines or with catheters 3.
Although some cases have been caused by swimming in pools or hot tubs with incorrect levels of chlorine. To avoid the spread and contamination of P. aeruginosa nurses and health care professionals should be sure to use aseptic technique because the bacteria is mostly spread through contaminated equipment and professionals hands. aeruginosa is quickly on its way to becoming resistant to antibiotics and becoming increasingly difficult to treat. Selecting the correct antibiotic for this particular bacteria is especially important. cpr louisville articles , cpr louisville , disease. The methods learned during previous lab exercises for identifying bacteria have been applied to these unknowns. All procedure performed were done so according to the […].
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To determine if the enzyme casease was produced to break down the milk enzyme casein. To determine if the bacteria reduced nitrogen to some further reduced form such as nitrite. Postive, the bacteria reduced the nitrate into nitrite or some further form of nitrogen.
4 rows · · The milk agar was incubated at 37 degrees Celsius for 48 hours. After observation there was a clear · For example, there are particular bacterial species that have the capacity of degrade red blood cells. This process of hemolysis is caused by exotoxins that are produced · Bacillus subtilis Staphylcoccus epidermidis Enterococcus faecalis Methyl Red (Positive) Bacillus cereus Milk Agar (positive) Glycerol Test (positive) Maltose Test (positive) An experiment using the aseptic technique to cultivate Staphylococcus aureus (S. aureus) on an agar plate to observe the isolation of its respective colonies 4,8/5(53) · Columbia CNA Agar (or C-CNA for short) is a classic example of a selective media enriched with two antibiotics, colistin and nalidixic acid, and a defibrinated sheep’s blood (5%) ... read more
Remember to take two samples from each of the six locations, each from a different colony. Looking for more fun ways to learn on wikiHow? At the same time, the Peptic digest of animal tissue is a source of both carbon and nitrogen necessary for bacterial growth. pg Although we still can found 3 visible colonies in the dilution of , it is not suitable to used in the calculation of the concentration of bacteria as only in the range of 30 to are statistically suitable. Repeat the previous seven steps for the other two groups of four slides. An isolation streak was done on a Nutrient Agar, and then was placed into an incubator at 37 degrees for two days to grow.Then, the CFU is multiplied with the corresponding dilution factor. This simple doubling of bacteria is observed in cultures that are classically conducted in microbiological laboratories. The two tests call for the bacterium to be in a liquid form when added to the test tubes, but the test was done from the bacterium being on an agar jell. The original 1 mL of sample has now been diluted 1 part in a total of 10, parts. Helpful 0 Not Helpful 0, examples of microbiology lab reports.