Measurement of Growth
Name : LEVENT
Surname : DEMiRAY
Student ID# : 140104012
Department : MOLECULAR BIOLOGY AND GENETICS
In the first lab section of semester, topic was measurement of microbial growth. At the beginning, demonstrator gave brief about six measurement way of microbial growth and phase of microbial growth. The most commonly used measurement techniques are serial dilution and colony counting, microscopic cell counts, membrane filtration, turbidity, mass determination and metabolic activity. In this lab two of them serial dilution and colony counting and turbidity were used only. In serial dilution and colony counting which is also named as a viable cell counts, concentrated samples are diluted by serial dilution and then diluted samples are plated on rich complete media. There are two plating methods, spread plating and pour plating. They both have some pros and cons. Moreover turbidity is based on the diffraction or scattering of light by bacteria. To measure absorbance rate spectrophotometer was used. The purpose of this experiment is learning measurement methods of microbial growth completely and practicing these techniques efficiently. Besides, plotting the graph of growth curve is one of the other purposes of this experiment.
In the laboratory, under convenient circumstances, a growing bacterial population doubles at regular intervals. Growth is by geometric progression: 1, 2, 4, 8, etc. or 20, 21, 22, 23………2n (where n = the number of generations). This is called exponential growth. In fact, exponential growth is only part of the bacterial life cycle, and not characteristic of the normal pattern of growth of bacteria in nature (1).
As it can be seen from the figure 1 the population of bacteria in a closed system such as test tube does not change initially. Then it will have a logarithmic increase. In the next phase the death and reproduction of bacteria will be equal to each other and it will be stationary. The next phase the rate of death of bacteria exceeds the rate of reproduction of bacteria, so the population decreases in the last phase. During lag phase, bacteria adapt themselves to growth conditions. It is the period where the individual bacteria are maturing and not yet able to divide. During the exponential phase (log phase), the number of new bacteria appearing per unit time is proportional to the present population. This gives rise to the classic exponential growth curve, in which the logarithm of the population density rises linearly with time. The actual rate of this growth depends upon the growth conditions, which affect the frequency of cell division events and the probability of both daughter cells surviving. Exponential growth cannot continue indefinitely, however, because the medium is soon depleted of nutrients and enriched with wastes. During stationary phase, the growth rate slows as a result of nutrient depletion and accumulation of toxic products. This phase is reached as the bacteria begin to exhaust the resources that are available to them. At death phase, bacteria run out of nutrients (2, 3).
When bacteria grow exponentially by binary fission, the increase in a bacterial population is by geometric progression. If we start with one cell, when it divides, there are 2 cells in the first generation, 4 cells in the second generation, 8 cells in the third generation, and so on. The generation time is the time interval required for the cells or population to divide (1, 4).
G (generation time) = (time, in minutes or hours)/n(number of generations)
G = t/n
t = time interval in hours or minutes
B = number of bacteria at the beginning of a time interval
b = number of bacteria at the end of the time interval
n = number of generations (number of times the cell population doubles during the time interval)
b = B x 2n (This equation is an expression of growth by binary fission)
Solve for n:
logb = logB + nlog2
n = logb – logB
n = logb – logB
n = 3.3 logb/B
G = t/n
Solve for G
G = t
3.3 log b/B
Measuring techniques involve direct counts, visually or instrumentally, and indirect viable cell counts.
