Food Microbiology Laboratory question, **********Question********** (PLease help
ID: 208464 • Letter: F
Question
Food Microbiology Laboratory question,
**********Question********** (PLease help me to figure out the answers after reading/ using following text, thx a lot)
1. Deeply analyse and explain the result of this lab (results is provide in the following text)
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Laboratory: Microbiological analysis of food products (Standard plate count)
Used media:
Nutrient Broth
Nutrient Broth is used for the cultivation of many species of non-fastidious microorganisms due to its content of peptone and beef extract. It is one of several nonselective media useful in routine cultivation of microorganisms.
Ingredients: Beef extract, peptone
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Food products used: 2 Char sui samples (Store at 4°C and room temperature for 20 hours)
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Most Probable Number (3-tube MPN)
The "most probable number" (MPN) method is a useful, if underutilized, tool for the microbiologist. The test is a method to estimate the concentration of viable microorganisms in a sample by means of replicate liquid broth growth in ten-fold dilutions and is particularly useful with samples that contain particular material that interferes with plate count enumeration methods.
For MPN test, the sample to be tested is prepared in 10-fold dilution series, and then 1mL samples of each dilution are inoculated into triplicate broth culture tubes for incubation. As the dilutions increase, the possibility that the broth tubes will fail to be inoculated with any microorganism increases. At some point therefore, very few of the replicate tubes will be inoculated with viable microorganisms.
Following incubation, all tubes are examined for turbidity and the pattern of growth in the tubes is scored against a table of such values. The MPN table from the US Food and Drug Administration's Bacterial Analytical Manual (BAM) (http://www.fda.gov) is provided above. A typical design uses three replicates with a three-log10 unit dilution series (although varying numbers of replicates and different dilution series may also be used). In this design, if all tubes showed growth, then the results will benotedas3,3,3. If only one tube in each replicate shows growth it would be denoted as 111. The pattern of growth is then read from the table to provide the most probable number and 95% confidence interval. By this, the result of 2,1,0 would reflect an MPN of 15, and a result of 3,2,2 would be interpreted as an MPN of 210.
Figures 1 and 2 show this in graphic depiction. As the incubated tubes would be read 3,2,1, the MPN would be recorded as 150. The MPN table normally only presents results for three dilutions in sequence (e.g. 10-1,10-2, 10-3), but the dilution series tested might have been from the 10-2 to 10-4 tubes. We will need to take the dilution factors in the table and in the actual experiment into account to derive the most probable number from this study.
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Procedure:
2.1 Label 5 tubes with Nutrient Broth (with 9 mL) as 10-3, 10-4, 10-5, 10-6 and 10-7 (1 set); prepare 3 sets for each food sample. (A total of 30 Nutrient Broth for 2 food samples).
2.2 Before pipetting, mix well each dilution by vortex.
2.3 Transfer 1ml of 10-3 dilution from part 1 to the tubes labeled as 10-3.
2.4 Transfer 1ml of 10-4 dilution from part 1 to the tubes labeled as 10-4.
2.5 Transfer 1ml of 10-5 dilution from part 1 to the tubes labeled as 10-5.
2.6 Transfer 1ml of 10-6 dilution from part 1 to the tubes labeled as 10-6.
2.7 Transfer 0.1ml of 10-6 dilution from part 1 to the tubes labeled as 10-7.
2.8 Incubate at 37oC for 24 hours.
2.9 Mix the broth by flipping and record the results as positive growth or no growth for each dilution.
2.10 Check and calculate MPN of your samples with reference to Table in page 13.
2.11 Make sure to take into consideration of dilution factor.
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Results:
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Table: MPN table for a three-replicate design from FDA's Bacterial Analytical Manual Positive Tubes 0.1 0.01 0.001 MPN 95% Confidence Range Positive Tubes 0.1 0.01 | 0.001 | MPN : 95% Confidence Range 3.0 0-9.5 4.5-42 0.15-9.6 2 21 28 8.7-94 35 8.7-94 29 8.7-94 36 8.7-94 23 4.6-94 38 8.7-110 0.15-11 6.1 1.2-18 6.2 12-18 9.4 3.6-38 3.6 0.17-18 7.2 1.3-18 3.6-38 1.3-20 3.6-38 3.6-42 15 4.5-42 4.5-42 9.2 1.4-38 3.6-42 20 4.5-42 3.7-42 4.5-42 27 8.7-94 0 0 20 0 30 2 3 0 2 3 1 3 0 0 17-180 3 0 2 0 64 43 9-180 0 7.4 75 17-200 120 37-420 160 40-420 93 18-420 150 37-420 21040-430 290 90-1000 240 42-1000 460 90-2000 2 3 2 0 16 2 0 0 3 22 14 3 30 3 31 3 32 20 1100 180-4100 1100 420-4000 Figure I: Three-tube design for MPN (unincubated) Figure 2: Three-tube design for MPN (incubated) 10 101 102 102 10- 103Explanation / Answer
Ans:
Results from table 3:
1. It is important to note that in case more than 3 dilutions have been used in a series of three tube MPN, results coming from only three consecutive dilutions are used to calculate the MPN. Here five different dilutions have been used, hence this qualifies for the same and only three dilutions will be taken to determine the MPN.
2.
Case 1: When one or more than one dilutions have all tubes showing positive results, then you have to select the highest dilution having the smallest sample quantity exhibiting positive results in all tubes and the next two higher dilutions,
Case 2: When none of the dilutions are showing 'all tubes positive', you have to select the three lowest dilutions where the mid dilution is showing a positive result.
Case 3: When all of the dilutions tested are showing 'all tubes positive', then you have to select the three highest dilutions.
This is a case 1, where samples stored at room temperature with 10-4 dilution has the highest dilution showing all three positives. Therefore the next two highest dilutions are chosen, making the combination 3-2-1.
Samples stored at 4degC at 10-5 dilution has all three positives being the highest dilution. Taking the next two highest dilutions the combination becomes 3-2-1.
Now, using FDA's Bacterial Analytical Model for three sample replicates we get that 3-2-1 combination has an MPN value of 150.
Results from Table 4:
Here the MPN is simply multiplied by the dilution factor to get MPN/g
eg: For samples stored at room temperature- MPN=150
Hence MPN/g = 150 * 10000 (104) =1500000