The aim of this experiment was to estimate the number of viable cells in a yeast suspension that was already provided. Estimations of the viable yeast cells were taken via two methods of plating; pour plating and spread plating, of which hot agar was used with the pour plate technique. The results that were obtained for this experiment show that overall; the spread plate method gives a higher yield of viable yeast cells compared to the total count value of 2.8 x 10^7.
This experiment was conducted to estimate the number of viable cells in a yeast suspension, already provided. The definition of a viable cell, as stated in the Collins English Dictionary, 2008, p991 is ‘capable of growth’. Therefore, the definition of a viable yeast cell is a yeast cell capable of living and being able to grow.
In industrial and research settings, there is a need to quantify the microbe content of microbial products. The method for doing this varies for different types of microbes. Traditionally, the first microbes to be used commercially were bacteria and yeasts. These are typically single-celled species that can be grown in natural and artificial media, and are well-suited to growth in agar gels on Petri plates. Using this method, individual cells or clumps of cells will form discrete colonies, which become visible to the naked eye as the colony grows. Counting the number of colonies provides a direct way to track the original number of discrete microbial units. A count determined this way been dubbed the number of “Colony-Forming Units” or “CFU” for short. CFU’s are only applicable to single-celled microbes that can be grown on nutrient media, such as bacteria, yeasts, or spore-forming moulds.
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As the total count for the number of yeast cells was so vast (2.8 x 10^7) dilutions were made in order for a characteristic estimate of the total count of yeast cells to be made. Having diluted the sample enabled the human eye to count an estimate of the yeast cells. If dilutions had not been carried out, the sample of yeast cells would have been far too large and it would have been extremely time consuming and impossible to count the number of yeast cells.
A haemocytometer enables for an estimate of the total number of yeast cells present. It has a known volume of chamber and area which is etched on the glass. A cell suspension is able to be above the known area. The chamber is then filled with a yeast suspension then covered with a cover slip. An average number of microbes can then be counted in the ruled area to give the number of yeast cells per cm³.
The aim of this experiment was essentially, to estimate the total number of yeast cells in a culture and to estimate the number of living (viable) yeast cells.
Materials and Methods
The total count of the yeast cells originally estimated by the haemocytometer was 3.8 x 10^7, however, it was later concluded that this was incorrect due to a mix up from another class. The new result for the estimated total count of the yeast cells was 2.8 x 10^7. This number was clearly too large and a series of ten fold dilutions were carried out in order to make it easier to estimate and investigate the viable yeast cells. A series of ten fold dilutions were needed as this is an important technique in identifying the viable cells. As a figure of 2.8 x 10^7 was established and it is vital that the number of colonies attained remains within the range of 30-300. So the dilution for a range of 30 – 300 is 1/100 (10^-2), however, it is essential that further dilutions, both above and below 1/100 are used; 1/10 (10^-1) and 1/1000 (10^-3). To make the estimation more accurate, dilutions of 10^-4, 10^-5 and 10^-6 were also used for both pour and spread plates.
For full method, please refer to introduction to biology, microbiology and pharmacology practical booklet, pp 13-14.
Results
The results obtained for the pour plate and spread plate methods were as follow:
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
Pour plate (ml)
TNTC
TNTC
TNTC
TNTC
83
8
Spread plate (ml)
TNTC
TNTC
TNTC
100
10
1
Key:
TNTC – Too numerous to count
To determine the number of colony forming units (CFU) cm^-3 this calculation was used:
Counts on plate x (1/dilution) x (1/volume inoculated (ml))
The calculations that were carried in order to determine the number of colony forming units (CFU) cm^-3 of the original culture for the pour plate and spread plate are shown below:
Calculations for pour plate method:
83 x 1/10^-5 a 1/1 = 8.3 x 10^6 CFU, ml
Calculations for spread plate method:
100 x 1/10^-4 x 1/0.1 = 1.0 x 10^7 CFU, ml
The volumes inoculated for the pour and spread plate were different, the pour plate was inoculated with 1.0cm^-3 and the spread plate with 0.1micrometer.
Discussion
The table in the results sections shows that the values of the colonies that were counted for each of the plating techniques show good continuation, especially with the spread plate as the figures are increasing by a factor of ten each time.
The figure obtained for the total count was 2.8 x 10^7, comparing this to the figure calculated for the pour plate method, 8.3 x 10^6 CFU, ml there has been a loss in the number of viable cells using this method, there has been a decrease of 1.97 x 10^7 of viable yeast cells. Comparing the total count value to the spread plate figure of 1.0 x 10^7 there was also a loss of viable yeast cells, with a loss of 1.8 x 10^7. This decrease in viable yeast cells compared to the pour plate loss is lower.
”The hot agar used in the pour plate technique may injure or kill sensitive cells; thus spread plates sometimes give higher counts than pour plates.”(p 130, Microbiology, Seventh Edition, Joanne M. Willey et al)
The above statement backs up the results of the experiment, as the spread plate technique has given a considerable higher count of viable yeast cells. Other factors that may have resulted in the smaller number of viable yeast cells in the pour plate method could have been that there is a much higher likelihood that clumps of the colonies may have formed together in portions of the plate, making it much more difficult to count. This occurs less in spread plating, as the clumps are broken up, and therefore there is a better distribution of the cells.
Other factors that may have affected the results obtained for this experiment were the techniques used for the serial dilutions. With each sequential serial dilution step, there may have been transfer inaccuracies that lead to less accurate and less precise dispensing. This meant that the highest dilutions had the highest number of inaccuracies.
Also, after every inoculation, the dilution must be thoroughly mixed; this was not carried during any of the dilutions, so this may have also affected the number of viable yeast cells.
Finally, when doing viable counts, the higher dilution is, the more error is found in “estimating” the count of the original volume. For example, there were 10 colonies growing on the 10^-5 spread plate, and it was estimated that there were approximately 500000 colonies in the original suspension, but this was only an estimation to the closest hundred thousand. Likewise, with higher dilutions, such as the 10^6 on the pour plate, it was only estimated to the closest million.
There were some limitations to the experiment, which may have altered the results slightly. Not having much experience in using the Gilson pipettes may have had an impact on the accuracy of the pipetting that was done during the serial dilutions.
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