From this experiment, I learnt how to enumerate the microorganisms in a given sample. There are many ways to determine the microbial growth. There are direct counting methods, chemical methods, measure the tubidity and the serial-dilution plate method. In this experiment, we do the serial-dilution plate method. From the given sample of E. coli, we carrries out a series of dilution. Then, we dilute the sample further by using pour plate and spread plate to get single colonies. After incubation, we count the single colonies present by using the Quebec colony counter. The number of colonies counted are taken for a range between 30 too 300. The number of colonies which is over 300 are labelled as TNTC while TFTC is labelled for plate which has colonies of less that 30.The procedures of the experiment are as followed. The procedures of the experiment are as followed. Six agar deep tubes are liquefied in an autoclave or by boiling. The molten agar tubes was cooled and maintained in a water bath at 45 °C . The E. coli culture tube are labelled with the number 1 and the seven 9-ml water blanks as numbers 2 through 8. The labelled tubes were placed in a test tube rack. The Petri dishes are labelled as 1A, 1B, 2A, 2B, 3A, and 3B. The E. coli culture was mixed by rolling the tube between the palms of the hands to ensure even dispersal of cells in the culture. Then, 1 ml from the bacterial suspension, Tube 1, was aseptically transferred to water blank Tube 2 with a sterile pipette. The pipette was discarded in the beaker of disinfectant. The culture has been diluted 10 times to 10-1. Tube 2 was mixed. With a fresh pipette, 1 ml from Tube 2 was transferred to Tube 3. The pipette was discarded. The culture has been diluted 100 times to 10-2. Tube 3 was mixed. With a fresh pipette, 1 ml from Tube 3 was transferred to Tube 4. The pipette was discarded. The culture has been diluted 1000 times to 10-3. Tube 4 was mixed. With a fresh pipette, 1 ml from Tube 4 was transferred to Tube 5. The pipette was discarded. The culture has been diluted 10000 times to 10-4. Tube 5 was mixed. With a fresh pipette, 0.1 ml from Tube 5 was transferred to Plate 1A. The pipette was returned to Tube 5 and 1 ml from Tube 5 is transferred to Tube 6. The pipette was discarded. The culture has been diluted 100000 times to 10-5. Tube 6 was mixed. With a fresh pipette, 1 ml of this suspension from Tube 6 was transferred to Plate 1B. The pipette was returned to Tube 6 and 0.1 ml was transferred from Tube 6 to Plate 2A. The pipette is returned to Tube 6 and1 ml from Tube 6 is transferred to Tube 7. The pipette was discarded. The culture has been diluted 1000000 times to 10-6. Tube 7 was mixed. With a fresh pipette, 1 ml of this suspension from Tube 7 was transferred to Plate 2B. The pipette was returned to Tube 7 and 0.1 ml was transferred from Tube 7 to Plate 3A. The pipette is returned to Tube 7 and1 ml from Tube 7 is transferred to Tube 8. The pipette was discarded. The culture has been diluted 10000000 times to 10-7. Tube 8 was mixed. 1 ml of this suspension was transferred from Tube 8 to Plate 3B with a fresh pipette. The pipette was discarded. The dilute procedure is now complete.
The temperature of the molten agar
medium was checked to be sure the temperature is 45 °C. A tube was removed from the water bath and the
outside surface was dried with a paper towel. The agar is pour into Plate 1A by
using the pour-plate technique. The plate was rotated gently to ensure uniform
distribution of the cells in the medium. This step is repeated for the addition
of molten nutrient agar to Plates 1B, 2A, 2B, 3A, and 3B. Once the agar
solidified, the plates is incubated in an inverted position for 24 hours at 37 °C.
The
spread plate was also carried out to get single colony. Bacterial suspensions
are prepared as described above and agar plates were labelled accordingly. The
bent glass rod was placed into a beaker and a sufficient amount of 95% ethyl
alcohol to cover the lower, bent portion. An appropriately labelled nutrient
agar was placed on the turntable. 0.1 ml of bacterial suspension was placed on
the center of the plate with a sterile pipette. The glass rod was removed from
the beaker and it was passed through the Bunsen burner flame with the bent
portion of the rod pointing downward to prevent the burning alcohol from
running down the arm. The alcohol was allowed to burn off the rod completely.
The rod was cooled for 10 to 15 seconds. The Petri dish cover was removed. In
the absence of a turntable, the Petri dish was turned manually and the culture
was spreaded with the sterile bent glass rod.
Using
a Quabec colony counter and a mechanical hand counter, all colonies were
observed on plates. Statistically valid plate counts are only obtained from
bacterial dilutions that yield between 30 and 300 colonies.
Picture of Quebec Colony Counter
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