Generation Time Of Bacteria

Name: Generation Time Of Bacteria
Uploaded: 2017-07-05T12:06:58+00:00
Duration: PTM34S10
Channel: Mark Burns
Description: Generation Time Of Bacteria

Generation Time Of Bacteria

220051215 ID: Prepared By: Prepared To : Tahreer Hassan Zourob
DR. Abdelraouf Elmanama MR. Mohammad Laqan

Contents 1 Introduction Generation Time 2 3
Methods Of Cell Mass Measurement 4 Methods Of Cell Number Measurement

Contents 5 The Bacterial Growth Curve Turbidity 6 7 The Plate Count 8
Direct Microscopic Method

Measurement of Bacterial Growth
Growth is an orderly increase in the quantity of cellular constituents. It depends upon the ability of the cell to form new protoplasm from nutrients available in the environment. In most bacteria, growth involves increase in cell mass and number of ribosome, duplication of the bacterial chromosome, synthesis of new cell wall and plasma membrane, partitioning of the two chromosomes, septum formation, and cell division. This asexual process of reproduction is called binary fission.

Measurement of Bacterial Growth

Generation time Time taken for a cell population to double in numbers and thus equivalent to the average length of the cell cycle

Generation Time (minutes) Escherichia coli Glucose-salts 17
Table 2. Generation times for some common bacteria under optimal conditions of growth. Bacterium Medium Generation Time (minutes) Escherichia coli Glucose-salts 17 Bacillus megaterium Sucrose-salts 25 Streptococcus lactis Milk 26 Lactose broth 48 Staphylococcus aureus Heart infusion broth 27-30

Lactobacillus acidophilus Milk 66-87
Table 2. Generation times for some common bacteria under optimal conditions of growth. Lactobacillus acidophilus Milk 66-87 Rhizobium japonicum Mannitol-salts-yeast extract Mycobacterium tuberculosis Synthetic Treponema pallidum Rabbit testes 1980

Methods for Measurement of Cell Mass
Methods for measurement of the cell mass involve both direct and indirect techniques. 1. Direct physical measurement of dry weight, wet weight, or volume of cells after centrifugation. . Wet and Dry Weights: A known volume of a microbial sample is centrifuged so that the cells form a pellet and are separated from the medium. The supernatant medium is discarded and the cell pellet can be weighed and the mg cells/ml of culture can be determined (wet weight).

- The cell pellet can be dried before weighing to get mg cell/ml (dry weight). Filtration ( preparation after staining with acridine orange SEM)

2. Direct chemical measurement of some chemical component of the cells such as total N, total protein, or total DNA content. 3. Indirect measurement of chemical activity such as rate of O2 production or consumption, CO2 production or consumption, etc. 4. Turbidity measurements employ a variety of instruments to determine the amount of light scattered by a suspension of cells. Particulate objects such as bacteria scatter light in proportion to their numbers.

The turbidity or optical density of a suspension of cells is directly related to cell mass or cell number, after construction and calibration of a standard curve. The method is simple and nondestructive, but the sensitivity is limited to about 107 cells per ml for most bacteria. Use colorimeter to measure turbidity of culture - the turbidity being, more or less, directly related to cell numbers or mass. In a colorimeter, light passing through a sample is measured by a photoelectric cell.

As cell density of the sample increases, i.e., becomes more turbid, a greater amount of light is scattered and fails to reach the photoelectric cell. This is measured in terms of optical density (OD) or absorbance(A) units. OD (A) = log Io/I where :- Io = incident light falling on the sample I = transmitted light; amount of light passing through sample on to the photoelectric cell .

Methods for Measurement of Cell Numbers
Measuring techniques involve direct counts, visually or instrumentally, and indirect viable cell counts. 1. 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.

2. Electronic counting chambers count numbers and measure size distribution of cells. Such electronic devices are more often used to count eukaryotic cells such as blood cells.

3. Indirect viable cell counts, also called plate counts, involve plating out (spreading) a sample of a culture on a nutrient agar surface. The sample or cell suspension can be diluted in a nontoxic diluent (e.g. water or saline) before plating. If plated on a suitable medium, each viable unit grows and forms a colony. Each colony that can be counted is called a colony forming unit (cfu) and the number of cfu's is related to the viable number of bacteria in the sample.

