- Advantage And Disadvantage Of The Serial Dilution Agar Plate Procedure Chart
- Advantage And Disadvantage Of The Serial Dilution Agar Plate Procedure Pdf
Tenover, in Encyclopedia of Microbiology (Third Edition), 2009. Agar dilution. The agar dilution method involves preparing a series of agar plates containing the antimicrobial agent to be tested in increasing concentrations, usually in doubling dilutions (i.e., 1, 2, 4, 8, 16, 32 μg ml −1, etc.). Study 5 Serial Dilution method flashcards from Mandy S. On StudyBlue. Advantages of serial dilution-agar plate; 1. Only viable cells counted 2. Allows isolation of discrete colonies that can be subcultured into pure colonies which can be studied/identified. Disadvantages of serial dilution-agar plate. Overnight incubation required. On StudyBlue. Advantages of serial dilution-agar plate; 1. Only viable cells counted 2. Spread Plate Technique- Principle, Procedure and Uses. It is the method. Spread Plate Technique- Principle, Procedure. Center of the surface of an agar plate. 2003).Before doing plate counts, serial dilutions are required.
Total Plate Count (TPC):
To enumerate bacteria present in a sample by serial dilution agar plating method or total plate count (TPC) method.
Purpose:
The extent of bacterial activity in a given sample in a definite set of conditions mainly depends on the total number of bacteria present in it irrespective of their species.
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Therefore, it is very often required to find out the total number of bacteria present in samples of food, water, soil, air and tissue during their microbiological analysis. This total number of bacteria includes both living and dead bacteria.’ Dead bacteria cannot grow and reproduce.
Advantage And Disadvantage Of The Serial Dilution Agar Plate Procedure Chart
It is only the living bacteria (viable bacteria), which can grow and multiply resulting in specific bacterial activity. Therefore, it is very often required to enumerate the viable bacteria cells in different samples. However, most of the enumeration methods like direct microscopic count, electronic cell count, chemical methods and spectrophotometric method count both living as well as dead cells.
These methods cannot discriminate between living and dead cells. Therefore, serial dilution-agar plating method, which enumerates only the viable bacteria cells, is the universally used method for counting living viable cells in different samples.
Principle:
A definite weight of solid sample is homogenised aseptically in nine volumes of sterile saline to get a homogenous suspension of bacteria. The liquid sample is directly used as homogenous suspension of bacteria. The suspensions of bacteria so obtained are diluted serially (10 times, 100 times, 1000 times etc.). Here 10-1, 10-2, 10-3 etc. are called dilutions.
Their reciprocals (101, 102, 103 etc.) are called dilution factors. A definite volume of the suspension of bacteria from each dilution is inoculated onto agar plates and spread properly, so as to space the individual bacteria cells wide apart and isolate them from each other.
The inoculation of bacteria for its enumeration is done in two techniques as follows:
1. Pour plate technique
2. Spread plate technique
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1. Pour Plate Technique:
In this technique, 1 ml of the bacteria suspension is dropped onto a sterilised petri dish and then liquefied nutrient agar medium is poured over it. The petri dish is swirled gently, so as to allow the suspension to mix with the medium uniformly. It is allowed to cool and solidify.
2. Spread Plate Technique:
Advantage And Disadvantage Of The Serial Dilution Agar Plate Procedure Pdf
In this technique 0.1 ml of the bacteria suspension is dropped onto a prepared agar plate. Enterprise architect 13 keygen. Then, the drop of suspension is spread uniformly on the agar plate by a sterilised glass spreader.
To minimise error, each diluted suspension is plated onto 2-5 replicate plates. The inoculated plates are incubated at 37°C for 24 hours. During this period, each isolated individual bacteria cell on the agar plate grows and multiplies rapidly to produce a macroscopic visible mass of bacteria cells called a ‘colony’. Thus, the number of colonies on the plate represents the number of bacteria in the sample.
However, very often, during spreading, some cells may not get separated properly and few such unseparated cells may give rise to a single colony. Moreover, few cells have tendency to remain in pairs, chains or clusters.
Here, each pair, chain or cluster produces a colony. Thus, each colony, in strict sense, does not represent a single bacterium. That is why, instead of expressing the counts of bacteria as ‘No. of bacteria/gm or ml of sample’, it is very often expressed as number of colony forming units per gm or ml (CFU/gm or ml).
The total plate count (TPC) in the original sample is calculated by multiplying the number of CPUs with the respective dilution factors. The ‘rules of enumeration’ are followed, while calculating the number of bacteria in the original sample.
