1. Using a toothpick, remove as much plaque as possible from between your molars. The sample will be placed into a mortar (be sure to get it the sticky plaque off of the toothpick). 2. Dispense 2 ml of the buffer into the mortar and grind the buffer-plaque with the pestle. Grind it up well. 3. You will make a 1/100 dilution (as near as we can, considering that we will not weigh out the plaque) of the plaque in phosphate buffer by adding the ground up plaque-buffer in the mortar back into the 99ml diluent buffer bottle and mixing it well . 4. Make 10 fold dilutions by using 9ml phosphate buffer solution dilutions. Be SURE to mix each dilution well, and use new pipettes. o Transfer 1ml of the 10-2 into 9ml phosphate buffer, using a fresh pipette: This is a 10 -3 . o Transfer 1ml of the 10-3 into 9ml phosphate buffer, using a fresh pipette: This is a 10-4. o Transfer 1ml of the 10-4 into 9ml phosphate buffer, using a fresh pipette: This is a 10-5. o Transfer 1ml of the 10-5 into 9ml phosphate buffer, using a fresh pipette: This is a 10-6. o Transfer 1ml of the 10-6 into 9ml phosphate buffer, using a fresh pipette: This is a 10-7. 5. Plate out 0.1 ml aliquots using the spread plate method, using sterile spreader sticks. Make blood plates (or BHI agar plates) from the dilutions labelled 10-2 through 10-7, as shown in the diagram below. TOOTHBRUSH 1. Cut the top off of the toothbrush with a pair of pliers and cutters or diagonal cutters, allowing it to drop into the glass container. Dispense 10ml of phosphate buffer solution into the bottle with the toothbrush head. This is your 1/10 dilution. 2. The sonicator is filled 2/3 with water, so place the bottle into the sonicator, and turn on for 30 seconds. Be sure to hold the bottle sonicator while it is sonicating. 3. Make 10 fold dilutions by using 9ml phosphate buffer solution dilutions. Be SURE to mix each dilution well, and use new pipettes. o Transfer 1ml of the 1/10 into 9ml phosphate buffer, using a fresh pipette: This is a 10 -2 . o Transfer 1ml of the 10-2 into 9ml phosphate buffer, using a fresh pipette: This is a 10-3. o Transfer 1ml of the 10-3 into 9ml phosphate buffer, using a fresh pipette: This is a 10-4. o Transfer 1ml of the 10-4 into 9ml phosphate buffer, using a fresh pipette: This is a 10-5. o Transfer 1ml of the 10-5 into 9ml phosphate buffer, using a fresh pipette: This is a 10-6. 4. Plate out 0.1 ml aliquots using the spread plate method (use sterile spreader sticks). Make blood plates (or BHI agar plates) from the dilutions labelled 1/10 through 10-6 , as shown in the diagram below. INCUBATION of plates: All BHI or blood plates inside of a candle jar, as well as Mitis-salivarius agar plates. The candle jar is lit, closed and placed in 37 C incubator. Mannitol salt agar and MacConkey’s plates are incubated in the regular 37 C incubator. Interpretation: 1. Remove all plates from the incubators, and separate them into 2 groups---plaque or toothbrush. 2. Determine the number of bacteria per plaque sample. This will not be per gram since you had much less than a gram sample of plaque. 3. Determine the number of bacteria per toothbrush head, using counts from blood or BHI. 4. Determine the number of bacteria per toothbrush head for each group of bacteria, based on each individual media type. 5. Analyze each type of medium for bacteria for BOTH toothbrush and plaque:  BAP or BHI - for total count of facultatives and aerobes  Mannitol salt - for salt-resistant Staphylococcus species, Staph aureus turns yellow   MacConkey's - for enteric gram - rods, coliforms turn red and non-coliforms are clearish  Mitis-salivarius agar - small blue colonies (Strep mitis), large blue gumdrop-like colonies (Strep salivarius) and dark blue/black, shiny colonies (Enterococcus faecalis) 6. Gram stain at least one colony on each type of medium and record the gram reaction, arrangement, and shape. Questions Q5. Why can’t the total CFU be determined from the selective media used in this lab? Q6. What is the purpose of each kind of medium? Q7. Generally, the plaque has higher counts than the toothbrush—WHY? Q4. Why are some plates placed into a candle jar?