Objectives of Serial dilution

• The objective of the serial dilution method is to estimate the concentration (number of organisms, bacteria, viruses, or colonies) of an unknown sample by enumerating the number of colonies cultured from serial dilutions of the sample.

• In serial dilution, the density of cells is reduced in each step so that it is easier to calculate the concentration of the cells in the original solution by calculating the total dilution over the entire series.

• Serial dilutions are commonly performed to avoid having to pipette very small volumes (1-10 µl) to make a dilution of a solution.

• By diluting a sample in a controlled way, it is possible to obtain incubated culture plates with an easily countable number of colonies (around 30–100) and calculate the number of microbes present in the sample.

Soil microorganisms live in thin films of water that surround soil particles. These tiny organisms include microflora (bacteria, fungi, and actinomycetes) and microfauna(protozoa and nematodes). In terms of numbers and biological activity, the microflora is dominant. Bacteria are small (about 1 - 10 µm) and occur in three general shapes rod(bacillus), spherical (coccus), and spiral (spirilla). Bacilli and cocci are more common in soil. Fungi are filamentous and much larger. The branched hyphae exhibit cell divisions and fungal mycelia (hyphal mass) are often macroscopic. Actinomycetes are also filamentous and branched but smaller.

Agar Plate Method for Microbial Count

In this method, soil is dispersed in an agar medium so that individual microbial cells, spores, or mycelial fragments develop into macroscopic colonies. The procedure involves successive dilutions of soil. Depending upon the extent of dilution, plates may be filled with a huge number of colonies or very few. Enumeration of colony-forming units initially present in the soil is from plates in between these extremes. This method requires a sterile technique to avoid the introduction of extraneous microbes.

Procedure

A homogenized, field-moist sample of topsoil and bottles containing 90 mL of sterilized water was taken to perform the experiment.

1. Add a 10 g sub-sample of topsoil to the bottle of sterilized water. Tightly cap and shake vigorously for 10 minutes to disperse the soil. This is the 10^-1 dilution.

2. Transfer 10 ml of the 10^-1 dilution to another bottle of sterilized water. Use a sterile pipette. Take the sample from the middle. Tightly cap and shake to uniformly mix. Thesis the 10^-2 dilution.

3. Repeat step 2 using the 10^-2 dilution to make a 10^-3 dilution and proceed similarly, making 10^-4, 10^-5, 10^{- 6}, and 10^-7 dilutions.

4. From the 10^-7 dilution, transfer 1 mL to each of 2 sterile Petri dishes using a sterile 1mL pipette. Make similar transfers from the 10^-6, 10^-5, and 10^-4 dilutions.

5. Into each seeded petri dish, pour enough sterile, melted agar to fill the dish. Immediately swirl it around to ensure good mixing of soil inoculant and agar.

6. After the agar has solidified, invert plates and incubate at 35^oC for 1 day.

7. Next week, count the number of colonies on plates from the dilution that contains from 30 to 300 colonies. Don't count from those plates that contain colonies larger than 2 cm diameter. Multiply by dilution, take the average, and correct to the oven-dry moisture content of the soil. This gives the number of colony-forming units (CFUs) per gram of soil.

A dilution problem such as the one shown above is relatively easy to solve if taken step by step. Follow the steps below.

1. First, determine which is the countable plate.

   • Count the number of colonies on each plate. If there are too many colonies on the plate, the colonies can run together and become indistinguishable from individual colonies. In this case, the plate is called confluent or Too Numerous To Count (TNTC). The countable plate has between 30 and 300 colonies. More than 300 colonies would be difficult to count, and less than 30 colonies are too small a sample size to present an accurate representation of the original sample. As stated above, the number of colonies is the number of Colony Forming Units which represents the number of microorganisms per ml.

2. Sample Dilution Factor (SDF)

   • A sample is often diluted prior to doing the serial dilutions. If it is, the sample dilution factor will be shown in the diagram as above (the 1/2 in the Erlenmeyer flask is the sample dilution factor). If the sample remains undiluted, use 1/1 as the Sample Dilution Factor.

3. Individual Tube Dilution Factor (ITDF)

   • The individual tube dilution factors are a calculation of how much the sample was diluted in each individual tube. This is just the amount of sample added to the tube divided by the total volume in the tube after adding the sample. In tube I above, 1 ml of sample was added to 9 ml of water, so the ITDF for tube-I is: 1ml/1ml + 9 ml = 1/10

4. Total Series Dilution Factor (TSDF)

   • The total series dilution factor is a calculation of how much the sample was diluted in all of the tubes combined. This is accomplished by multiplying each of the appropriate ISDF. This series does not include any dilutions after the countable plate. In the example above, since the countable plate was plate C, tube IV is not included in the TSDF. The TSDF for the example above is 1/10 (ITDF for tube I) x 1/10 (ITDF for tube II) x 1/6 (ITDF for tube III) = 1/600.

5. Plating Dilution Factor (PDF)

   • When the sample is plated, a dilution factor must also be calculated for this transfer. Since the object of these calculations is to determine CFU/ml, the amount plated for the countable plate is divided by 1 ml to get the PDF. In the example above, 0.3 ml from tube III was plated onto plate C, so the PDF is 0.3ml/1.0 ml = 0.3ml/1.0ml x 10/10 = 3/10.

6. Final Dilution Factor (FDF)

   • The FDF takes into account all of the above dilution factors, giving you the total dilution from the original sample to the countable plate. The FDF = SDF x TSDF x PDF, so in this example, the FDF = 1/2 x 1/600 x 3/10 = 3/12000 = 1/4000. This means that the original sample was 4000 times as concentrated as the plated sample from tube III. In other words, it would take 4 L of the sample in tube III to contain the same number of bacteria as 1 ml of the original sample.

7. Colony Forming Units/ml (CFU/ml) in the original sample

   • To find out the number of CFU/ ml in the original sample, the number of colony-forming units on the countable plate is multiplied by 1/FDF. This takes into account all of the dilutions of the original sample. For the example above, the countable plate had 200 colonies, so there were 200 CFU, and the FDF was 1/4000.

   • 200 CFU x 1/1/4000 = 200 CFU x 4000 = 800000 CFU/ml = 8 x 10

   • CFU/ml in the original sample.

Total no. of cfu = colony count on agar plate / (total dilution of tube used to make plate for colony count X amount plated)

Example

Determine the amount of c.f.u.

1. The countable plate is the one with 71 colonies.

2. The total dilution of 3^rd tube from which the above pour plate was made = 1/10 X 1/10 X 1/10 = 1/10³

3. The amount used to make that pour plate = 1ml

4. Total no. of cfu = colony count on agar plate / (total dilution of tube used to make plate for colony count X amount plated)

71 colonies/ (1/10³ X 1) = 71 X 10³ = 7.1 X 10⁴ (scientific notation) OR 71,000/ml

Rules for Scientific Notation

For a number to be in correct scientific notation, the following conditions must be true:

1. The coefficient must be greater than or equal to 1 and less than 10.

2. The base must be 10.

3. The exponent must show the number of decimal places that the decimal needs to be moved to change the number to standard notation. A negative exponent means that the decimal is moved to the left when changing to standard notation.

Questions:

Q.1: What is the dilution factor if you add 0.2 mL of a stock solution to 3.8 mL of diluent?

Q.2: Determine the number of bacteria per ml. of water specimen.

Q.3: Determine the number of bacterial cells per ml. in the original culture.

Q.4: Determine the number of bacterial cells per gram of meat.