Advantages of the technique are its sensitivity (theoretically, a single cell can be detected), and it allows for inspection and positive identification of the organism counted. Disadvantages are (1) only living cells develop colonies that are counted; (2) clumps or chains of cells develop into a single colony; (3) colonies develop only from those organisms for which the cultural conditions are suitable for growth.

Table 1. Some Methods used to measure bacterial growth
Application Comments Direct microscopic count Enumeration of bacteria in milk or cellular vaccines Cannot distinguish living from nonliving cells Viable cell count (colony counts) Enumeration of bacteria in milk, foods, soil, water, laboratory cultures, etc. Very sensitive if plating conditions are optimal Turbidity measurement Estimations of large numbers of bacteria in clear liquid media and broths Fast and nondestructive, but cannot detect cell densities less than 107 cells per ml

Table 1. Some Methods used to measure bacterial growth
Measurement of total N or protein Measurement of total cell yield from very dense cultures only practical application is in the research laboratory Measurement of Biochemical activity e.g. O2 uptake CO2 production, ATP production, etc. Microbiological assays Requires a fixed standard to relate chemical activity to cell mass and/or cell numbers Measurement of dry weight or wet weight of cells or volume of cells after centrifugation Measurement of total cell yield in cultures probably more sensitive than total N or total protein measurements

The Bacterial Growth Curve

Four characteristic phases of the growth cycle are recognized. 1. Lag Phase. Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity.

The length of the lag phase is apparently dependent on a wide variety of factors including the size of the inoculum; time necessary to recover from physical damage or shock in the transfer; time required for synthesis of essential coenzymes or division factors; and time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium.

2. Exponential (log) Phase. The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation. The rate of exponential growth of a bacterial culture is expressed as generation time, also the doubling time of the bacterial population. Generation time (G) is defined as the time (t) per generation (n = number of generations).

3. Stationary Phase. Exponential growth cannot be continued forever in a batch culture (e.g. a closed system such as a test tube or flask). Population growth is limited by one of three factors: 1. exhaustion of available nutrients; 2. accumulation of inhibitory metabolites or end products; 3. exhaustion of space, in this case called a lack of "biological space".

During the stationary phase, if viable cells are being counted, it cannot be determined whether some cells are dying and an equal number of cells are dividing, or the population of cells has simply stopped growing and dividing. The stationary phase, like the lag phase, is not necessarily a period of quiescence. Bacteria that produce secondary metabolites, such as antibiotics, do so during the stationary phase of the growth cycle (Secondary metabolites are defined as metabolites produced after the active stage of growth). It is during the stationary phase that spore-forming bacteria have to induce or unmask the activity of dozens of genes that may be involved in sporulation process.

4. Death Phase. If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. (Note, if counting by turbidimetric measurements or microscopic counts, the death phase cannot be observed.). During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase

TURBIDITY When you mix the bacteria growing in a liquid medium, the culture appears turbid. This is because a bacterial culture acts as a colloidal suspension that blocks and reflects light passing through the culture. Within limits, the light absorbed by the bacterial suspension will be directly proportional to the concentration of cells in the culture. By measuring the amount of light absorbed by a bacterial suspension, one can estimate and compare the number of bacteria present.

TURBIDITY The instrument used to measure turbidity is a spectrophotometer (see Fig. 1). It consists of a light source, a filter which allows only a single wavelength of light to pass through, the sample tube containing the bacterial suspension, and a photocell that compares the amount of light coming through the tube with the total light entering the tube.

Figure 1

TURBIDITY The ability of the culture to block the light can be expressed as either percent of light transmitted through the tube or the amount of light absorbed in the tube (see Fig. 2). The percent of light transmitted is inversely proportional to the bacterial concentration. (The greater the percent transmittance, the lower the number of bacteria.) The absorbance (or optical density) is directly proportional to the cell concentration. (The greater the absorbance, the greater the number of bacteria.)

