LABORATORY REPORT
MICROBIAL PHYSIOLOGY
MIC 310
EXPERIMENT
|
: LABORATORY 1
|
|
TITTLE
|
: MEASUREMENT OF MICROBIAL GROWTH
|
|
NAME
|
: WAN MUHAMAD QUSYAIRI BIN WAN MAZLAN
|
|
STUDENT ID
|
: 2013xxxxxx
|
|
GROUP
|
: AS1144A1
|
|
LECTURER’S NAME
|
: MADAM IWANA IZNI BINTI ZAINUDDIN
|
|
TITLE: MEASUREMENT OF MICROBIAL GROWTH
OBJECTIVE:
To learn and assess different techniques for monitoring the growth of bacteria in liquid culture. At the end of the experiment, students should be able to correlate the findings and construct a typical growth pattern.
INTRODUCTION
Bacterial population growth studies require inoculation of viable cells into a sterile broth medium and incubation of the culture under optimum temperature, pH, and gaseous conditions. Under these conditions, the cells will reproduce rapidly and the dynamics of the microbial growth can be charted by means of a population growth curve, which is constructed by plotting the increase in cell numbers versus time of incubation and can be used to delineate stages of the growth cycle. It also facilitates measurement of cell numbers and the rate of growth of a particular organism under standardized conditions as expressed by its generation time, the time required for a microbial population to double.
Construction of a complete bacterial growth curve requires that aliquots of a 24-hour shake-flask culture be measured for population size at intervals during the incubation period, however, such a procedure does not lend itself to a regular laboratory session. This experiment is designed to include only the lag, log and possibly stationary phases of population growth. Upon completion of this experiment, you will plot the data collected during this experiment by using two values for the measurement of growth. The direct method requires that you use serial dilution to plate out cells at 30-minute intervals in order to calculate the number of colony forming units (CFU) at a given time. The indirect method uses spectrophotometric measurements of the developing turbidity at the same 30-minute intervals, as an index of increasing cellular mass (assumed to correlate with an increase in the number of cells).
The overnight culture of E. coli prepared prior to the practice is growing in log phase. Therefore, at the end of the practical student will come out with a Standard Growth Curve of E. coli which will be used in the next practical in order to determine the dry mass of a single E. coli cell.
METHOD
A. Haemocytometer count
The hemocytometer consists of thick glass slide specially constructed to hold 0.1 mm3 liquid (when the proper coverslip is used) in a grid 0f 1mm x 1mm x 0.1mm. The grid was divided into 400 small squares grids for ease of counting. A drop of culture was shaken and placed at the center of the platform using a sterile pipette, and covered by a coverslip. Since the volume of culture in the grid was 0.1mm3, and 1 ml was made up of 1000 mm3 the numbers of cells counted from all 400 squares (0.1 mm3) was multiplied by 104 to give the cell population in 1 ml. in this experiment, we discover it is tedious and almost impossible to count all cells from 400 squares. Therefore it is more practical to get a representative c, count from 2, 4, 8 or equivalent squares. If this done then the cells population in 1 ml if a count is taken from 4 squares is:
Cell count x 400/4 x 10000
Counting the cells:
1. At least 50-100 cells in the large square were checked.
2. The average count from 3 different locations was taken.
3. The cell that lies on a boundary was counted where counted once only.
B. TURBIDITY
When the light of approx. 650nm wavelength was shone through bacterial suspension, some of the light was scattered, deflected and refracted. In a spectrophotometer, this light does not reach the defector, and so this suspension was said to have an optical density compared to a clear standard solution. The amount of light scattered is proportional to the number of bacterial cells in suspension.
1. The spectrophotometer was switched on and 650 nm was selected as the wavelength. 30 min warm up time was allowed.
2. The reading was zeroed using a tube of sterile Nutrient broth.
3. The turbidity of the culture was measured at every designated interval.
C. Dilution plating
1. The culture was ensured mixed thoroughly. Using a sterile pipette, the sample was sucked up and down to wet the pipette. Then, 0.1ml of the sample was withdrawn into 0.9 ml diluent. The pipette must not touch the dilution water. The sample was mixed and continued diluting into tube 2 and 3.
2. Using a fresh pipette, 0.5 ml of each dilution was plated onto plates (duplicate). One pipette was used for each dilution. The plating was done with the highest dilution. The plates were incubated at the designated temperature.
