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Bacteria Enumeration

Bacteria Enumeration.

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Bacteria Enumeration

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  1. Bacteria Enumeration IF YOU CAN SEE THIS MESSAGE YOU ARE NOT IN “SLIDE SHOW” MODE. PERFOMING THE LAB IN THIS MODE WILL NOT ALLOW FOR THE ANIMATIONS AND INTERACTIVITY OF THE EXERCISE TO WORK PROPERLY. TO CHANGE TO “SLIDE SHOW” MODE YOU CAN CLICK ON “VIEW” AT THE TOP OF THE PAGE AND SELECT “SLIDE SHOW” FROM THE PULL DOWN MENU. YOU CAN ALSO JUST HIT THE “F5” KEY. Instructor Terry Wiseth

  2. Click on the blackboard to view a larger board for discussion. Incubator 370C Agar Plates Swabs Loops Antiseptic Dispenser Microbe Samples Loops Pencil Bunsen burner

  3. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C ENUMERATION OF BACTERIA As part of daily routine, the laboratory microbiologist often has to determine the number of bacteria in a given sample as well as having to compare the amount of bacterial growth under various conditions. Enumeration of microorganisms is especially important in dairy microbiology, food microbiology, and water microbiology. Knowing the bacterial count in drinking water, fresh milk, buttermilk, yogurt, can be useful in many aspects of industrial microbiology. Bacteria are so small and numerous, counting them directly can be very difficult. Some of the methods used involve diluting the sample to a point at which the number of bacteria has been reduced to very small numbers. This enables an estimate to be established for quantifying the bacteria. Direct counts ofbacteria require a dye to be introduced to thepopulations of bacteria to allow the observerto view the bacteria. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

  4. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 350C 500C 1000C Observe the three links given below to bring you to the VIRTUAL LAB that you wish to perform. If you have performed all of the exercises, you can click on END LAB. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Viable Plate Count Direct Count End Lab Turbidity Count

  5. VIABLE PLATE COUNT

  6. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C VIABLE PLATE COUNT Viable Plate Count (also called a Standard Plate Count) is one of the most common methods, for enumeration of bacteria. Serial dilutions of bacteria are plated onto an agar plate. Dilution procedure influences overall counting process. The suspension is spread over the surface of growth medium. The plates are incubated so that colonies are formed. Multiplication of a bacterium on solid media results in the formation of a macroscopic colony visible to naked eye. It is assumed that each colony arises from an individual viable cell. Total number of colonies is counted and this number multiplied by the dilution factor to find out concentration of cells in the original sample. Counting plates should have 30-300 colonies at least. Since the enumeration of microorganisms involves the use of extremely small dilutions and extremely large numbers of cells, scientific notation is routinely used in calculations. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

  7. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C A major limitation in this method is selectivity. The nature of the growth medium and the incubation conditions determine which bacteria can grow and thus be counted. Viable counting measures only those cells that are capable of growth on the given medium under the set of conditions used for incubation. Sometimes cells are viable but non-culturable. The number of bacteria in a given sample is usually too great to be counted directly. However, if the sample is serially diluted and then plated out on an agar surface in such a manner that single isolated bacteria form visible isolated colonies, the number of colonies can be used as a measure of the number of viable (living) cells in that known dilution. The viable plate count method is an indirect measurement of cell density and reveals information related only to live bacteria. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

  8. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Normally, the bacterial sample is diluted by factors of 10 and plated on agar. After incubation, the number of colonies on a dilution plate showing between 30 and 300 colonies is determined. A plate having 30-300 colonies is chosen because this range is considered statistically significant. If there are less than 30 colonies on the plate, small errors in dilution technique or the presence of a few contaminants will have a drastic effect on the final count. Likewise, if there are more than 300 colonies on the plate, there will be poor isolation and colonies will have grown together. Generally, one wants to determine the number of (colony forming units) CFUs per milliliter (ml) of sample. To find this, the number of colonies (on a plate having 30-300 colonies) is multiplied by the number of times the original ml of bacteria was diluted (the dilution factor of the plate counted). For example, if a plate containing a 1/1,000,000 dilution of the original ml of sample shows 150 colonies, then 150 represents 1/1,000,000 the number of CFUs present in the original ml. Therefore the number of CFUs per ml in the original sample is found by multiplying 150 x 1,000,000 as shown in the formula given on the next page. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

