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Microbiology, pharmacy. Dr. Amal Bakri. th The 6 lecture Done by: Deema Mohammad. --------------------------------------------------------------------------------------------------------------------------------------- At this stage the mother cell will be able to divide into two daughter cells; these daughter cells are small Chapter 6 …. * Growth and Culturing Of comparing to the mother cell; because the mother cell was in the stage of increase-in-size. The time needed for the mother cell to undergo division is called the "Generation Time" which is about 20 minutes to 20 hours, it is genetically defined and it depends on the environment conditions that the microorganism grows in. The daughter cells that resulted from the binary fission are independent and each of them can also undergo binary fission in order to give another two daughter cells and these daughter cells can also divide, so the type of growth here is an exponential growth which will give straight line; if drawn on a semi-log paper. But, sometimes total separation will not occur , if this happened we will have the phenomena of arrangement such as : diplococcic , tetracocci, staphylococci ,streptococci . Bacteria: Bacteria is one of the organisms that can be cultured in the lab , and in order to be cultured we have to put it in a medium , like solid medium "Agar" that we mentioned when we were talking about Robert Koch . If we bring a fresh nutrient medium (like source of carbon and nitrogen …); which is suitable for a certain microorganism and we put this microorganism in a suitable environment conditions such as: PH, temperature, presence of oxygen …. Etc, the M.O or Bacteria will start to grow. The concept of growth in human being means the increase in size , but regarding to microorganisms it indicates the increase in number , when we talk about growth in bacteria we don't mean the process of meiosis nor mitosis but instead we mean the processes of Binary Fission or Budding. * Binary Fission : Binary fission is the predominant way of microorganism increase in number. BUT How does binary fission occur??? First there is a mother cell, this cell will undergo a certain type of growth within a specific time , it doubles in size and duplicates its contents (especially the genetic material ) Then it makes the septum; which is a partition that grows between the two daughter cells that will be separated. 1 Microbiology, pharmacy. Dr. Amal Bakri. th The 6 lecture Done by: Deema Mohammad. --------------------------------------------------------------------------------------------------------------------------------------- * Budding: * Traces of Growth: Binary fission is an equal cell division , the mother cell will give two daughter cells , but we have some microorganisms ( mainly the yeast which is a fungus that is eukaryotic not prokaryotic ) that have the phenomena of budding . Budding is an unequal cell division in which every cell will not give rise to two daughter cells only, instead it will give many buds and every bud will separate and starts as a new microorganism. BUT How does Budding Occurs ?? If we want to culture a certain type of bacteria in the lab we will put it in a fresh nutrient medium and in a favorable physical conditions, then we will look for an indication of growth. In fluid medium the indication of growth is the turbidity, sometimes we may have turbidity along with certain odor ,the presence of bubbles in the gas test tubes and the production of certain colors may also be an indication of growth . In solid medium or agar the indication of growth is the formation of colonies. Small cells ( buds ) are developed from the surface of the stem cell and then they will separate to form new cells that can then undergo the same method of increasing in number which is budding . Budding may be found in certain bacteria but it is basically found in "yeast". * Phases of Growth: If we constructed an experiment to see how many bacterial cells are increasing with time, we will notice that: " Budding " The number of bacteria is increasing significantly with time then we can conclude that the conditions are favorable for the growth of bacteria. If we took several samples in flasks from the culture at different times and enumerated the number of microorganisms or the amount of turbidity (any indication for the quantity of growth) in each sample at each time. Then, by drawing a graph between the time (x-axis) and the number of microorganisms or any indication of the growth like turbidity ( Y-axis ) we will end up by a graph called growth curve , this curve has four 2 Microbiology, pharmacy. Dr. Amal Bakri. th The 6 lecture Done by: Deema Mohammad. --------------------------------------------------------------------------------------------------------------------------------------- phases which are : lag phase , log phase (exponential phase ) , stationary phase and decline phase (death phase) . The length of the phase and density of the microorganisms in each phase are dependent on: a) The microorganism itself (genetically). b) And on the medium where the microorganism is cultured, if it is favorable or not >>>> if the medium is unfavorable then the rate of growth will be slow, but if it is favorable we will have the maximum growth. The same microorganism in different conditions would give different growth phases and different number of cells. The microorganisms in this phase are metabolically very active; they increase in size and incorporate molecules from the medium, they synthesize enzymes and produce large quantities of energy in the form of ATP in order to help the microorganism for doubling of the components and then for entering the division phase. Lag phase's length depends on : 1. The microorganism itself. 