Microbiology Dilution Worksheet and Problems Objectives Understand how to quantify bacterial cells. Learn how to solve a dilution problem. It is a common practice to determine microbial counts for both liquid and solid specimens such as suspensions of E. coli in nutrient broth. Most specimens have high enough numbers of microorganisms that the specimen has to be serially diluted to quantitate effectively. The following is a step-by-step procedure to working dilution problems, and includes some practice problems at the end. It is specific for bacterial counts (colony-forming units, CFUs). To produce a set of pour plates from many dilutions, a set of serial dilutions is made from the original sample. A sample of each is placed into a liquefied agar medium, and the medium poured into a petri dish. The agar solidifies, with the bacterial cells locked inside of the agar. Colonies grow within the agar, as well as on top of the agar and below the agar (between the agar and the lower dish). The agar plate allows accurate counting of the microorganisms, resulting from the equal distribution across the agar plate. Note: MICROBIAL COUNT = colony count on agar plate (CFU) dilution factor of dilution tube X volume plated (mL) To work the problem, you need 3 values---a colony count from the pour plates, a dilution factor for the dilution tube from which the countable agar plate comes, and the volume of the dilution that was plated on the agar plate. STEP 1: Determine the appropriate plate for counting Look at all plates and find the one with 30-200 colonies, preferably. Greater than 200 and less than 30 is a high degree of error. If duplicate plates (with same amount plated) have been made from one dilution, average the counts together. STEP 2: Determine the total dilution for the dilution tubes Dilution = amount of specimen transferred [amount of specimen transferred + amount already in tube] Total dilution factor = previous dilution of tube X dilution of next container 1 ml added to 9ml = 1/10 for 1st tube 1ml added to 9ml = 1/10 for 2nd tube Therefore total dilution factor = previous dilution of 1/10 X 1/10 = total dilution of 1/100 (=10-2) STEP 3: Determine the amount plated The amount plated is the amount of dilution used to make the particular pour plate. Solve the above problem by completing the following: 1. The countable plate is the one with ______ colonies. 2. The total dilution of 3rd tube from which the pour plate was made = 1/10 X 1/10 X 1/10 = ____________ 3. The amount used to make that pour plate was ____ mL. Microbial count = colony count on agar plate (CFU) dilution factor of dilution tube X volume plated (mL) Microbial count = = ___________ bacterial/mL X (Answer should be 71,000/ml or 7.1 X 104 in scientific notation – how many bacteria would have been in the original sample?) Now calculate the number of E.coli bacteria in the sample below after studying the diagram. Show all working. MICROBIOLOGY WORKSHEET 1. Complete the following sentences regarding aseptic techniques in a microbiology lab. (a) Wash your hands with soap both __________ and _________ a microbiology lab. (b) Always __________ the tables before and after a lab. (c) Always work _____________ away from a _______________________ flame. (d) Flame the _____________ of the glass bottles used to create an updraft that eliminates potential air contamination. (e) Agar plates are always taped and stored ________________________ in the incubator to reduce possibility of water condensation dropping onto agar. 2. What is the meniscus? 3. How much fluid is in the pipette on the right? 4. When drawing up a volume of solution, place a disposable, sterile pipette ______ into a micropipette. Place the tip into the solution and rotate the pipump wheel so that the fluid _________________. 5. What is the dilution factor for the following dilutions? (a) 0.5ml of sample into the 4.5ml of water (b) 1ml of sample into the 4ml of water (c) 0.5ml of sample into the 9.5ml of water (d) 0.1ml of sample into the 99.9ml of water 6. Determine the number of bacteria per ml. of water specimen. 7. Determine the number of bacterial cells per ml. in the original culture.