Bacteria Enumeration Instructor Terry Wiseth

Bacteria Enumeration Instructor Terry Wiseth
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

Click on the blackboard to view a larger board for discussion.
Agar Plates Antiseptic Dispenser Swabs Loops Microbe Samples Pencil Loops Bunsen burner Incubator 37 0C

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 of bacteria require a dye to be introduced to the populations of bacteria to allow the observer to view the bacteria. Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C

Viable Plate Count Direct Count End Lab Turbidity Count
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 = 7 pH = 9 pH = 11 pH = 5 pH = 3 Viable Plate Count Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C Direct Count End Lab Turbidity Count

VIABLE PLATE COUNT

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 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 = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C

A major limitation in this method is selectivity
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 = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C

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 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 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 = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C

The # of colonies X The dilution factor of the plate counted
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 = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C

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 fountain 3) Water from the local river 4) 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 = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C DATA TABLE 1

1) Take 6 dilution tubes, each containing 9 ml of sterile saline
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 = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C

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 = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C

The # of colonies X The dilution factor of the plate counted
7) Count the exact number of colonies on that plate 8) Calculate the number of CFUs per ml of original sample as follows: CFUs per ml of sample = The # of colonies X The dilution factor of the plate counted Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C

Sterile Dilution Tubes Sample #1 Fountain Water
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. Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Incubator 37 0C Bunsen burner 4 1 2 3 4 1 2 3 6 5 Sample #1 Fountain Water

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. Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Incubator 37 0C Bunsen burner 4 1 2 3 4 1 2 3 6 5 Sample #1 Fountain Water

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 Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Incubator 37 0C Bunsen burner 4 1 2 3 4 1 2 3 6 5 Sample #1 Fountain Water

Lactose Broth Culture Tubes
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. Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 37 0C Bunsen burner 10,000 100,000 1,000,000 4 1 2 3 4 1 2 3 6 5 Sample #1 Fountain Water

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. Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner 10,000 100,000 1,000,000 4 1 2 3 4 1 2 3 6 5 Sample #1 Fountain Water

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. Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes 1) Fountain Water 2) Boiled Fountain Water 3) River Water 4) Boiled River Water Eye Droppers Incubator 370C Bunsen burner #1 #2 #3 #4

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

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”. Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner 1) Fountain Water 2) Boiled Fountain Water 3) River Water 4) Boiled River Water #1 #2 #3 #4 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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 Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner #2 #3 #4 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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. 100,000 Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner #2 #3 #4 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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 Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner #2 #3 #4 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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”. Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner 1) Fountain Water 2) Boiled Fountain Water 3) River Water 4) Boiled River Water #1 #2 #3 #4 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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 10,000. 10,000 Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner #1 #3 #4 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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 100,000. 100,000 Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner #1 #3 #4 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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 Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner #1 #3 #4 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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

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”. Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner 1) Fountain Water 2) Boiled Fountain Water 3) River Water 4) Boiled River Water #1 #2 #3 #4 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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 Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner #1 #2 #4 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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 Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner #1 #2 #4 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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. 1,000,000 Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner #1 #2 #4 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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”. Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner 1) Fountain Water 2) Boiled Fountain Water 3) River Water 4) Boiled River Water #1 #2 #3 #4 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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 Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner #1 #2 #3 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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. 100,000 Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner #1 #2 #3 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

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 Agar Plates Antiseptic Dispenser Swabs Loops Water Samples Pencil Lactose Broth Culture Tubes Eye Droppers Incubator 370C Bunsen burner #1 #2 #3 10,000 100,000 1,000,000 Click here if you have viewed all the agar plates from all four of the samples

Viable Plate Count Direct Count End Lab Turbidity Count
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 = 7 pH = 9 pH = 11 pH = 5 pH = 3 Viable Plate Count Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C Direct Count End Lab Turbidity Count

Direct Count

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 = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C

Petroff-Hausser counting chamber
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 = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C Petroff-Hausser counting chamber

Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C The Petroff-Hausser counting chamber as viewed through low power of the microscope

= X # bacteria per cc (ml) # of bacteria per large square
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) dilution factor (dye) Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C

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 = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C DATA TABLE 2

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 square X The dilution factor of the large square (1,250,000) X The dilution factor after mixing it with dye (2 in this case) Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C

Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C 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.

