Generation Time Of Bacteria LOGO LOGO Pre ed

Generation Time Of Bacteria LOGO

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

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

Measurement of Bacterial Growth LOGO 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 LOGO

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

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

Table 2. Generation times for some common bacteria under optimal conditions of growth. Lactobacillus acidophilus Milk 66 -87 Rhizobium japonicum Mannitol-saltsyeast extract 344 -461 Mycobacterium Synthetic tuberculosis 792 -932 Treponema pallidum 1980 Rabbit testes LOGO

Methods for Measurement of Cell Mass LOGO 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).

Methods for Measurement of Cell Mass LOGO - The cell pellet can be dried before weighing to get mg cell/ml (dry weight). Filtration ( preparation after staining with acridine orange SEM)

Methods for Measurement of Cell Mass LOGO 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 O 2 production or consumption, CO 2 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.

Methods for Measurement of Cell Numbers LOGO 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.

Methods for Measurement of Cell Numbers 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. LOGO

Methods for Measurement of Cell Numbers LOGO 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.

Table 1. Some Methods used to measure bacterial growth Method Application Direct microscopic count Enumeration of Cannot distinguish living bacteria in milk or from nonliving cells cellular vaccines Viable cell count (colony counts) Enumeration of bacteria in milk, Very sensitive if plating foods, soil, water, conditions are optimal laboratory cultures, etc. Turbidity measurement Estimations of large numbers of bacteria in clear liquid media and broths LOGO Comments 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 LOGO Measurement of total only practical application cell yield from is in the research very dense laboratory cultures Measurement of Biochemical activity e. g. O 2 uptake CO 2 production, ATP production, etc. Microbiological assays Measurement of dry weight or wet weight of cells or volume of cells after centrifugation Measurement of total probably more sensitive cell yield in than total N or total cultures protein measurements Requires a fixed standard to relate chemical activity to cell mass and/or cell numbers

The Bacterial Growth Curve LOGO

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. LOGO

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. LOGO

Figure 1 LOGO

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. ) LOGO

Figure 2 LOGO

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TURBIDITY LOGO 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 LOGO 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).

Mc. Farland 0. 5 Standard LOGO

Figure 3 LOGO

Figure 4 LOGO

TURBIDITY LOGO MATERIALS Spectrophotometer, sample test, broth tubes , pipette PROCEDURE 1. Let the spectrophotometer (opposite) "warm up" for at least 15 minutes so you get the correct reading. 2. Adjust the wavelength to 600 nm (green light). Place a cuvette containing a blank of medium in the machine and adjust so the reading is zero. 3. 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 LOGO

THE PLATE COUNT (VIABLE COUNT) LOGO 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.

THE PLATE COUNT (VIABLE COUNT) LOGO 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.

THE PLATE COUNT (VIABLE COUNT) LOGO 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 30300 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.

THE PLATE COUNT (VIABLE COUNT) LOGO 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 30 -300 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 dilution of the original ml of sample shows 150 colonies, then 150 represents 1/1, 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 as shown in the formula below:

THE PLATE COUNT (VIABLE COUNT) LOGO The number of CFUs per ml of sample = The number of colonies (30 -300 plate) X The dilution factor of the plate counted In the case of the example above, 150 x 1, 000 = 150, 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 LOGO

Figure 6 & 7 LOGO

THE PLATE COUNT (VIABLE COUNT) 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 LOGO

1. 0 Milliliter (ml) Pipette LOGO

Using a Pipette to Remove Bacteria from a Tube LOGO

Using a Vortex Mixer to Mix Bacteria Throughout a Tube LOGO

Using a Pipette to Transfer Bacteria to an Agar Plate LOGO

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

Dilution of Bacterial Sample, Step 1 LOGO

Dilution of Bacterial Sample, Step 2 LOGO

Dilution of Bacterial Sample, Step 3 LOGO

Dilution of Bacterial Sample, Step 4 LOGO

Dilution of Bacterial Sample, Step 5 LOGO

Dilution of Bacterial Sample, Step 6 LOGO

Dilution of Bacterial Sample, Step 8 LOGO

Dilution of Bacterial Sample, Step 9 LOGO

Result LOGO 1. Choose a plate that appears to have between 30 and 300 colonies. ØSample 1/100, 000 dilution plate (Figure a. ( ØSample 1/1, 000 dilution plate (Figure b. ( ØSample 1/10, 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

LOGO Result Choice a b c

Result Of Viable Count And O. D. Time (h): 8 O. D 600 : 3. 8 Viable Count: 10 -1 dilution: 10 -2 dilution: LOGO

Result Of Viable Count And O. D. 10 -3 dilution: 10 -4 dilution: LOGO

Result Of Viable Count And O. D. 10 -5 dilution: 10 -6 dilution: LOGO

Result Of Viable Count And O. D. Time (h): Viable cells/ml: O. D. 600: 0. 5 3. 1 x 101 0. 64 1. 0 3. 2 x 101 0. 65 1. 5 8. 4 x 101 0. 85 2. 0 2. 58 x 102 1. 10 2. 5 8. 57 x 102 1. 36 3. 0 2. 11 x 103 1. 55 3. 5 9. 80 x 103 1. 89 4. 0 2. 04 x 104 2. 05 4. 5 5. 92 x 104 2. 28 5. 0 1. 90 x 105 2. 53 5. 5 3. 15 x 105 2. 64 6. 0 1. 71 x 106 3. 01 6. 5 4. 33 x 106 3. 21 7. 0 1. 42 x 107 3. 47 7. 5 3. 11 x 107 3. 64 8. 0 8. 70 x 107 3. 77 8. 5 3. 39 x 108 4. 16 9. 0 1. 04 x 109 4. 40 9. 5 1. 04 x 109 4. 40 10. 0 9. 68 x 108 4. 39 10. 5 1. 04 x 109 4. 40 11. 03 x 109 4. 40 11. 5 1. 04 x 109 4. 40 12. 0 4. 45 x 108 4. 22 18. 0 3. 49 x 102 1. 16 24. 0 3 0. 13 LOGO

Calculation of Generation Time LOGO 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).

Calculation of Generation Time the log plot LOGO

Calculation of Generation Time LOGO 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.

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

Calculation of Generation Time LOGO 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

Calculation of Generation Time LOGO Example: What is the generation time of a bacterial population that increases from 10, 000 cells to 10, 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) LOGO 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

DIRECT MICROSCOPIC METHOD (TOTAL CELL COUNT) 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. LOGO

DIRECT MICROSCOPIC METHOD (TOTAL CELL COUNT) LOGO 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

DIRECT MICROSCOPIC METHOD (TOTAL CELL COUNT) LOGO

DIRECT MICROSCOPIC METHOD (TOTAL CELL COUNT) 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. LOGO

Methods for Measurement of Cell Numbers LOGO

The MPN Method LOGO

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