Figure 2

TURBIDITY Turbidimetric measurement is often correlated with some other method of cell count, such as the direct microscopic method or the plate count. In this way, turbidity can be used as an indirect measurement of the cell count. For example: 1. Several dilutions can be made of a bacterial stock. 2. A Petroff-Hausser counter can then be used to perform a direct microscopic count on each dilution. 3. Then a spectrophotometer can be used to measure the absorbance of each dilution tube.

TURBIDITY 4. A standard curve comparing absorbance to the number of bacteria can be made by plotting absorbance versus the number of bacteria per cc (see Fig. 3). 5. Once the standard curve is completed, any dilution tube of that organism can be placed in a spectrophotometer and its absorbance read. Once the absorbance is determined, the standard curve can be used to determine the corresponding number of bacteria per cc (see Fig. 4).

McFarland 0.5 Standard

Figure 3

Figure 4

TURBIDITY MATERIALS Spectrophotometer, sample test, broth tubes , pipette PROCEDURE Let the spectrophotometer (opposite) "warm up" for at least 15 minutes so you get the correct reading. Adjust the wavelength to 600nm (green light). Place a cuvette containing a blank of medium in the machine and adjust so the reading is zero. Place a cuvette containing your sample in the machine and read the optical density (O.D.). N.B. You can only accurately read OD up to a value of about 2.0. Above this level readings are not accurate. If the reading from your sample is higher than 2.0, make a 10-fold dilution and record the OD of this. (Don't forget to multiply the reading by 10 to take account of the dilution).

TURBIDITY

THE PLATE COUNT (VIABLE COUNT)
The number of bacteria in a given sample is usually too great to be counted directly. However, if the sample is serially diluted (see Fig. 5) and then plated out on an agar surface in such a manner that single isolated bacteria form visible isolated colonies (see Fig. 6), the number of colonies can be used as a measure of the number of viable (living) cells in that known dilution.

However, keep in mind that if the organism normally forms multiple cell arrangements, such as chains, the colony-forming unit may consist of a chain of bacteria rather than a single bacterium. In addition, some of the bacteria may be clumped together. Therefore, when doing the plate count technique, we generally say we are determining the number of Colony-Forming Units (CFUs) in that known dilution. By extrapolation, this number can in turn be used to calculate the number of CFUs in the original sample.

Normally, the bacterial sample is diluted by factors of 10 and plated on agar. After incubation, the number of colonies on a dilution plate showing between 30 and 300 colonies (see Fig. 7) is determined. A plate having colonies is chosen because this range is considered statistically significant. If there are less than 30 colonies on the plate, small errors in dilution technique or the presence of a few contaminants will have a drastic effect on the final count. Likewise, if there are more than 300 colonies on the plate, there will be poor isolation and colonies will have grown together.

Generally, one wants to determine the number of CFUs per milliliter (ml) of sample. To find this, the number of colonies (on a plate having colonies) is multiplied by the number of times the original ml of bacteria was diluted (the dilution factor of the plate counted). For example, if a plate containing a 1/1,000,000 dilution of the original ml of sample shows 150 colonies, then 150 represents 1/1,000,000 the number of CFUs present in the original ml. Therefore the number of CFUs per ml in the original sample is found by multiplying 150 x 1,000,000 as shown in the formula below:

The number of CFUs per ml of sample = The number of colonies ( plate) X The dilution factor of the plate counted In the case of the example above, 150 x 1,000,000 = 150,000,000 CFUs per ml. For a more accurate count it is advisable to plate each dilution in duplicate or triplicate and then find an average count.

Figure 5

Figure 6 & 7

MATERIALS 6 tubes each containing 9.0 ml sterile saline, 3 plates of Trypticase Soy agar, 2 sterile 1.0 ml pipettes, pipette filler, turntable, bent glass rod, dish of alcohol ORGANISM Trypticase Soy broth culture of Escherichia coli PROCEDURE

1.0 Milliliter (ml) Pipette

Using a Pipette to Remove Bacteria from a Tube

Using a Vortex Mixer to Mix Bacteria Throughout a Tube

Using a Pipette to Transfer Bacteria to an Agar Plate

Using a Bent Glass Rod and a Turntable to Spread a Bacterial Sample

Dilution of Bacterial Sample, Step 1

Result 1. Choose a plate that appears to have between 30 and 300 colonies. Sample 1/100,000 dilution plate (Figure a). Sample 1/1,000,000 dilution plate (Figure b). Sample 1/10,000,000 dilution plate (Figure c). 2. Count the exact number of colonies on that plate using the colony counter 3. Calculate the number of CFUs per ml of original sample

