BTE 2211 BIOTECHNOLOGY ENGINEERING LAB 1

EXPERIMENT 5:
THE BACTERIAL GROWTH CURVE

DATE:
23 AUGUST 2007
th

NAME: SITI FARHAANA BT SHAMSUDDIN MATRIC N0 : 0535788 SECTION: 1 ( THURSDAY 2-5 PM) INSTRUCTOR: PROF DR ISMAIL

1. OBJECTIVES:
a. To acquaint students with the population growth dynamics of bacterial cultures. b. To plot a growth curve and determine the generation time of bacterial cultures. 2. RESULT AND OBSERVATION.

Incubation time (hour) 0 1 1 2

Optical Density @ 600 nm 0.010 0.028 0.055 0.094 0.127 10-4 0.99 x 106 1.03 x 106 1.22 x 106 1.31 x 106 1.55 x 106

Plate counts Cells/ml 10-5 0.27 x 107 0.45 x 107 0.53 x 107 0.69 x 107 0.85 x 107 10-6 0.05 x 108 0.19 x 108 0.37 x 108 0.49 x 108 0.60 x 108 10-4 5.9956 6.0128 6.0864 6.1173 6.1903

Log of Cells/ml 10-5 6.4314 6.6532 6.7243 6.8388 6.9294 10-6 6.6990 7.2788 7.5682 7.6902 7.7782

Calculation of Generation Time: 1. Direct method g= 2 log 2 (7.5682 7.2788)

= 2.0804 hours 2. Indirect method GT = t (OD 0.4) t (OD 0.2) = 0.75 hour 0.25 hour = 0.50 hours

3. DISCUSSION.
A typical growth curve for a population of bacterial cells illustrates some of the dynamics that effect the population over the course of time. The population may begin when several bacteria enter the human respiratory tract or transferred to a tube of growth medium in the laboratory. Four distinct phases of the curve are recognized: the lag phase, the logarithmic phase, the stationary phase and the decline phase. The lag phase encompasses the first few hours of the curve. During this time, bacteria adapt to their new environment. Although cell division does not take place right away and there is no net increase in mass, the cell is synthesizing new components. The lag phase varies considerably in length with the condition of the microorganism and the nature of the medium. During the exponential or log phase, microorganism are growing and dividing at the maximal rate possible given their genetic potential, the nature of the medium and the condition under which they are growing. Their rate of growth is constant during the exponential phase that is the microorganisms are dividing and doubling in number at regular interval. Exponential growth is balanced growth. If nutrient levels or other environmental conditions change, unbalanced result. This is growth during which bacteria are transferred from a nutritionally poor medium to a richer one. There is a lag while the cells first construct new ribosome to enhance their capacity for protein synthesis. Stationary phase is where the population growth curves become horizontal. This stationary phase usually is attained by bacteria at a population level of around 109 cells

per ml. Microbial populations enter the stationary phase for several reasons. One obvious factor is nutrient limitation. If an essential nutrient is severely depleted, population growth will slow. The death of a microbial population, like its growth during the exponential phase, may be logarithmic. This pattern in viable cell count holds even when the total cell number remains constant because the cells simply fail to lyses after dying. Often the only way of deciding whether a bacterial cell is viable is by incubating it in fresh medium .If it is not grow and reproduce it is assumed to be dead. In this experiment, the generation time is determined using direct and indirect method. The direct method requires enumeration of viable cells using serially diluted samples of the test culture taken at two hour intervals. The indirect method uses spectrophotometer measurement of the developing turbidity at the same two hour intervals as an index of increasing cellular mass. The viable counting technique involves plating diluted samples onto suitable growth media and monitoring colony formation. This type of technique counts only those cells that are reproductively active. Because it is not possible to be certain that each colony arose from a single cell, the results are usually expressed as colony forming units (CFU) rather than the number of microorganisms. The spread plate technique is used because it is more accurate than pour plate technique. In the spread plate technique, the mixture of cells is spread out on an agar surface so that every cell grow into a completely separate colony, a macroscopically visible growth or cluster of microorganisms on a solid medium, each colony represents a pure culture. In the pour plate technique, the hot agar used may injure or kill sensitive cells. While completing the experiment, I have recognized a few weaknesses that make my groups result not as wanted in the theoretical result. First and foremost, during the calculation of amount of cells in the plate. We had made wrong calculation. Supposedly we should only calculate the single amount of colony but we had included the calculation of double colony as well. The other factor that contributed to this weakness is that our method of spreading is not successfully done. Hence the bacteria s is not well mixed up with the nutrient. Apart from that, the place to run of the experiment also is important. This is due to avoid the culture sample from getting contaminated. All of above weakness has been analyzed and action to improve has been recognized for better future experiment. By referring to the first weakness, I should fully understand what the objective of the experiment is and manual should be read before doing the experiment. Any confusion should be asked to the demonstrator or lectures so that the flowing of the experiment run out smoothly. Because not fully understand, I have made wrong calculation and my method of spreading not cover all the nutrient. According to second weakness, the experiment should be done in the laminar flow. Therefore the sample is not contaminated. Apart from the strictly precaution that are being highlighted in the manual is that the practice sterile techniques should be apply. The hockey stick should immersed in the ethanol first and then flame it to kill any bacteria that present. Besides that, the sample in the test tube should be vortex well in order to ensure the diluted bacteria are mixed up. So that, while in the spectrophotometer the bacteria can be seen. From my understanding, in the early of the experiment, in preparing the E.Coli . It is mix with fresh nutrient broth. Then it is incubated. The purpose of incubated is that it can double up the growth.

4. CONCLUSION.
As conclusion, the objectives of this experiment were achieved as we finally understand the four stages of the growth curve and the population growth dynamics of bacterial cultures. Besides that, we are now able to conduct the experiment and more confident to make other bacterial growth since we are familiarize with the technique (spreading technique). From the mistakes that we had done, we now are more careful and aware while conducting an experiment. The growth curve is plotted and the generation time of bacterial cultures is determined by direct and indirect methods.

5. QUESTIONS
1. Explain what is occurring in the culture during the lag phase. During the lag phase, the cell is synthesizing new components. A lag phase prior to the start of cell division. New enzymes would be needed to use different nutrients. The cells retool, replicate their DNA, begin to increase in mass and finally divide. Inoculation of a culture into a chemically different medium also results in a longer phase. 2. Cite the factors responsible for the stationary phase. a. Nutrient Limitation- if an essential nutrient is severely depleted, population growth is slow. b. Accumulation of toxic waste product- To limit the growth of many anaerobic cultures. 3. Define generation time. Generation time is defined as the double in number of population during a specific length of time. 4. Can it (generation time) be calculated at any phase of the grow curve? Explain. Yes, the generation time can be calculated only at log phase of the grow curve. This is because during the log phase, the organisms are growing at the maximal rate possible given their genetic potential, the nature of the medium, and the conditions under which they are growing. The population is most uniform in terms of chemical and physical properties. Thus there is a rapid exponential increase in population, which doubles regularly until a maximum number of cells are reached.