Aseptic Techniques and Enumeration of Bacteria by Viable Count - Assignment Example

This experiment was aimed at enabling the student to practice the proper handling of microorganisms. Handling of microorganisms requires safety and effectiveness. It was also aimed at refreshing the student’s memory on the aseptic technique and how bacteria in suspension are enumerated.

Materials

The materials used included: nutrient agar plate, E.coli broth, wire loop, Bunsen burner, sterile saline, glass spreader, pipette, and 95% ethanol.

Procedure

By use of the cultures and some of the plates availed, transfers were done aseptically. Transfers done included: broth to broth transfer, broth to agar plate transfer using a loop, agar to agar plate transfer, and broth to broth transfer again. The numbers of the viable bacteria count were then estimated by colony count. The E. coli culture was diluted using sterile saline as a diluent. Spread plate inoculation was then used. This technique enables the quantification of the number of bacteria in a solution (Wise, 2013). Miles and Misra method was also used. Its advantage is that it is very economical with agar media (Markey, Leonard, Archambault, Cullinane, and Maguire, 2013). The laboratory environment was then sampled for bacterial contaminants. The two environments sampled included air and the individuals in the laboratory. In the case of humans in the laboratory, the student in person was sampled through swabbing.

Practical Two

Aims

The aim of this experiment was to motivate the students to utilize the power of observation. It was also aimed at ensuring the students could register and record accurately what they observed.

Gathering results

Aseptic Transfer and the Streak Plate

Turbidity was observed in the broth-to broth transfer in one of the broths while the other one was clear. Hence, it was inferred that the wanted bacteria grew throughout the sediment at the bottom of the bottle.

As the E.coli were transferred into the agar plate, the growth of a single colony was observed on the spread plate. The colony had cells that were small, white, and round in shape. The agar plate did not show any characteristics of bacterial growth. It was also noted that the bacteria that were collected were not enough with no contamination occurring.

Estimating the Numbers of Viable Bacteria by Colony Count

Table 1

     Colony count

      Spread Plate

Miles and Misra Plate

           10-1

        TMTC

           10-2

        TMTC

             30

           10-3

         TMTC

    Average equalling 20

           10-4

         116

             3

           10-5

         13

           None

           10-6

          2

           None

The average is the total number of colonies divided by 3.

Calculating the Colony

CFU/ml of original culture = number of colonies on plate × 10/dilution. This is the formula for calculating colonies on Spread Plate. CFU = Colony Forming Unit.

CFU/ml of original culture = Average number of colonies per spot × 100/dilution. This is the formula for calculating colonies on the Miles and Misra Plate. CFU = Colony Forming Unit.

Table 2

          10-4

10-3

Spread and Plate

       11,600,000

Miles and Misra

        200,000

Table 3 below shows the results of the standard deviation and the mean of the class data. The formula:
               

Table 3

Standard Deviation

Mean

Spread Plate

21,596,407

9,141,120

Miles and Misra

2,570,291

2,2216,171

Sampling the Environment for Bacterial Contaminants

At 25 and 37 degrees Celsius, the air plate that was placed away from the Bunsen burner showed the growth of a small single colony, unlike the plate near the burner which expressed the growth of five colonies of bacteria. From the body swabs, the plate that had the wet swab showed bacterial growth on the plate, unlike the plate that had the dry swab which showed no growth of bacteria.

Discussion

The growth of bacteria in the broths and plate coupled with contamination showed there was minimal difference between the cultured bacteria. The lack of growth of bacteria during the agar to agar plate transfer may have been due to contamination of the culture and a large number of collected bacteria collected. For bacteria to survive, an optimum temperature, moisture, and pH have to be present as was shown in the difference between bacterial growth near and far from the burner and also those from the wet and dry body swabs. Variation in the data collected by the class may have been caused by the improper spreading of plates and inaccurate measuring using the pipette by various students. Also, some students mixed the bacteria as they did not follow the instructions. The class should consider repeating the experiments to collect the variations observed. The Miles and Misra method showed less variation than the Spread plate method hence it is the better method.