Serial Dilutions in Microbiology :
🤔 Why this is called a serial dilution ?
A serial dilution is a series of sequential dilutions used to reduce a dense culture of cells to a more usable concentration. Each dilution will reduce the concentration of bacteria by a specific amount.
#Advantages of the serial dilution–agar plate technique are as follows:
1. Only viable cells are counted.
2. It allows isolation of discrete colonies that can be subcultured into pure cultures, which may then be easily studied and identified.
#Disadvantages of this method are as follows:
1. Overnight incubation is necessary before colonies develop on the agar surface.
2. More glassware is used in this procedure.
3. The need for greater manipulation may result in erroneous counts due to errors in dilution or plating.
Materials :
a) Culture :
24- to 48-hour nutrient broth culture of Escherichia coli.
b) Media :
The six 20-ml nutrient agar deep tubes and seven sterile 0.9-ml water blanks.
c) Equipments :
Hot plate, water bath, thermometer, test tube rack, micro incinerator or Bunsen burner, vortex mixer, micropipette tips, mechanical pipetting device, sterile Petri dishes, Quebec colony counter, manual hand counter, disinfectant solution in a 500-ml beaker, glassware marking pencil, turntable, bent glass rod, and beaker with 95% alcohol.
Procedure :
1. Liquefy six agar deep tubes in an autoclave or by boiling. Cool the molten agar tubes and maintain in a water bath at 45°C.
2. Label the E. coli culture tube with the number 1 and the seven 9-ml water blanks as numbers 2 through 8. Place the labeled tubes in a test tube rack. Label the Petri dishes 1A, 1B, 2A, 2B, 3A, and 3B.
3. Mix the E. coli culture (Tube 1) by rolling the tube between the palms of your hands to ensure even dispersal of cells in the culture.
4. With a sterile pipette, aseptically transfer 1 ml from the bacterial suspension, Tube 1, to water blank Tube 2. Discard the pipette in the beaker of disinfectant. The culture has been diluted 10 times to 10−1.
5. Mix Tube 2 and, with a fresh pipette, transfer 1 ml from Tube 2 to Tube 3. Discard the pipette. The culture has been diluted 100 times to 10−2.
6. Mix Tube 3 and, with a fresh pipette, transfer 1 ml from Tube 3 to Tube 4. Discard the pipette. The culture has been diluted 1000 times to 10−3.
7. Mix Tube 4 and, with a fresh pipette, transfer 1 ml from Tube 4 to Tube 5. Discard the pipette. The culture has been diluted 10,000 times to 10−4.
8. Mix Tube 5 and, with a fresh pipette, transfer 0.1 ml of this suspension from Tube 5 to Plate 1A. Return the pipette to Tube 5 and transfer 1 ml from Tube 5 to Tube 6. Discard the pipette. The culture has been diluted 100,000 times to 10−5.
9. Mix Tube 6 and, with a fresh pipette, transfer 1 ml of this suspension from Tube 6 to Plate 1B. Return the pipette to Tube 6 and transfer 0.1 ml from Tube 6 to Plate 2A. Return the pipette to Tube 6 and transfer 1 ml from Tube 6 to Tube 7. Discard the pipette. The culture has been diluted 1,000,000 times to 10−6.
10. Mix Tube 7 and, with a fresh pipette, transfer 1 ml of this suspension from Tube 7 to Plate 2B. Return the pipette to Tube 7 and transfer 0.1 ml to from Tube 7 Plate 3A. Return the pipette to Tube 7 and transfer 1 ml from Tube 7 to Tube 8. Discard the pipette. The culture has been diluted 10,000,000 times to 10−7.
11. Mix Tube 8 and, with a fresh pipette, transfer 1 ml of this suspension from Tube 8 to Plate 3B. Discard the pipette. The dilution procedure is now complete.
12. Check the temperature of the molten agar medium to be sure the temperature is 45°C. Remove a tube from the waterbath and wipe the outside surface dry with a paper towel. Using the pour-plate technique, pour the agar into Plate 1A as shown in Figure 18.2 and rotate the plate gently to ensure uniform distribution of the cells in the medium.