  9. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C CFUs per ml of sample = The # of colonies X The dilution factor of the plate counted In the case of the example given on the previous page:150 x 1,000,000 = 150,000,000 CFUs per ml At the end of the incubation period, select all of the agar plates containing between 30 and 300 colonies. Plates with more than 300 colonies cannot be counted and are designated too numerous to count (TNTC). Plates with fewer than 30 colonies are designated too few to count (TFTC). Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

  10. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 350C 500C 1000C • PROCEDURE: VIABLE PLATE COUNT • We will be testing four samples of water for the Viable Count. The samples include: • 1) Water from a drinking fountain • 2) Boiled water from a drinking fountain3) Water from the local river4) Boiled water from the local river • You will need DATA TABLE 1 to input your data and calculate the number of CFU per ml.Use the link given below to access a printable version of DATA TABLE 1. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 DATA TABLE 1

  11. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C 1) Take 6 dilution tubes, each containing 9 ml of sterile saline. 2) Dilute 1 ml of a sample by withdrawing 1 ml of the sample and dispensing this 1 ml into the first dilution tube.3) Using the same procedure, withdraw 1 ml from the first dilution tube and dispense into the second dilution tube. Subsequently withdraw 1 ml from the second dilution tube and dispense into the third dilution tube. Continue doing this from tube to tube until the dilution is completed. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

  12. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C 4) Transfer 1 ml from each of only the last three dilution tubes onto the surface of the corresponding agar plates.5) Incubate the agar plates at 37°C for 48 hours.6) Choose a plate that appears to have between 30 and 300 colonies. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

  13. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C 7) Count the exact number of colonies on that plate8) Calculate the number of CFUs per ml of original sample as follows: CFUs per ml of sample= The # of coloniesXThe dilution factor of the plate counted Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

  14. Click on the DILUTION TUBE rack of test tubes to bring them to the table. Each of the dilution tubes contain 9 ml of sterile saline solution. Next Click on the WATER SAMPLES to bring the samples to the table. Now Click on the Eye Droppers to withdraw 1 ml of sample #1 (Fountain Water) and dispense this to the first dilution tube. Click on NEXT when this initial transfer is finished. 4 2 1 3 Incubator 370C 6 5 4 3 2 1 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Sterile Dilution Tubes Pencil Bunsen burner Eye Droppers Sample #1 Fountain Water

  15. Click again on the EYE DROPPER to withdraw 1 ml from the first dilution tube and dispense into the second dilution tube and subsequently withdraw 1 ml from the second dilution tube and dispense into the third dilution tube. Continue doing this from tube to tube until the dilution is completed through dilution tube #6. Click on NEXT when the dilutions are complete. 4 2 1 3 Incubator 370C 6 5 4 3 2 1 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Sterile Dilution Tubes Pencil Bunsen burner Eye Droppers Sample #1 Fountain Water

  16. The dilutions for each of the 6 dilutions tubes can be summarized in the image below.Dilution tube #1 has a 1/10 dilution with a dilution factor of 10. The dilution factor for each of the tubes is listed below.Tube #1 = 10 Tube #2 = 100 Tube #3 = 1000 Tube #4 = 10,000 Tube #5 = 100,000 Tube #6 = 1,000,000 Click on NEXT when you are ready for the next step in the exercise 4 2 1 3 Incubator 370C 6 5 4 3 2 1 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Sterile Dilution Tubes Pencil Bunsen burner Eye Droppers Sample #1 Fountain Water

  17. Next we will be inoculating agar plates with the last three broth culture dilutions. Click on the agar plates on the shelf to bring them to the table. Now click on the EYE DROPPERS to transfer 1 ml of dilution #4 to plate # 1, 1 ml of dilutions #5 to plate #2 and 1 mil of dilution #6 to plate #3. Next click on the pencil to label agar plate #1 with a dilution factor 10,000; plate #2 with a dilution factor 100,000 and plate #3 with a dilution factor 1,000,000. Click on NEXT when finished. 1 4 3 2 Incubator 370C 6 5 4 3 2 1 Sample #1 Fountain Water Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers 10,000 100,000 1,000,000

  18. Click on the agar plates to place them in the incubator at 37 0C for 48 hours.We will now need to perform these same dilution and inoculation steps for each of the test samples. The process is the same for each sample and we will assume the process of dilution and inoculation has been completed for all four of the water samples and the 48 hours of incubation time has now been completed. Click on NEXT when you are ready to view the incubated plates. 1 3 2 4 Incubator 370C 10,000 100,000 1,000,000 6 5 4 3 2 1 Sample #1 Fountain Water Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers

  19. Click on the incubator to bring all of the inoculated agar plates to the table. Each of the groups of inoculated plates is labeled with the source of their respective samples. A key for the sample #s is given below. Click on one of the sample groups to view the bacterial growth of the individual dilutions. Incubator #4 #3 #2 #1 370C Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil 1) Fountain Water2) Boiled Fountain Water3) River Water4) Boiled River Water Bunsen burner Eye Droppers

  20. 10,000 1,000,000 100,000 Sample 1Viable Plate Count

  21. You are viewing the agar plates that were inoculated with FOUNTAIN WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. Incubator #4 #3 #2 #1 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers 1) Fountain Water2) Boiled Fountain Water3) River Water4) Boiled River Water Click here if you have viewed all the agar plates from all four of the samples

  22. You are viewing the agar plates that were inoculated with FOUNTAIN WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. The dilution factor for the plate you are viewing is 10,000. 10,000 Incubator #4 #3 #2 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers Click here if you have viewed all the agar plates from all four of the samples

  23. You are viewing the agar plates that were inoculated with FOUNTAIN WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. The dilution factor for the plate you are viewing is 100,000. Incubator #4 #3 #2 100,000 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers Click here if you have viewed all the agar plates from all four of the samples

  24. You are viewing the agar plates that were inoculated with FOUNTAIN WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. The dilution factor for the plate you are viewing is 1,000,000. 1,000,000 Incubator #4 #3 #2 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers Click here if you have viewed all the agar plates from all four of the samples

  25. 10,000 1,000,000 100,000 Sample 2Viable Plate Count

  26. You are viewing the agar plates that were inoculated with BOILED FOUNTAIN WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. Incubator #4 #3 #2 #1 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers 1) Fountain Water2) Boiled Fountain Water3) River Water4) Boiled River Water Click here if you have viewed all the agar plates from all four of the samples

  27. You are viewing the agar plates that were inoculated with BOILEDFOUNTAIN WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. The dilution factor for the plate you are viewing is 10,000. 10,000 Incubator #4 #3 #1 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers Click here if you have viewed all the agar plates from all four of the samples

  28. You are viewing the agar plates that were inoculated with BOILEDFOUNTAIN WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. The dilution factor for the plate you are viewing is 100,000. Incubator #4 #3 #1 100,000 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers Click here if you have viewed all the agar plates from all four of the samples

  29. You are viewing the agar plates that were inoculated with BOILED FOUNTAIN WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. The dilution factor for the plate you are viewing is 1,000,000. 1,000,000 Incubator #4 #3 #1 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers Click here if you have viewed all the agar plates from all four of the samples

  30. 10,000 100,000 1,000,000 Sample 3Viable Plate Count

  31. You are viewing the agar plates that were inoculated with RIVER WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. Incubator #4 #3 #2 #1 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers 1) Fountain Water2) Boiled Fountain Water3) River Water4) Boiled River Water Click here if you have viewed all the agar plates from all four of the samples

  32. You are viewing the agar plates that were inoculated with RIVER WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. The dilution factor for the plate you are viewing is 10,000. 10,000 Incubator #4 #2 #1 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers Click here if you have viewed all the agar plates from all four of the samples

  33. You are viewing the agar plates that were inoculated with RIVER WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. The dilution factor for the plate you are viewing is 100,000. 100,000 Incubator #4 #2 #1 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers Click here if you have viewed all the agar plates from all four of the samples

  34. You are viewing the agar plates that were inoculated with RIVER WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. The dilution factor for the plate you are viewing is 1,000,000. Incubator #4 #2 #1 1,000,000 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers Click here if you have viewed all the agar plates from all four of the samples

  35. 10,000 1,000,000 100,000 Sample 4Viable Plate Count

  36. You are viewing the agar plates that were inoculated with BOILED RIVER WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. Incubator #4 #3 #2 #1 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers 1) Fountain Water2) Boiled Fountain Water3) River Water4) Boiled River Water Click here if you have viewed all the agar plates from all four of the samples

  37. You are viewing the agar plates that were inoculated with BOILED RIVER WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. The dilution factor for the plate you are viewing is 10,000. 10,000 Incubator #3 #2 #1 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers Click here if you have viewed all the agar plates from all four of the samples

  38. You are viewing the agar plates that were inoculated with BOILED RIVER WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. The dilution factor for the plate you are viewing is 100,000. Incubator #3 #2 #1 100,000 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers Click here if you have viewed all the agar plates from all four of the samples

