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Generation Time Of Bacteria

Generation Time Of Bacteria. ID :. Prepared By:. Prepared To :. 220051215. 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.

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Generation Time Of Bacteria

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  1. Generation Time Of Bacteria

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

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

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

  5. 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.

  6. Measurement of Bacterial Growth

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

  8. Table 2. Generation times for some common bacteria under optimal conditions of growth.

  9. Table 2. Generation times for some common bacteria under optimal conditions of growth.

  10. 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).

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

  12. Methods for Measurement of Cell Mass 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.

  13. 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.

  14. 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.

  15. Methods for Measurement of Cell Numbers 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.

  16. Table 1. Some Methods used to measure bacterial growth

  17. Table 1. Some Methods used to measure bacterial growth

  18. The Bacterial Growth Curve

  19. The Bacterial Growth Curve

  20. 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.

  21. 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.

  22. Figure 1

  23. 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.)

  24. Figure 2

  25. 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.

  26. 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).

  27. McFarland 0.5 Standard

  28. Figure 3

  29. Figure 4

  30. 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).

  31. TURBIDITY

  32. TURBIDITY

  33. 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.

  34. THE PLATE COUNT (VIABLE COUNT) 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.

  35. THE PLATE COUNT (VIABLE COUNT) 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 30-300 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.

  36. THE PLATE COUNT (VIABLE COUNT) 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 1/1,000,000 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:

  37. THE PLATE COUNT (VIABLE COUNT) 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 1,000,000 = 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.

  38. Figure 5

  39. Figure 6 & 7

  40. 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

  41. 1.0 Milliliter (ml) Pipette

  42. Using a Pipette to Remove Bacteria from a Tube

  43. Using a Vortex Mixer to Mix Bacteria Throughout a Tube

  44. Using a Pipette to Transfer Bacteria to an Agar Plate

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

  46. Dilution of Bacterial Sample, Step 1

  47. Dilution of Bacterial Sample, Step 2

  48. Dilution of Bacterial Sample, Step 3

  49. Dilution of Bacterial Sample, Step 4

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