3. The result was tabulated based on plates having between 30-300 cfu.
RESULT
A. TURBIDIMETRY
Time(min)
|
Optical density at 650nm
|
Blank
|
0.000
|
0
|
0.112
|
20
|
0.154
|
40
|
0.186
|
60
|
0.192
|
C. DILUTION PLATING
Time(min)
|
Number of colonies for each dilution factor
|
||
102
|
104
|
106
|
|
0
|
252
|
3
|
0
|
20
|
TMTC
|
31
|
1
|
40
|
TMTC
|
TMTC
|
18
|
60
|
TMTC
|
TMTC
|
66
|
Data processing
The number of colony forming units of bacteria is calculated based on the following equation:
Microbial growth (CFU/ml) = Number of CFU x dilution factor x aliquot factor
Based on our result on dilution plating, the data is all statistically unreliable at every dilution since none produced the colony-forming units between 30 t0 300. Therefore, we pick the most feasible one which is the 106 dilution. The calculation is shown in the table below
Time(min)
|
Calculation
|
Microbial growth (CFU/ml)
|
0
|
0
|
|
20
|
2,000,000
|
|
40
|
36,000,000
|
|
60
|
132,000,000
|
Finally, we tabulate the turbidity with the colony-forming unit:
Time(min)
|
Microbial growth(CFU/ml)
|
Optical density at 650nm
|
0
|
0
|
0.112
|
20
|
2,000,000
|
0.154
|
40
|
36,000,000
|
0.186
|
60
|
132,000,000
|
0.192
|
Graph: Microbial growth against optical density at 650nm
DISCUSSION
Based on our result we manage to plot the typical growth pattern for E.coli and therefore it’s Standard Growth Curve. However, there are several matters which should be considered. The general relationship between the turbidity and plate count shows that turbidity increases and the plate count increases and support the theories underlying the method. However, the growth pattern shown here does not really fit a straight line. In fact, it resembles more of an exponential growth. Theoretically, the graph will form an increasing straight line indicating a positive proportional relation between the plate count and turbidity. This is because the culture prepared by the lab technician was supposed to be in its log phase, in which the bacteria divide at a logarithmic rate and the number of bacteria increases at a constant rate, therefore forming a straight line.
This gives rise to whether or not actually the bacteria culture when inoculated is still in the lag phase and was just entering log phase. If this is the case then it explains why the graph plotted would fit an exponential line better. However, this suggestion can easily be dismissed because it seems that the exponential shape if the curve is attributed to several factors. The first reason is that the turbidity measured at 0 minutes gives 0.112optical density but when pour plated it gives zero colonies. This is wrong. What happens is actually because the dilution is too small (one millionth) and the bacterial number is reduced to an undetected level. It is not correct because, at lower dilution, there are colonies present. Secondly, the turbidity and plate count was done separately which can contribute to error. Thirdly, dilution is not done gradually, which will not provide a wider array of numbers so that we can choose ones which are more reliable statistically. The dilution is done at 1:100 dilution at every pipette transfer instead of 1:10. Finally, none of the data obtained is statistically reliable and this causes the points plotted to scatter farther away from the line of best fit showing the erroneous result.
There are several ways we can improve our methods so that our outcome will better fit a straight. Firstly, the turbidity measurement and pour plating should not be done separately. Instead, every inoculum should be measured its turbidity first before undergoing serial dilution to give a more coherent result. Secondly, serial dilution should be done gradually, at the rate of 1:10 at every pipette transfer. Notice that in our result, the numbers fluctuate greatly between every dilution meaning that maybe we can find more reliable data at the intermediate dilution for example at 105 . By comparing the procedure and result of each method, we can say that there is actually no best method to measure bacterial growth. Rather, the suitability of each method actually depends on the time, type of bacteria, the purpose and also the situation.
Based on our observation every method of measuring the bacterial growth has its pros and cons. For example, the hemocytometer is frequently used to directly count the cell number in blood count and sperm count in clinical labs. However, it is a very tedious process. Turbidity is a very fast and easy method and often used in industrial microbiology. However, it cannot differentiate between live cells and dead cells and cannot detect below 107 dilutions. When it comes to counting viable cells, plate count is used for example to test milk and dairy product. However, it requires incubation time.
CONCLUSION
Students have managed to construct the Standard Growth Curve of E. coli. Students have also performed different methods of measuring microbial growth and concluded that the suitability of each method depends on many factors such as the type of microbe, purpose, time taken and situation.
QUESTIONS
1. Which method is judged more appropriate to measure the growth of bacteria?
This is a rather debatable topic. Each method has its own pros and cons and the suitability depends on several factors such type of bacteria and purpose. In the context this experiment, turbidity is seen to be the most appropriate method to measure the growth of bacteria in its most active phase. This is because turbidity does not require incubation time and takes very little. Plus, we use a machine which can reduce human error. Turbidity is also used to measure bacterial growth mostly in industrial microbiology for example to plot the standard growth curve. On the other hand, plate count is used to measure the bacterial growth when viability in concern for example in food product testing. Haemocytometer is often used in the direct count of the cells such as in blood count and sperm count in clinical labs.
2. If this experiment is repeated do you think you can produce the same graphs you obtained? Explain.
Theoretically, if were to repeat the exact same procedure we will able to produce the exact same graph as the three aspects of growth which are genetic makeup, media used and environmental variable remain intact.
However, in reality, human error and some environmental variables are hard to control and this will always alter the result.
3. What valuable data can you derive and conclude from the results you obtained
Among the valuable data, we can obtain from the standard growth curve is the doubling time or the generation time for E. coli. Generation time is the time taken by the bacteria to double in number during a specified time period.
REFERENCE
Cappucino, J. G., & Sherman, N. (2001). Microbiology: A Laboratory Manual. San Francisco: Pearson Education, Inc.
Gladwin, M., Trattler, W., & Mahan, C. (2013). Clinical Microbiology Made Ridiculously Simple (Ed. 6). Pittsburgh: Medmaster.
Sutton, S. (2011). Measurement of Microbial Cells with Optical Density. Journal of Validation Technology, 46-49.
Talaro, K. P., & Chess, B. (2012). Foundations in Microbiology. New Yowk: McGraw-Hill Companies.
vlab.amrita.edu. (2011). Bacterial Growth Curve. Retrieved July 27, 2015, from Virtual Lab Amrita: http://vlab.amrita.edu/?sub=3&brch=73&sim=1105&cnt=1
No comments:
Post a Comment