  39. You are viewing the agar plates that were inoculated with BOILED RIVER WATER. Click on each of the three inoculated agar plates to view the bacterial colony growth. Count the number of colonies that are present and enter the data in DATA TABLE 1. If the count is less than 30 colonies, the notation will be “TFTC”. If the count is more than 300 colonies, the notation will be “TNTC”. The dilution factor for the plate you are viewing is 1,000,000. 1,000,000 Incubator #3 #2 #1 370C 10,000 100,000 1,000,000 Agar Plates Swabs Loops Antiseptic Dispenser Water Samples Lactose Broth Culture Tubes Pencil Bunsen burner Eye Droppers Click here if you have viewed all the agar plates from all four of the samples

  40. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 350C 500C 1000C Observe the three links given below to bring you to the VIRTUAL LAB that you wish to perform. If you have performed all of the exercises, you can click on END LAB. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Viable Plate Count Direct Count End Lab Turbidity Count

  41. Direct Count

  42. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C DIRECT MICROSCOPIC COUNT In the direct microscopic count, a counting chamber with a ruled slide is employed. It is constructed in such a manner that the ruled lines define a known volume. The number of bacteria in a small known volume is directly counted microscopically and the number of bacteria in the larger original sample is determined by extrapolation. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

  43. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 350C 500C 1000C The Petroff-Hausser counting chamber for example, has small etched squares 1/20 of a millimeter (mm) by 1/20 of a mm and is 1/50 of a mm deep. The volume of one small square therefore is 1/20,000 of a cubic mm or 1/20,000,000 of a cubic centimeter (cc). There are 16 small squares in the large double-lined squares that are actually counted, making the volume of a large double-lined square 1/1,250,000 cc. The normal procedure is to count the number of bacteria in five large double-lined squares and divide by five to get the average number of bacteria per large square. This number is then multiplied by 1,250,000 since the square holds a volume of 1/1,250,000 cc, to find the total number of organisms per ml in the original sample. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Petroff-Hausser counting chamber

  44. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 The Petroff-Hausser counting chamber as viewed through low power of the microscope

  45. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C If the bacteria are diluted, such as by mixing the bacteria with dye before being placed in the counting chamber, then this dilution must also be considered in the final calculations. The formula used for the direct microscopic count is: # bacteria per cc (ml) = # of bacteria per large square X dilution factor of large square (1,250,000) X dilution factor (dye) Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

  46. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 350C 500C 1000C • PROCEDURE: DIRECT MICROSCOPIC COUNT • We will be testing four samples of water for the Direct Microscopic Count. The samples include: • 1) water from a drinking fountain • 2) boiled water from a drinking fountain • 3) water from the local river • 4) boiled water from the local river • You will need DATA TABLE 2 to input your data and calculate the number of bacteria per ml. Click below to access a printable version of Data Table 2. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 DATA TABLE 2

  47. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C 1) Add 1 ml of the sample into a tube containing 1 ml of the dye methylene blue. This gives a 1/2 dilution of the sample. 2) Fill the chamber of a Petroff-Hausser counting chamber with this 1/2 dilution. 3) Place the chamber on a microscope and focus on the squares using 400X. 4) Count the number of bacteria in one of the large double-lined squares. Count all organisms that are on or within the lines. 5) Calculate the number of bacteria per cc (ml) as follows: The number of bacteria per cc (ml)= The number of bacteria per large squareXThe dilution factor of the large square (1,250,000)XThe dilution factor after mixing it with dye (2 in this case) Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

  48. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 The large, double-lined square holds a volume of 1/1,250,000 of a cubic centimeter. Using a microscope, the bacteria in the large square are counted. Count all organisms that are on or within the darker double lines.

  49. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Printable Version ofDATA TABLE 2

  50. Click on the WATER SAMPLES to bring the samples to the table. Next, click on the Methylene Blue bottle to bring the dye to the table. Now Click on the top of the Methylene Blue dye to withdraw 1 ml of the dye and dispense this to 1 ml of each of the Water Samples. Click on NEXT when dye has been added to all of the Water Samples. 3 2 4 1 Incubator 370C Methylene Blue Microscope Agar Plates Slides Loops Antiseptic Dispenser Water Samples Sterile Dilution Tubes Pencil Bunsen burner Eye Droppers 1) Fountain Water2) Boiled Fountain Water3) River Water4) Boiled River Water

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