Printable Version of DATA TABLE 2
Data Table 2 Direct Count Sample # of Bacteria Dilution Factor (Large Square) Dilution Factor (Dye) DF (large square) X DF (Dye) X # of Colonies # of Bacteria / ml Faucet Water 1,250,000 2 1,250,000 X 2 X ______ River Water Boiled Faucet Water Boiled River Water # bacteria per ml = # of bacteria in square X dilution factor (Large Square) (1,250,000) X dilution factor (dye) Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C Printable Version of DATA TABLE 2

Sterile Dilution Tubes
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. Methylene Blue Microscope Agar Plates Antiseptic Dispenser Slides Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Incubator 37 0C Bunsen burner 1) Fountain Water 2) Boiled Fountain Water 3) River Water 4) Boiled River Water 4 1 2 3

Click on any one of the numbered WATER SAMPLES to add 1 ml of the sample to the Petroff-Hausser counting chamber for viewing and counting using the microscope under 400 X. You will need to view all four of the Water Samples. Methylene Blue Microscope Agar Plates Antiseptic Dispenser Slides Loops Water Samples Pencil Sterile Dilution Tubes 1) Fountain Water 2) Boiled Fountain Water 3) River Water 4) Boiled River Water Eye Droppers Incubator 37 0C Bunsen burner 4 1 2 3 Click Here if You Have Viewed All of the Water Samples

Water Sample 1

Click on the Microscope to bring it to the table. Next click on the SLIDES to bring one of them to the microscope. Now click on the EYE DROPPERS to transfer 1 ml of Water Sample #1 to the slide. Click on NEXT when you have added the sample to the slide on the microscope. Methylene Blue Microscope Agar Plates Antiseptic Dispenser Slides Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Incubator 37 0C Bunsen burner 4 1 2 3

Sterile Dilution Tubes Click Here to View a Different Water Sample
You are viewing bacteria from Sample #1 (Fountain Water). Count all organisms that are on or within the darker double lines. Record your count in TABLE 2. Calculate the number of bacteria per ml. Click on the EYEPIECE of the microscope to view the slide under High Power (400 X). Methylene Blue Microscope Agar Plates Antiseptic Dispenser Slides Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Incubator 37 0C Bunsen burner 4 1 2 3 Click Here to View a Different Water Sample

Water Sample 2

You are viewing bacteria from Sample #2 (Boiled Fountain Water). Count all organisms that are on or within the darker double lines. Record your count in TABLE 2. Calculate the number of bacteria per ml. Click on the EYEPIECE of the microscope to view the slide under High Power (400 X). Methylene Blue Microscope Agar Plates Antiseptic Dispenser Slides Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Incubator 37 0C Bunsen burner 4 1 2 3 Click Here to View a Different Water Sample

Water Sample 3

You are viewing bacteria from Sample #3 (River Water). Count all organisms that are on or within the darker double lines. Record your count in TABLE 2. Calculate the number of bacteria per ml. Click on the EYEPIECE of the microscope to view the slide under High Power (400 X). Methylene Blue Microscope Agar Plates Antiseptic Dispenser Slides Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Incubator 37 0C Bunsen burner 4 1 2 3 Click Here to View a Different Water Sample

Water Sample 4

You are viewing bacteria from Sample #4 (Boiled River Water). Count all organisms that are on or within the darker double lines. Record your count in TABLE 2. Calculate the number of bacteria per ml. Click on the EYEPIECE of the microscope to view the slide under High Power (400 X). Methylene Blue Microscope Agar Plates Antiseptic Dispenser Slides Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Incubator 37 0C Bunsen burner 4 1 2 3 Click Here to View a Different Water Sample

Turbidity Count

TURBIDITY COUNT When you mix the bacteria growing in a liquid medium, the culture appears turbid. This is because a bacterial culture acts as a colloidal suspension that blocks and reflects light passing through the culture. Within limits, the light absorbed by the bacterial suspension will be directly proportional to the concentration of cells in the culture. By measuring the amount of light absorbed by a bacterial suspension, one can estimate and compare the number of bacteria present. Spectrophotometric analysis is based on turbidity and indirectly measures all bacteria (cell biomass), dead and alive. Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C The Spectrophotometer used to analyze turbidity of bacteria

A Description of How the Spectrophotometer Works
The instrument used to measure turbidity is a spectrophotometer. It consists of a light source, a filter which allows only a single wavelength of light to pass through, the sample tube containing the bacterial suspension, and a photocell that compares the amount of light coming through the tube with the total light entering the tube. The ability of the culture to block the light can be expressed as the amount of light absorbed in the tube. The absorbance (or optical density) is directly proportional to the cell concentration. (The greater the absorbance, the greater the number of bacteria.) Light entering a cloudy solution will be absorbed. A clear solution will allow almost all of the light through. Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C A Description of How the Spectrophotometer Works

The amount of absorbance measures what fraction of the light passes through a given solution and indicates on the absorbance display the amount of light absorbed compared to that absorbed by a clear solution. Inside, a light shines through a filter (which can be adjusted by controlling the wavelength of light), then through the sample and onto a light-sensitive phototube. This produces an electrical current. The absorbance meter measures how much light has been blocked by the sample and thereby prevented from striking the phototube. A clear tube of water or other clear solution is the BLANK and has zero absorbance. The amount of substance in the solution is directly proportional to the absorbance reading. A graph of absorbance vs. concentration will produce a straight line. As the number of bacteria in a broth culture increases, the absorbance increases. Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C