2. The suitability of the medium for this microorganism >>>> when this medium is more suitable for the microorganism the length of lag phase will be shorter , and when it is less suitable, the length of lag phase will be longer because bacteria will need more time to adapt to the medium. #2# Log phase (exponential phase) : It comes after the lag phase, once the microorganism has been adapted to the medium it will start to duplicate and increase in number by binary fission. At this phase the growth is at its maximal level, this phase appears like straight line when we draw the growth curve on a semi-log paper (time is a linear scale on "x-axis" and the indicator of growth is a logarithmic scale on "y-axis"). The Phases : #1# Lag phase : It is an adaptational phase. The number of microorganisms in it doesn't increase or they increase slightly. 3 Why we want straight line ??? Microbiology, pharmacy. Dr. Amal Bakri. th The 6 lecture Done by: Deema Mohammad. --------------------------------------------------------------------------------------------------------------------------------------- In order to be able to calculate the doubling time or generation time or the growth rate for certain bacteria in the log phase. For example , if we have 200 microorganisms we take the time needed for them to be 400 from the curve by making extrapolation , then we can calculate the doubling time (or generation time) which is genetically determined for this certain bacteria , some bacteria have long generation time and others have short generation time. Mycobacterium; which is an acid fast bacteria, has a waxy layer and this waxy layer impedes penetration of nutrients, and because of lack of nutrient the growth rate will be very low in the cell , as well as; this bacteria makes large expenditure for the amount of ATP in order to synthesize the waxy layer so the growth rate of it will be very low . will be inoculated at the same time ( zero time ) so all of them will be in the same age. Then when we take from this medium an inoculum size of bacteria ( certain number of bacteria ) and put it in a flask ,all of the bacterial cells will duplicate together after the generation time finishes . This Type of growth is called Synchronous growth. Let's take this example to understand the stair step pattern: Consider that at zero time the number of microorganisms was 10 and after 20 min (the generation or doubling time ) it became 20 , after another 20 min it would become 40 , then 60 , 80 , 160 … etc. At the period between the numbers 20 and 40 for example , the number of microorganisms will be 20 because the number of microorganisms is constant in the generation time , so the shape of the curve will be in a stair step pattern. We might do this hypothetical situation in the lab, by applying freezing for the microorganisms and then putting them in the medium at the same time in order to make the growth of all of them starts at the same time. Remember that; in lag phase the microorganism have adapted to the growth medium and at log phase growth occurs and microorganisms are at their most rapid growth rate . we can calculate from log phase the genetically determined interval which is called the generation time . Synchronous growth: Theoretically if the microorganisms divide together at the same rate and the generation time was exactly 20 minutes the shape of the curve will be in a stair step pattern. This means we are assuming that when we put bacteria in the medium (we make inoculation for the bacteria in the medium) all of them 4 Microbiology, pharmacy. Dr. Amal Bakri. th The 6 lecture Done by: Deema Mohammad. --------------------------------------------------------------------------------------------------------------------------------------- Nonsynchronous Growth :In reality adapt to these conditions so they will start to die and the culture will reach to the stationary phase. o This phase appears as a plateau; the number of cells in it is not increasing nor decreasing, it instead reaches to a steady state, at this phase cell division decreases to a point in which the rate of cell division will be equal to the rate of cell dying so the number of cells which are dividing becomes equal to the number of cells which are dying because the growth conditions start to be not suitable for the bacteria in the closed flask . o In exponential phase we have death but the rate of it is slower than the rate of growth , but in stationary phase they become equal because the conditions will be unfavorable for the bacteria . (in life) and even in the lab not all the bacteria are doubled exactly after 20 min , some of them are doubled in a shorter duration and some other in a longer duration. Also not all of them were at the same age from the beginning , some of them may be passed 5 minutes from the generation time when were put in the flask. This means that growth in reality is a Nonsynchronous