Result Choice a b c

Result Of Viable Count And O.D.
Time (h): 8 O.D600 : 3.8 Viable Count: 10-1 dilution: 10-2 dilution:

10-3 dilution: 10-4 dilution:

10-5 dilution: 10-6 dilution:

Results: Result Of Viable Count And O.D. Time (h): Viable cells/ml: O.D. 600: 0.5 3.1x101 0.64 1.0 3.2x101 0.65 1.5 8.4x101 0.85 2.0 2.58x102 1.10 2.5 8.57x102 1.36 3.0 2.11x103 1.55 3.5 9.80x103 1.89 4.0 2.04x104 2.05 4.5 5.92x104 2.28 5.0 1.90x105 2.53 5.5 3.15x105 2.64 6.0 1.71x106 3.01 6.5 4.33x106 3.21 7.0 1.42x107 3.47 7.5 3.11x107 3.64 8.0 8.70x107 3.77 8.5 3.39x108 4.16 9.0 1.04x109 4.40 9.5 10.0 9.68x108 4.39 10.5 11.0 1.03x109 11.5 12.0 4.45x108 4.22 18.0 3.49x102 1.16 24.0 3 0.13

Calculation of Generation Time
Because of the very large differences in the number of cells present at the peak and at the start/end of the experiment, it's hard to see what's going on from this graph. It's much easier to see the whole experiment if you plot the number of viable cells on a logarithmic scale (or more simply, plot the log of cell number).

the log plot

As you can see, the indirect method of counting (optical density) closely parallels the direct method (viable count). (At later time points, you can see that the number of viable cells declines faster than the optical density of the culture. It will be even easier to see the results if we concentrate on the first 12 hours of the experiment.

The graph of the results reveals FOUR distinct phases which occur during the growth of a bacterial culture.

When growing 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. B = number of bacteria at the beginning of a time interval b = number of bacteria at the end of the time interval G = t 3.3 log b/B

Example: What is the generation time of a bacterial population that increases from 10,000 cells to 10,000,000 cells in four hours of growth? G = t_____ 3.3 log b/B G = 240 minutes 3.3 log 107/104 G = 240 minutes 3.3 x 3 G = 24 minutes

DIRECT MICROSCOPIC METHOD (TOTAL CELL COUNT)
In the direct microscopic count, a counting chamber consisting of a ruled slide and a coverslip is employed.. It is constructed in such a manner that the coverslip, slide, and ruled lines delimit a known volume. The number of bacteria in a small known volume is directly counted microscopically and the number of bacteria in the larger original sample is determined by extrapolation

The Petroff-Hausser counting chamber for example, has small etched squares 1/20 of a millimeter (mm) by 1/20 of a mm and is 1/50 of a mm deep.

The formula used for the direct microscopic count is:
DIRECT MICROSCOPIC METHOD (TOTAL CELL COUNT) If the bacteria are diluted, such as by mixing the bacteria with dye before being placed in the counting chamber, then this dilution must also be considered in the final calculations. The formula used for the direct microscopic count is: The number of bacteria per cc = The average number of bacteria per large double-lined square X The dilution factor of the large square (1,250,000) X The dilution factor of any dilutions made prior to placing the sample in the counting chamber, e.g., mixing the bacteria with dye

The volume of one small square therefore is 1/20,000 of a cubic mm or 1/20,000,000 of a cubic centimeter (cc). There are 16 small squares in the large double-lined squares that are actually counted, making the volume of a large double-lined square 1/1,250,000 cc. The normal procedure is to count the number of bacteria in five large double-lined squares and divide by five to get the average number of bacteria per large square. This number is then multiplied by 1,250,000 since the square holds a volume of 1/1,250,000 cc, to find the total number of organisms per cc in the original sample.

A variation of the direct microscopic count has been used to observe and measure growth of bacteria in natural environments. In order to detect and prove that thermopiles 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.

The MPN Method

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Generation Time Of Bacteria

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