13. Repeat Step 12 for the addition of molten nutrient agar to Plates 1B, 2A, 2B, 3A, and 3B.
14. Once the agar has solidified, incubate the plates in an inverted position for 24 hours at 37°C.
The spread-plate technique requires that a previously diluted mixture of microorganisms be used. During inoculation, the cells are spread over the surface of a solid agar medium with a sterile, L-shaped bent glass rod while the Petri dish is spun on a lazy Susan/turntable.
The step-by-step procedure for this technique is as follows :
Spread Plating Technique :
1. Prepare bacterial suspensions as described above and label agar plates accordingly.
2. Place the bent glass rod into a beaker and add a sufficient amount of 95% ethyl alcohol to cover the lower, bent portion.
3. Place an appropriately labeled nutrient agar plate on the turntable. With a sterile pipette, place 0.1 ml of bacterial suspension on the center of the plate.
4. Remove the glass rod from the beaker, and pass it through the Bunsen burner flame with the bent portion of the rod pointing downward to prevent the burning alcohol from running down your arm. Allow the alcohol to burn off the rod completely. Cool the rod for 10 to 15 seconds.
5. Remove the Petri dish cover and spin the turntable.
6. While the turntable is spinning, lightly touch the sterile bent rod to the surface of the agar and move it back and forth. This will spread the culture over the agar surface.
7. When the turntable comes to a stop, replace the cover. Immerse the rod in alcohol and reflame.
8. In the absence of a turntable, turn the Petri dish manually and spread the culture with the sterile bent glass rod.
Results :
Once the agar has solidified, incubate the plates in an inverted position for 24 hours at 37°C. The spread-plate technique requires that a previously diluted mixture of microorganisms be used. During inoculation, the cells are spread over the surface of a solid agar medium with a sterile, L-shaped bent glass rod while the Petri dish is spun on a lazy Susan/turntable. The step-by-step procedure for this technique.
Procedure After Serial dilution and Spread plate plate :
Now Colony Counting Will be there :
1. Using a Quebec colony counter and a mechanical hand counter, observe all colonies on plates. Statistically valid plate counts are only obtained from bacterial cell dilutions that
yield between 30 and 300 colonies. Plates with more than 300 colonies cannot be counted and are designated as too numerous to count— TNTC; plates with fewer than 30 colonies are designated as too few to count—TFTC. Count only plates containing between 30 and 300 colonies. Remember to count all subsurface as well as surface colonies. 2. The number of organisms per ml of original culture is calculated by multiplying the number of colonies counted by the dilution factor: number of cells per ml = number of colonies * dilution factor Examples: a. b. Colonies per plate = 50 Dilution factor = 1:1 * 106 (1:1,000,000) Volume of dilution added to plate = 1 ml 50 * 1,000,000 = 50,000,000 or (5 * 107) CFUs/ml (colony -forming units) Colonies per plate = 50 Dilution factor = 1:1 * 105 (1:1,00,000) Volume of dilution added to plate = 0.1 ml 50 * 100,000 = 50,000,000 (5 * 106) cells/0.1 ml 5,000,000 * 10 = 50,000,000 (5 * 107) CFUs/ml
3. Record your observations and calculated bacterial counts per ml of sample in the Lab Report.
4. Since the dilutions plated are replicates of each other, determine the average of the duplicate bacterial counts per ml of sample and record in the chart provided in the Lab Report.
You see The Notable Observation Table and Results Table for Reference Regarding Your Experiment Serial Dilutions.
The dilution factor of each tube in a set:
dilution factor of each tube.
The Following is just for understanding Calulation & Formula :
The dilution factor of each tube in a set:
dilution factor of each tube
For a ten-fold dilution, 1 ml of sample is added to 9 ml of diluent. In this case, the dilution factor for that test tube will be:
Dilution factor formula
After the first tube, each tube is the dilution of the previous dilution tube.
Now, for total dilution factor ,
Total dilution factor for the second tube = dilution of first tube × dilution of the second tube.
Example:
For the first tube, dilution factor = 10-1 (1 ml added to 9 ml)
For the second tube, dilution factor = 10-1 (1ml added to 9 ml)
Total dilution factor = previous dilution × dilution of next tube
= total dilution of 10-1 × 10-1 = 10-2
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