growth , it is an exponential growth ( appears as a straight line on semi-log paper ). So ,the nonsynchronous growth is the natural situation in which an actual culture has some cells dividing at slightly slower rate from the generation time and some other dividing at slightly higher rate from the generation time (there is differences between cells in the dividing rate ). for example if the generation time is 20 min , some bacteria will divide in 20 min , some other on 19 min and some on 21 min , so there is slight difference between them and the result is a continuous growth curve . #3# Stationary phase: o If we put the microorganism in a flask (closed flask); which is called closed environment or batch culture , then when the microorganism starts to grow , it will consume the nutrients and the metabolites will be accumulating within the flask , these metabolites are toxic ,so the presence of toxins and the lack of nutrients will cause the growth rate to decrease ,and some microorganisms are not capable to 5 #4# Decline phase ( death phase ) : In this phase the growth rate will decrease very much to a point in which the number of cells which are dying would exceed the number of existing cells , so the total number of cells is declining. The number of live cells stayed constant in the previous phase (stationary phase) , but because they stayed in that closed environment , the nutrients will continue to deplete and toxins will keep accumulating then we will reach to this phase (decline phase) in which the conditions of the environment become less and less supportive for cell division and cells lose their ability to divide and thus die. The number of living cells decreases Microbiology, pharmacy. Dr. Amal Bakri. th The 6 lecture Done by: Deema Mohammad. --------------------------------------------------------------------------------------------------------------------------------------- exponentially; this means if the decline phase started at 400 bacterial cells it will become 200 after certain time and so on …. . Now we talked about the growth phases (lag , log , stationary and decline phases ) , and we said that we reaches the stationary and decline phases; because microorganisms are growing in a closed environment ( Batch culture ) which will result in nutrient depletion and toxins accumulation. We can prevent the microorganism from reaching the decline phase by using the chemostat which is a device helps the microorganism to grow continuously. What is the principle of the Chemostat ? There is a part used for the continuous adding of the medium to help for the continuous growing of the microorganism and there is also another part to remove the toxins ( remove a part from the medium that contains toxins ), so we add a part of the medium and we remove another part . This means that we are refreshing the medium all the time by supplying it with nutrients and removing the toxins from it , so we can help the microorganism to continue its growing. we use this device in the fermentation processes and in the production of penicillin in industries , so when we want to produce antibiotics in continuous culture and we don't want them to die we will use the Chemostat . Chemostat ,also known as Biostat 6 If we took microorganism from a culture even if they were in the stationary phase and we put them in a suitable medium these microorganisms would start growing . Now what are the characteristics of the microorganisms in decline phase? The microorganisms in this phase are under stressful conditions , if the microorganisms were from the species of clostridium and bacillus ( G+ bacteria ) they would form endospores, the formation of endospores will be mainly in the decline phase because it is a stressful phase .When we bring a microorganism from a culture in the decline phase and we put it in a nutrient depleted conditions , this microorganism will be more adapted to these conditions than other microorganisms; because it was in a decline phase . Microbiology, pharmacy. Dr. Amal Bakri. th The 6 lecture Done by: Deema Mohammad. --------------------------------------------------------------------------------------------------------------------------------------- * Methods of measuring CFU/ml CFU stands for colony forming unit. Bacterial Growth: In order to be able to count the colonies in the plate, the plate must not be crowded with colonies nor containing very low number of them . Because if the plate was very crowded the colonies would be very close from each other and we will be confused if this is a one colony or they are two colonies , so the recommended number of colonies in the plate is from 30-300 , more than 300 the plate will be crowded , less than 30 the number of colonies will be statistically not valid . When we talked about drawing the growth curve we said that we put the time on x-axis and the indication of growth on y- axis , and we said that the indication of growth can be a measure of how many bacterial cells are increased in number ( viable bacteria) or can be the amount of turbidity . Now we will learn the methods used to assess the number of bacteria (the viable bacteria ) , sometimes we need to know the number of viable and nonviable bacteria. These methods are : (1) Standard Plate Count. (2) Counting Bacteria Using a Bacterial Colony Counter. (3) Direct Microscope