Materials Required:
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Petri dishes (15 nos.), 2-ml pipettes (10 nos.), 10-ml pipette (1 no.), test tubes (10 nos.), conical flasks (500 ml and 1 liter-1 no. each), 500 ml beaker (2 nos.), glass spreader, stainless steel pipette case, craft paper, thread (or rubber band), non-absorbent cotton, ethyl alcohol, sodium chloride (NaCl), 0.1N hydrochloric acid (HCI), 0.1N sodium hydroxide (NaOH), distilled water, nutrient agar, liquid sample (e.g. pond water/sewage water), solid sample (e.g. soil/fish meat/oyster meat/processed food), pH paper (or pH meter), pestle and mortar (or homogeniser), bunsen burner, hot air oven, autoclave, incubator, laminar flow chamber, Quebec colony counter.
Procedure:
1. Ten pipettes (in a stainless steel pipette case), 15 petri dishes and a pair of pestle and mortar (or one homogeniser cup) are sterilised in hot air oven at 180° C for 3 hours. Alternatively, they can be covered with craft paper, tied with thread or rubber band and sterilised in autoclave along with the medium (Figure 6.6).
The number of petri dishes and accordingly the amount of medium to be used is calculated depending on the number of replications and dilutions required. Here, the glassware’s and the medium have been taken for single replication and dilution upto 10-6. The number of glasswares and the amount of medium taken for sterilisation is slightly more to avoid any incidental error, because sterilisation is a lengthy process.
2. 4.25 g of NaCl is dissolved in 500 ml of distilled water to get physiological saline (0.85%). 225 ml of this saline solution is poured into a 500 ml conical flask. Its mouth is cotton-plugged, covered with craft paper and tied with thread or rubber band. It is used as the first diluents to dilute the solid sample.
3. Advanced serial port monitor. 9.0 ml of the left over saline is also pipetted into each of the 10 test tubes. Their mouths are cotton-plugged, covered with craft paper and tied with thread or rubber band. These are used as diluents for serial dilution.
4. The ingredients of nutrient agar medium or its ready-made powder required for 500 ml of the medium is weighed and dissolved in 500 ml of distilled water in a 1 liter conical flask by shaking and swirling.
Its pH is determined using a pH paper or pH meter and adjusted to 7.0 using 0.1N HC1 if it is more or using 0.1N NaOH if it is less. The flask is heated to dissolve the agar in the medium completely. Then, it is cotton-plugged, covered with craft paper and tied with thread or rubber band.
5. The 500 ml conical flask containing 225 ml of saline, the 10 test tubes containing 9 ml of saline each and the 1 liter conical flask containing 500 ml of nutrient agar medium are sterilized at 121°C (15 psi pressure) for 15 minutes in an autoclave.
6. After sterilisation, the sterilised materials are removed from the autoclave and allowed to cool for some time, without allowing the medium to solidify. Cooling of the medium prevents condensation and accumulation of water droplets inside the plates. If the medium has already been prepared and solidified during storage, it has to be liquefied by heating carefully till it melts completely.
7. To prepare agar plates, before the sterilised nutrient agar medium cools and solidifies, in warm molten condition, it is poured aseptically into the 6 sterilised petri dishes (approximately 20 ml each), so that the molten medium covers the bottom of the petri dishes completely.
Then, the plates are covered with their lids and allowed to cool, so as to solidify the medium in them. Water vapour that may condense on the inner surface of the plates and lids is evaporated by keeping the plates and lids in inverted position in an incubator at 37°C for about 1 hour.
8. 25 g of the solid sample (e.g. fish meat/oyster meat/processed food) is weight and homogenised in 225 ml sterilised saline (diluent) aseptically (Figure 6.7). This gives a 10 times dilution (dilution = 10-1). For the liquid sample, 1ml of sample is pipetted aseptically into a 9 ml sterilised saline tube. This also gives a 10 times dilution (dilution = 10-1).
9. 1 ml of the 10-1 dilution is transferred to 9 ml sterilised saline in another test tube. This gives 100 times dilution (dilution =10-2). From the 10-2 dilution, 1 ml is dropped into a sterilised petri dish and 0.1 ml onto an agar plate, from the same pipette. For each dilution a separate sterilised pipette is used. After use it is dipped in the dispose jar.