A Standard Curve Chart For Bacterial Count
A standard curve comparing absorbance to the number of bacteria can be made by plotting absorbance versus the number of bacteria per ml. Once the standard curve is completed, any dilution tube of that organism can be placed in a spectrophotometer and its absorbance read. Once the absorbance is determined, the standard curve can be used to determine the corresponding number of bacteria per ml. Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C A Standard Curve Chart For Bacterial Count

PROCEDURE: TURBIDITY COUNT
We will be testing only two samples of water for the turbidity enumeration test. One of the samples has been drawn from a drinking water faucet while the other was taken from the local river. You will need DATA TABLE 3 and a printable version of the STANDARD CURVE CHART to enumerate your samples bacteria. Click on NEXT when you have the DATA TABLE 3 and STANDARD CURVE CHART. Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C Click Here for a Printable Version of DATA TABLE 3 Click Here for a Printable Version of the STANDARD CURVE CHART

Turbidity dilution tubes
1) Place the ORIGINAL tube of the sample and four tubes of the sterile broth in a test-tube rack. Each tube of broth contains 5 ml of sterile broth. 2) Use four of these tubes (tubes 2 to 5) of broth to make four serial dilutions of the culture. 3) Transfer 5ml of the ORIGINAL sample to the first broth tube. Transfer 5ml from that tube to the next tube, and so on until the last of the four tubes has 5ml added to it. These tubes will be 1/2, 1/4, 1/8, and 1/16 dilutions. Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C Turbidity dilution tubes

5) Set the wavelength to 520 nm by using the WAVELENGTH dial.
4) Set the display mode on the Spectrophotometer to ABSORBANCE by pressing the MODE control key until the appropriate red LED is lit. 5) Set the wavelength to 520 nm by using the WAVELENGTH dial. 6) Standardize the spectrophotometer by using a BLANK. The BLANK used to standardize the machine is sterile nutrient broth: it is called the BLANK because it has a sample concentration equal to zero (# of bacteria = 0). 7) Place the original bacterial specimen into the spectrophotometer. 8) Next insert the 1/2 dilution and read it. Repeat this with the 1/4, 1/8, and 1/16 dilutions. Read to the nearest thousandth (0.001) on the absorbance digital display. Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C Spectrophotometer

Click Here for a Printable Version of DATA TABLE 3
9) Record your values in DATA TABLE 3 for each of the individual samples, along with the dilutions that they came from. 10) Using the standard curve table given below, calculate the number of bacteria per milliliter for each dilution. Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C Click Here for a Printable Version of DATA TABLE 3 Click Here for a Printable Version of the STANDARD CURVE CHART

Dilution factor X Bacteria # DATA TABLE 3 TURBIDITY COUNT
**Review the example of absorbance counts acquired and the determinations of # of bacteria for the dilutions using the STANDARD CURVE CHART given on the next page. Be sure to keep track of all of the zeros in your calculations of the subsequent calculations for average bacteria per ml. Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 SAMPLE NAME: EXAMPLE Dilutions Absorbance # of Bacteria Dilution Factor Dilution factor X Bacteria # Original 0.130 26,000,000 1 1 X 26,000,000 = 26,000,000 1/2 0.066 12,900,000 2 2 X 12,900,000 = 25,800,000 1/4 0.034 6,500,000 4 4 X 6,500,000 = 26,000,000 1/8 0.018 3,200,000 8 8 X 3,200,000 = 25,600,000 1/16 0.010 1,750,000 16 16 X 1,750,000 = 28,000,000 Total = 131,400,000 Average # of Bacterial Cells per ml (Total / 5) = 26,306,280 bacteria per ml EXAMPLE DATA TABLE 3 TURBIDITY COUNT Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C

Click Here for a Printable Version of the STANDARD CURVE CHART
Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Standard Curve for Bacterial Count Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C Click Here for a Printable Version of the STANDARD CURVE CHART

Click on the WATER SAMPLES to bring the rack of samples to the table. There are only two samples of water we will test. Sample A is Faucet or Fountain Water and Sample B is River Water. Click on the STERILE DILUTION TUBES to bring them to the table. Click on NEXT when both of the test tube racks are on the table. Spectrophotometer Antiseptic Dispenser Swabs Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Bunsen burner A B 1/8 1/2 1/4 1/16 Blank

Next click on Sample A to move the sample to the Dilution rack. Next we will perform the dilution by transferring 5 ml of the original sample to the first dilution tube labeled as 1/2. Then we will transfer 5 ml of the 1/2 dilution to the tube labeled as 1/4 and so on until the last tube labeled as 1/16 has 5 ml added to it. Click on the blue EYE DROPPERS to perform the dilution. There is a tube labeled as BLANK which contains only pure sterile broth with no bacteria (population = 0). Click on NEXT when the dilutions have been made. Spectrophotometer Antiseptic Dispenser Swabs Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Bunsen burner B 1/16 Blank A A A 1/8 1/4 1/2