Counts. (4) Most probable Number (MPN ) Method. If we brought a culture and put it directly on a plate we would have the confluent growth; confluent growth means that the plate would be full of the grown bacteria which would be seen as a layer and no obvious colonies could be seen. Because we don't want confluent growth and we want discrete colonies (separated colonies having a number from 30-300) to enable us for counting them , we have to make dilution . the standard method of dilution that is used in microbiology is called Ten fold serial dilution ; ten fold means that every dilution process will be by decreasing the concentration of cells by ten times , so if we started by 10^4 cells , after the first dilution we would end up by 10^3 cells and after the second dilution the number of cells would be 10^2 …etc. (1) Standard Plate Count : It is a method of measuring bacterial growth , it is not the sole method , we have other methods but it is the most commonly used method .It is medicinal valuable method , we use in it agar plate which is a Petri dish containing a nutrient medium solidified with agar. The principle of this method : we bring a microbial culture and take a volume from it and put it on agar , the bacteria will grow in the form of colonies , after that we count these colonies per the volume that we took from the culture and we can then calculate the number of colonies in the original culture , when we apply this method we use the unit of 7 How this dilution is performed ? 1. We bring the culture and we put a 9 ml of saline or nutrient growth in a test tube. Microbiology, pharmacy. Dr. Amal Bakri. th The 6 lecture Done by: Deema Mohammad. --------------------------------------------------------------------------------------------------------------------------------------- 2. We take 1 ml from the culture and we put it on the 9 ml so the 9 ml saline or nutrient growth becomes 10 ml. 3. Then we make agitation for the content in the test tube because bacteria is a suspension and the medium must be homogenous for the next dilution process. 4. We repeat these steps again several times in order to prepare several test tubes each of them has a different concentration from the other. 5. Now we need to know which one contains the appropriate concentration for counting the colonies (having a number of colonies between 30 and 300). 6. In order to determine the appropriate concentration for counting, we have to make plating out for all test tubes , so the determination is by trial and error , but after a period the determination of the appropriate concentration (turbidity) for counting the colonies will be experienced and we will be able to know if this amount of turbidity is appropriate to count colonies or not without making plating out for all test tubes. 7. After choosing the appropriate concentration we calculate the number of colonies from the test tube that we selected and we multiply the result by the dilution factor to know the real number of colonies in the original culture. 8. After performing the dilution and selecting the appropriate concentration we will transfer the bacteria to a Petri plate, the transfer can be done by either the pour plate method or the spread plate method. "In this figure it is shown that the color differs according to the difference in concentration but in fact the color doesn't change the change will be in the turbidity" . # Pour Plate Method: a) It is made by first adding 1.0 ml of a diluted culture from a serial dilution to 9 ml of melted nutrient agar. b) After the medium is mixed, it is poured into an empty Petri plate. c) Once the agar medium cools, solidifies, and is incubated, colonies will develop both within the medium and on its surface. d) Cells suspended in the melted agar during preparation may be heatdamaged, and then they will not form colonies. e) Those that do grow inside the agar will form smaller colonies than those growing on the surface. f) The growing of bacteria inside agar make the counting of colonies somehow difficult , so the counting using spread plate method will be is easier . 8 Microbiology, pharmacy. Dr. Amal Bakri. th The 6 lecture Done by: Deema Mohammad. --------------------------------------------------------------------------------------------------------------------------------------- # Spread Plate Method: Eliminates such problems because all cells remain on the surface of the solid medium. a. 0.1 ml of the diluted sample is first placed on the center of a solid, cooled agar medium. b. The sample is then spread evenly over the medium’s surface with a sterile, bent glass rod. c. After incubation, colonies develop on the agar surface. d. In spread plate method we add 0.1 ml not 1 ml because if we added 1 ml of the diluted culture on the surface of agar which is in the solid state , agar would not absorb all of the fluid because 90% of agar is water and there is too much fluid was added ,so fluid would remain and the colony would not be grown in the form of discrete colonies . But in pour plate method we add the diluted culture which is a fluid to molten agar , so incorporation will happen between these too fluid states and we will not face a problem such like that. e. Then we make incubation upside down , why ?? Because when the bacteria die respiration happens , and there will be a fluid on the top and if this fluid