Ingeniera

Lab #1-Total Plate Count (TPC), Most Probable Number (MPN) and Selective Media
Short Report

Purpose: To become familiar with the total plate count (TPC) and most probable number techniques (MPN) for counting bacteria in a given sample. To learn the characteristics of selective media by determining which microorganisms are likely to be present in a variety of media.
TPC
An E. coli stockculture was used to make serial dilutions; two LB agar plates were inoculated (using the spread plates technique) for each of the following dilutions: 10-4, 10-5 and 10-6. The volume of the inoculum was 100 µL.
After the incubation period, it was observed that the 10-4 plate exhibited more bacterial growth than the 10-5 and even less in the 10-6. By visually examining the plates, it was determinedthat the one containing between 25-250 colonies was the 10-5 plate. Colonies were counted for each of the two 10-5 plates, resulting a total of 480 and 1,638 colonies, respectively. The CFU/ml was determined as follows:
CFUmL=average # of colonies from duplicatedilution factorx (volume plated)=480 CFU+1638 CFU210-5(0.1 mL)=1.059x109 CFU/mL
MPN
Eleven tubes, each with 5 mL of LST broth, wereprepared. An inverted Durham tube was inserted into each tube; once autoclaved and cooled, the Durham tubes were completely filled with liquid. Three tubes were inoculated for each of the following inoculum (E. coli) volumes: 0.1 mL, 0.01 mL, and 0.001 mL. The other two tubes served as positive and negative controls, respectively. After the incubation period, the appearance of bubbles was observed insome of the tubes. The positive control, of course, was one of those tubes, while the negative control exhibited no bubble. The following table summarizes the number of positive tubes (meaning they had bubbles) for each dilution.
Table 1. Results of the MPN technique |
Volume of inoculum (mL) | 0.1 | 0.01 | 0.001 |
# of positive tubes | 3 | 0 | 3 |

The combination 3-0-3 wasn’t found inthe table of results provided by the instructor. The closest combination was 3-0-2, which corresponded to 64 organisms/mL. Knowing the volumes of inoculum in each tube and the volume of the stock sample (5 mL), the total number of organisms was calculated for each of the diluted samples and the stock sample. The calculations and results are presented in the following table.
Table 2. Number oforganisms in the diluted and stock samples (MPN technique) |
Sample | # of organisms |
0.1 sample | 64 organisms/mLx0.1 mL= 6.4 organisms |
0.01 sample | 64 organisms/mLx0.01 mL= 0.64 organisms |
0.001 sample | 64 organisms/mLx0.001 mL= 0.064 organisms |
Stock sample | 64 organisms/mLx5 mL= 320 organisms |

Diffusion Disc Assay
Each of three MEA agar plates was inoculated with either ofthree stock cultures labeled “A”, “B”, and “C” (volume of inoculum: 100 µL). The process was repeated with three TSA plates. Four discs were placed on the agar’s surface of each of the nine plates. The discs contained: Chlortetracycline HCL (Chlor), Ethanol (the blank of Chlor), Nystatin (Nys), or DMSO (the blank of Nys).
After the incubation period, fungal and/or bacterial growth was observedin the plates. A photograph of each plate is presented in Table 3. Based on the results, the following observations can be done:
* There is fungal growth in MEA “A” and a zone of inhibition only in the Nys quadrant.
* There is fungal growth in TSA “A” and a zone of inhibition only in the Nys quadrant.
* There is fungal growth in MEA “B” and a zone of inhibition only in the Nysquadrant; however, slight bacterial growth can be observed in the same quadrant around the Nys disc.
* There is no inhibition zone in any of the quadrants of TSA “B”.
* There is no fungal growth in either of the TSA or MEA plates inoculated with stock culture “C”.
* There is little bacterial growth in MEA “C”, almost imperceptible.
* There is visible bacterial growth in TSA “C”; a zone...