Direct microscopic counts are possible using special slides known as counting chambers. Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted (107 cells per ml), but samples can be concentrated by centrifugation or filtration to increase sensitivity. A variation of the direct microscopic count has been used to observe and measure growth of bacteria in natural environments. In order to discover and prove that thermophilic bacteria were growing in boiling hot springs, T.D. Brock immersed microscope slides in the springs and withdrew them periodically for microscopic observation. The bacteria in the boiling water attached to the glass slides naturally and grew as microcolonies on the surface. Microscopic cell counts are generally not convenient for bacteria since cells tend to move in or out of counting field. (1)
In addition to that, there are some particular counting chambers such as the phase hemacytometer used to counting blood cells, the Petroff Hausser counting chamber used for bacteria counting, the sperm counting chambers used primarily in fertility testing and the Howard Mold Counter and the Sedgewick Rafter counting chamber. Moreover, sometimes special stanins can us efor specific purpose.(5)
The other technique is serial dilution and colony counting which is also known viable cell counts. This method is widely used to estimate numbers of viable bacteria in various fluids such as milk, water, etc. In typical procedure 1 ml quantities of sample diluted in decimal, four fold, two fold or other convenient series are placed in tubes of nutrient broth. after preparing diluted solution, 100 µl of sample is taken from each tube and inoculated on a sterile Petri dish. After that this plate culture is held in an incubator at about 35C for 24 hours and is then examined for the presence of colonies distributed throughout the agar. With continued incubation of the broth culture, the number of bacteria per ml increases quickly toward several thousand or hundreds of thousands. The 1 ml of material remove removed for plate count is diluted so that plates are obtained which show only about 50 to 300 colonies. These numbers are optimal for colony counts. Deathly crowding of the colonies is avoided and the colonies are separated so that counting is more accurate. (6)
Moreover, there are two plating techniques named spreading method and pouring method. The spreading method is employed for bacterial-cell enumeration and isolation. In the spreading method of addition of cells to solid medium, a small volume of culture is dropped onto the surface of agar that has already hardened in a petri dish. This technique is advantageous particularly when cells are sensitive to exposure to relatively high temperatures plus the method does not require a prior melting of the solid medium. Just like spreading method, pouring method is employed for bacterial-cell enumeration and isolation. In the pouring method of addition of cells to solid medium contained within a petri dish, cells are added to melted (but not too hot) solid medium. The melted solid medium is then poured into a petri dish and allowed to harden. Colonies appear both within, beneath, and on top of the agar. (7)
Tubidometric method is other commonly used technique measures turbidity or scattering of light in the culture due to accumulation of evenly scattered cells hung in it. A measured volume of culture is placed in a special, clear glass tube known cuvvette. This is interposed between a unit source of light and a photoelectric unit, which is attached to a galvanometer. The reading on a galvanometer depends on the passage of light through culture from the unit source. Of the total light from the unit source, the percentage transmitted through the tube will be reduced in proportion to the turbidity. The method is subject to errors due to variation in size and shape and clumping of cells, as well as to different degrees of translucency of various species and other materials in culture. However the method is one of the quickest, simplest and is reasonable accuracy. (6)
Furthermore, membrane filtration which is convenient for extra diluted fluids is widely used method. The membrane filtration method provides a direct count of E.coli in fluid based on the development of colonies that grow on the surface of a membrane filter. A fluid sample is filtered through the membrane, which keep in the bacteria. After filtration, the membrane containing the bacteria is placed on a selective and differential medium, modified mTEC Agar, incubated at 35C for 2 h to restore the life the injured or stressed bacteria, and then incubated at 44C for 22 h. The modified method eliminates the transfer of the membrane filter to another substrate. The target colonies on modified mTEC agar are red or magenta in color after the incubation period. After that the membrane filter with acceptable number (20-80) of magenta or red colonies is selected and then number of E.coli is calculated with following formula (8)
E. coli / 100ml =( number of E. coli colonies / volume of sample filtered) x 100
The rate of use of substrates or liberation of metabolic products also can be employed as methods of enumeration, though just as with turbidity, prior standardization is necessary. A time-consuming method of culture-mass determination involves drying cells (removing water) and then weighing them. (7)
The purpose of this experiment is learning measurement methods of microbial growth completely and practicing these techniques efficiently. Besides, plotting the graph of growth curve is one of the other purposes of this experiment.