reached to bacteria, colonies would be damaged . Now , after performing any method of these (pour and spread plate methods ) and after making incubation ( usually for 18 hours ) , the microorganism will be grown in the form of colonies , we calculate these colonies and we multiply the result by the dilution factor to determine the number of colonies in the original culture , the result will be in CFU/ml . But a single measurement is not very reliable, so the procedure is repeated at least three times, and the results are averaged. The average number of colonies is multiplied by the dilution factor to ascertain the total number of organisms per milliliter of the original culture. (2) Counting Bacteria Using a Bacterial Colony Counter: It is the second method of counting bacteria, in this method bacterial colonies are viewed through a magnifying glass against a colony-counting grid . Which of these plates would be the correct one to count ? Why ? 9 Microbiology, pharmacy. Dr. Amal Bakri. th The 6 lecture Done by: Deema Mohammad. --------------------------------------------------------------------------------------------------------------------------------------- The second one , because it is not crowded nor containing low number of colonies . (3) Direct Microscope Counts - It is another way to measure bacterial growth. - It is also used to count blood cells. - In this method a known volume of medium is introduced into a specially calibrated, etched glass slide called a Petroff-Hausser counting chamber also known as a hemocytometer.Bacterial suspension is introduced onto chamber with a calibrated pipette. - - We count under the microscope. - The accuracy of direct microscopic counts depends on the presence of more than 10 million bacteria per milliliter of culture .This is because counting chambers are designed to allow accurate counts only when large numbers of cells are present. An accurate count also requires that the bacteria be homogeneously distributed throughout the culture . - This technique has the disadvantage of generally not distinguishing between living and dead cells. So we can't use it do determine the number of microorganisms exists in a suspension because the suspension contains many particle so we can't distinguish between bacteria and other particles. When we use this method ?? We use it when we want to count the number of microorganisms rapidly and directly , because it is a rapid method for counting microorganisms directly without waiting for 18 hours as in the previous methods . After the bacteria settle and the liquid currents have slowed, the microorganisms are counted in specific calibrate areas. Their number per unit volume of the original suspension is calculated by using an appropriate formula we use here the unit of cell/ml not colony per ml (CFU/ml) . (4) Most probable Number (MPN) Method : 10 Microbiology, pharmacy. Dr. Amal Bakri. th The 6 lecture Done by: Deema Mohammad. --------------------------------------------------------------------------------------------------------------------------------------- contain an organism will display growth by producing gas bubbles and/or by becoming cloudy, when incubated . And those tubes in which gas bubbles are visible will be ( labeled + ) and those that don't contain an organism ( there is no gas bubbles ) in them will (labeled - ). Then the number of organisms in the original culture is estimated from a most probable number table. As the name indicates this method depends on the probability, so the number that will result will not be accurate . So , we will use it only when samples contain too few organisms to give reliable measures of population size by the standard plate count method, as in food and water sanitation studies, or when organisms will not grow on agar. With this method, the technician observes the sample, estimates the number of cells in it, and makes a series of progressively greater dilutions. As the dilution factor increases, a point will be reached where some tubes will contain a single organism and others none. To understand that let's take this example; [ If we have a 10 ml volume of a culture , and if this culture contains 1000 microorganism , and we took 1 ml from it , the probable number of bacterial content might be 100 or 99 or 98 because we are talking about suspension , and when this 1 ml is put in a nutrient medium and incubated for 18 hours we will have a growth . If the culture contains 100 microorganism and we took 1 ml from it , the probable number of microorganisms might be 10 or 9 or 8 and when we put the 1 ml in a nutrient medium we will have an indication of growth (turbidity) . If the number of microorganisms in the culture is 10 and we took 1ml from it , then the probable number of microorganisms might be 1 or 0 , so if we put the 1 ml in a nutrient medium we might have turbidity and we might not.] #REMEMBER# that we can use this method to estimate the number of microorganisms content in food , because when a certain type of food contains a very low number of microorganisms we can't use the previous methods . for example , we use this method to determine the presence of salmonella in food which might exist in a very low number and cause infection. A typical MPN test consists of five tubes of each of three volumes (using 10, 1, and 0.1 ml) of a dilution. Those that Huda 11 ®