Serial Dilution & Spread Plate PDF

# Serial Dilution A series of sequential dilutions that are performed to convert a dense solution into a more usable concentration. ## Objectives of Serial Dilution 1. Estimate the concentration of an unknown sample by enumeration of the number of colonies cultured from serial dilutions of sample...

# Serial Dilution A series of sequential dilutions that are performed to convert a dense solution into a more usable concentration. ## Objectives of Serial Dilution 1. Estimate the concentration of an unknown sample by enumeration of the number of colonies cultured from serial dilutions of sample. 2. Reduce the density of cells in each step so that it is easier to calculate the concentration of the cells in the original solution by calculating the total dilution over the entire series. 3. Perform to avoid having to pipette very small volumes (1-10µl) to make a dilution of a solution. 4. Obtain incubated culture plates with easily countable number of colonies (around 30-100) and calculate the number of microbes present in sample. ## Formulas/Calculations - Serial dilution involves the process of taking a sample and diluting it through a series of standard volumes of sterile diluent, which can either be distilled water or 0.9% saline. - Then, a small measured volume of each dilution is used to make a series of pour or spread plates. Depending on the estimated concentration of cells/organisms in a sample, the extent of dilution is determined. - The dilution factor in a serial dilution can be determined either for an individual test tube or can be calculated as a total dilution factor entire series. - The dilution factor of each tube in a set: $VOS / (VOS + VD)$ For a ten-fold dilution, 1 ml of sample is added to 9ml of diluent. In this case, the dilution factor for that test tube will be: Dilution Factor = $1ml / (1ml + 9ml) = 1 / 10 = 10^-1$ After the first tube, each tube is the dilution of the previous dilution tube. Total dilution factor for the second tube = dilution of the first tube x dilution of the second tube ## Procedure of Serial Dilution 1. The sample/culture is taken in a test tube and six test tubes, each with 9 ml of sterile diluents, which can either be distilled water or 0.9% saline, are taken. 2. A sterile pipette is taken. 3. 1 ml of properly mixed sample/culture is drawn into the pipette. 4. The sample is then added to the first tube to make the total volume of 10 ml. This provides an initial dilution of $10^-1$. 5. The dilution is thoroughly mixed by emptying and filling the pipette several times. 6. The pipette tip is discarded, and a new pipette tip is attached to the pipette. 7. Now, 1ml of mixture is taken from the $10^-1$ dilution and is emptied into the second tube. The second tube now has a total dilution factor of $10^-2$. 8. The same process is then repeated for the remaining tubes, taking 1 ml from the previous tube and adding it to the next 9 ml diluents. 9. As six tubes are used, the final dilution for the bacteria/cells will be $10^-6$ (1 in 1,000,000). ## Application and Uses - In microbiology, to estimate the concentration or number of cells/organisms in a sample to obtain an incubated plate with an easily countable number of colonies. - In biochemistry, serial dilution is used to obtain the desired concentration of reagents and chemicals from a higher concentration. - In pharmaceutical laboratories, serial dilution is performed to receive the necessary concentration of chemicals and compounds as this method is more effective than individual dilutions. - In homeopathy, homeopathic dilutions are used where a substance is diluted in distilled water or alcohol. It is believed that dilution increases the potency of the diluted substance by activating its vital energy. ## Limitations and Problems 1. An error might occur during the propagation of the sample, and the transfer inaccuracies lead to less accurate and less precise transfer. This results in the highest dilution to have the most inaccuracies and the least accuracy. 2. Because serial dilution is performed in a stepwise manner, it requires a more extended period of time which limits the efficiency of the method. 3. Serial dilution only allows the reduction of bacteria/cells but not the separation of bacteria/cells like in other techniques like flow cytometry. 4. This technique also requires highly trained microbiologists and experts in aseptic techniques. ## Spread Plate Spread plates, also known as lawn plates, should result in a heavy, often confluent growth of culture spread evenly over the surface of the growth medium. This means that they can be used to test the sensitivity of bacteria to many antimicrobial substances, for example mouthwashes, garlic, disinfectants and antibiotics. The spread plate can be used for quantitative work (colony counts). If the dilution and volume of the inoculum, usually 0.1 cm³, are known, the viable count of the sample, i.e. the number of bacteria or clumps of bacteria per cm³, can be determined. The dilutions chosen must be appropriate to produce between 30 and 100 separate countable colonies. ## Preparing Serial dilutions There are various ways of counting or monitoring microbial growth in a culture. Serial dilution involves taking a sample and diluting it through a series of standard volumes of sterile diluent, e.g. distilled water or 0.9% saline. Then a small measured volume of each dilution is used to make a series of pour or spread plates. By diluting the sample in this controlled way it is possible to obtain an incubated plate with an easily countable number of colonies (30-100) and calculate the number of microbes present in the sample. ### Materials - Culture of bacteria or yeast or sample of natural material - 6 sterile test tubes containing 9 cm³ sterile diluent, fitted with a cap or cotton wool plug, labelled 1→6 and with the dilution factor as shown in the diagram - 12 sterile, plugged Pasteur pipettes - 1 cm³ syringe barrel fitted with rubber tubing - Pot of Virkon disinfectant ### Procedure 1. Take a sterile pipette. 2. Place the syringe onto the plugged end of the pipette. 3. Draw up 1 cm³ of a well-mixed sample/culture into the pipette. 4. Add this sample to the first tube. The volume of this tube is now 10 cm³. This provides an initial dilution of $10^-1$. 5. Mix the dilution thoroughly, by emptying and filling the pipette several times. 6. Discard this pipette into the pot of disinfectant, but keep the syringe for making the next dilution. 7. Take a new pipette, fit it to the syringe and draw up a 1 cm³ sample of the $10^-1$ dilution and place it in the second tube. 8. Mix well as before. This gives a $10^-2$ dilution. 9. Discard the pipette in disinfectant. 10. Repeat this for the remaining tubes, removing 1cm³ from the previous dilution and adding it to the next 9 cm³ of diluent. If 6 tubes are used, the final dilution for the bacteria will be $10^-6$ (1 in 1,000,000). ### Plating and counting procedure Use a known volume of each dilution to make either pour plates or spread plates. By starting with the highest dilution, the same pipette may be used throughout. For statistical purposes, replicate plates should be prepared. After incubation the plates will show a range of numbers of colonies. Choose the plate that has an easily countable number (about 30-100) and carefully count every colony. Using a marker pen helps to avoid counting the same colony twice. Then calculate the number of micro-organisms in the sample: **Number of microbes/cm³ = number of colonies x dilution of sample** The image contains a grid pattern.