Materials and methods
• Tube, Petri dish, bent glass rod
• E. coli
• Micropipette, vortex
• Spectrophotometer, cuvvette
• Incubator, shaker
• Bunsen burner, alcohol
To begin with, ten tubes containing 80 µl of fresh culture prepared before and 9 ml LB broth were prepared and placed in shaker. LB broth including tryptone, yeast extract and NaCl was prepared before the lab by demonstrator and some students. At 0th minute 100 µl of fresh culture was inoculated in 3 different plates by the help of bent glass rod and alcohol. Bent glass rod was dipped into alcohol then fired. After made glass rod cool, sample was spread around the agar surface with that. To make glass rod cool, it was touched for 1-2 seconds on the upside of plate. Meanwhile, 2-3 ml of fresh culture was put into cuvette and measured at 0th minute. After that, at every 20 minutes one of prepared tube in shaker was taken and diluted. At each stage of dilution, vortex was used. After dilution, 100µl of sample taken from last 3 tubes containing diluted solution was inoculated in 6 separate plates with spreading method. By the way, 3 ml of sample was added into cuvette before dilution for spectrophotometric measurement for each tube involving fresh culture at every 20 min. To dilute samples 1 ml of sample was taken by the help of micropipette and then added into 9 ml LB broth and it was vortexed. Following that 1 ml of sample was taken again from previous dilution tube and added in to tube containing 9 ml LB broth. This dilution procedure was followed for each sample. For first six tubes, samples were diluted 6 times. For 7th and 8th it was diluted 7 times and for 9th and 10th it was diluted 8 times. After inoculations, all of plates were placed in incubator. One day later, they were taken from incubator and colonies in plates were counted and noted.
The number of the colonies after incubating is shown in this table.
Time (min) 0 20 40 60 80 100 120 140 160 180
Not diluted Above 300 – – – – – – – – –
– 286-236 248-above 300 Above 300 84-2 – – – – –
– 40-43 68-68 39-53 19-32 76-68 130-161 – – –
– 1-4 5-5 7-4 1-6 7-0 15-21 10-14 50-49 48-33
– – – – – 1-0 2-1 1-1 6-4 5-4
– – – – – – – 0-0 0-1 1-0
Time (min) Cfu (N) Log(N)
20 23600000 7,372912
40 24800000 7,394452
60 31800000 7,502427
80 60000000 7,778151
100 76000000 7,880814
120 100000000 8
140 140000000 8,146128
160 400000000 8,60206
180 480000000 8,681241
The table above shows the colony forming unit (N) and log(N).
Colony forming unit can be found with this formula;
CFU= number of colonies Ã· (dilution factor Ã— volume in ml)
This table above shows the absorbance rate that we got from spectrophotometer.
At 0th minute colony amount is more then 300 because we didn’t dilute the sample. We just inoculated fresh colony.
There might be several reasons for counting no colony in some plates. Bacteria might have been killed by hot bent glass rod or some error was done during transferring bacteria from tube to plate. It is possible to be forgotten about vortex after adding bacteria into tubes during the dilution process.
In the growth curve, we couldn’t observe all phase of bacterial growth. According to graph, between 20th and 80th minutes everything looks normal and we can observe stationary phase between 20th and 40th minutes. Then, there is a sudden increase as we expect between 40th and 80th minutes. However, the strange thing is that graph keeps on increasing after 80th minute therefore we can’t observe stationary phase. After 140th minute we should have observed death phase, but there is a sharp increase between 140th and 180th minutes. Taking curve into consideration, it is clear that some error occurred during pleating process, inoculation or dilution.
The curve plotted according to spectrophotometric readings shows an unexpected dramatic increase during 20th minute. This may stem from holding the cuvvette from its lined sides. This leads to see bacteria more than usual. In addition the number (density) of bacteria increases sharply when we use a spectrophotometer. The reason of this is death and living bacteria is counted with spectrophotometer. Even if the bacteria goes to the stationary phase the number of them counted by a spectrophotometer increase because the death ones are still in the sample tube.
When counting the bacteria with spectrophotometer the number of cells will be more than the number of cells counted with the method of plate counting. Because the death bacteria is also counted with spectrophotometer as it mentioned above. Viable cell count is more sensitive than spectrophotometric cell count if the conditions are optimal
3- Biology lab manual
6- Fundamentals of microbiology, Frobisher, Hinsdill, Crabtree, Goodheart, ninth edition .