Click on the SPECTROPHOTOMETER to bring the machine to the table. Next click on the MODE button to set the machine to ABSORBANCE mode. Then click on the DIAL to set the wavelength to 520 nm. Click on the BLANK to insert it into the Spectrophotometer. Next click on READ to view the ABSORBANCE for the BLANK. The BLANK will read “0” as there are no bacteria in the solution and thus no absorbance of light. Click on NEXT when the BLANK has been read. Spectrophotometer Antiseptic Dispenser Swabs Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Bunsen burner SPECTROPHOTOMETER MODE Absorbance Transmittance 000 READ Blank 1/8 1/16 1/4 A 1/2 0.000 520

Click on one of the dilutions in the dilution rack. Once the dilution has been inserted into the Spectrophotometer, click on READ to view ABSORBANCE for that dilution. Record your value for the dilution that you have selected in DATA TABLE 3 for FOUNTAIN WATER. One at a time click on each of the other dilutions and then click on READ to view each of the ABSORBANCE values for the individual dilutions. Click on NEXT when you have viewed all of the dilutions. Spectrophotometer Antiseptic Dispenser Swabs Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Bunsen burner SPECTROPHOTOMETER MODE Absorbance Transmittance 000 READ 1/8 1/16 1/4 A 1/2 0.014 0.008 0.023 0.044 0.005 0.000 520

Click on Sample B to move the sample to the Dilution rack. Next we will perform the dilution by transferring 5 ml of the original sample to the first dilution tube labeled as 1/2. Then we will transfer 5 ml of the 1/2 dilution to the tube labeled as 1/4 and so on until the last tube labeled as 1/16 has 5 ml added to it. Click on the blue EYE DROPPERS to perform the dilution. There is a tube labeled as BLANK which contains only pure sterile broth with no bacteria (population = 0). Click on NEXT when the dilutions have been made. Spectrophotometer Antiseptic Dispenser Swabs Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Bunsen burner B B 1/16 Blank A B 1/8 1/4 1/2

Click on the SPECTROPHOTOMETER to bring the machine to the table. Next click on the MODE button to set the machine to ABSORBANCE mode. Then click on the DIAL to set the wavelength to 520 nm. Click on the BLANK to insert it into the Spectrophotometer. Next click on READ to view the ABSORBANCE for the BLANK. The BLANK will read “0” as there are no bacteria in the solution and thus no absorbance of light. Click on NEXT when the BLANK has been read. Spectrophotometer Antiseptic Dispenser Swabs Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Bunsen burner SPECTROPHOTOMETER MODE Absorbance Transmittance 000 READ Blank 1/8 1/16 1/4 B 1/2 0.000 520

Click on one of the dilutions in the dilution rack. Once the dilution has been inserted into the Spectrophotometer, click on READ to view ABSORBANCE for that dilution. Record your value for the dilution that you have selected in DATA TABLE 3 for FOUNTAIN WATER. One at a time click on each of the other dilutions and then click on READ to view each of the ABSORBANCE values for the individual dilutions. Click on NEXT when you have viewed all of the dilutions. Spectrophotometer Antiseptic Dispenser Swabs Loops Water Samples Pencil Sterile Dilution Tubes Eye Droppers Bunsen burner SPECTROPHOTOMETER MODE Absorbance Transmittance 000 READ 1/8 1/16 1/4 B 1/2 0.035 0.018 0.073 0.121 0.010 0.000 520

Viable Plate Count End Lab Direct Count Turbidity Count
You have now entered the Data required for DATA TABLE #3. Calculate the number of bacteria for each of the two water samples by using the formulas given. If you have performed all of the enumeration exercises you can click on END LAB given below. If you would like to review or perform any of the other exercises for this lab click on the appropriate link given below. Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Freezer -10 0C Refrigerator 0 0C Incubator 35 0C Incubator 50 0C Incubator 100 0C Viable Plate Count End Lab Direct Count Turbidity Count

STUFF A 4 1 2 3 2 1

READ READ 520 000 MODE 722-2000 SPECTROPHOTOMETER MODE 722-2000
Transmittance 520 Absorbance SPECTROPHOTOMETER SPECTROPHOTOMETER MODE Absorbance Transmittance 000 READ

1/8 1/2 1/4 1/16 A Blank

Boiled River Water

River Water

Faucet Water

Boiled Faucet Water

Square in which bacteria are counted
Mouth Culture Square in which bacteria are counted

Bacteria Enumeration Instructor Terry Wiseth

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Bacteria Enumeration Instructor Terry Wiseth

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