10. 1 ml of the 10-2 dilution is transferred to 9 ml sterilised saline in another test tube. This gives 1000 times dilution (dilution =10-3). From the 10-3 dilution, 1 ml is dropped into a sterilised petri dish and 0.1 ml onto an agar plate, from the same pipette. In a similar way, dilution is continued upto 10-6 serially, each time transferring 1ml to a sterilised petri dish and 0.1 ml to an agar plate from the same pipette.
11. Then, the drops of suspension on the agar plates are spread aseptically by a sterilised glass spreader. After spreading in each plate, it is flame-sterilised by dipping in alcohol and showing over a flame. This is the ‘spread plate technique’.
12. The petri dishes containing 1ml of bacteria suspension each are taken and sterilised liquefied nutrient agar is poured into them. They are swirled gently, so as to allow the suspension to mix with the medium uniformly. The plates are allowed to cool till the medium solidifies. This is ‘pour plate technique’.
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13. Then, the plates are incubated in inverted position, top down, at 37°C for 24 hours in an incubator (Figure 6.7).
14. An un-inoculated agar plate is incubated as control to ensure proper sterilisation as shown by no growth on it.
Observations:
The number of colonies of bacteria on the plates is counted directly or with the help of a Quebec colony counter. From this, the number of bacteria present per gram or ml of the original sample is calculated. This is called enumeration.
Rules of Enumeration:
1. Petri dishes with 30 to 300 colonies should be considered.
2. Average numbers of duplicates and triplicates (R1, R2 R3…) are considered only if one count is not more than double of the other. If one is more than double of the other lower value is taken.
3. For pour plate technique, bacterial count is No.X 10c/gm, where c = dilution factor. For spread plate technique, bacterial count is No. X10c+1/gm, where c = dilution factor. The number is converted to two decimal places in the form of (x.yz X 10m). For example, 288 X 104 is expressed as 2.88 X 106.
4. If, in all dilutions, colony number is more than 300, count for highest dilution and if in all dilutions, it is less than 30, count for the lowest dilution is considered. In both cases count is represented as: Estimated No. X 10c/gm or ml for pour plate technique and Estimated No. X 10c+1/gm or ml for spread plate technique.
5. If no colony is observed in any dilution taken, it is represented as: Estimated <1 x lowest dilution.
6. As the serial dilution takes place in terms of 10 times, mathematically it is obvious that no two dilutions can have colonies between 30 and 300. For example, if 10-3 has 50 colonies, 10-2 should have 500 (i.e. >300) and 10-4 should have 5 (i.e. <30) colonies.
However, this does not occur in reality, as bacteria do not occur as a homogenous solution; rather occur as a suspension in the diluents. If there are two dilutions having countable colonies (between 30 and 300) first calculate the number of colony forming units/gm or ml using each dilution.
If one value is more than double of the other, report the lower value. If not, take the average of the two values and report that value.
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Part C: UV Experiments Serial Dilutions and Viable Cell Counts The experiment Observing the Effects of Solar Ultraviolet Radiation on Cells shows that when cells are exposed to sunlight all, some, or none of them may be killed. Many experimental questions can be answered with qualitative answers like 'all, some, or none.' Other questions may require quantitative answers. For example, in the next experiment you will use the sensitive yeast strain to measure the intensity of solar UV radiation by measuring the fraction of cells exposed that survive. To get quantitative answers about yeast survival you must put a known numbers of viable (living) cells onto the agar plates and then count the number that remain after being exposed.
What Are Some Advantages And Disadvantages Of The Serial Dilution Agar Plate Technique
What are the advantages of the serial dilution agar plate procedure. Disadvantage: the method requires an incubation periods so it takes longer to get results. Serial dilution method for bacterial. Diluted bacteria samples then spread onto the agar plate by L-shaped. The advantages and disadvantages of serial.
You can determine the number of viable cells by counting the colonies that grow up on the agar growth medium in a Petri plate by assuming that each colony grows from a single viable cell. This is usually a reasonable assumption. Experiment: In the experiment that follows you will learn how to measure the number of viable cells on a Petri plate. You will be able to use this procedure whenever you need to measure the number of cells that survive an exposure to radiation or some other treatment. First you will estimate the number of cells in a liquid suspension in order to plate a reasonable number of cells. For this you will use one of the most sophisticated and sensitive optical instruments in existence, the human eye.
With surprisingly little practice you can learn to estimate the number of cells in a suspension by just looking at it. You can estimate cell density because of your eyes' fairly sharp threshold for observing turbidity (cloudiness). When viewed in a standard 13 100 mm glass tube, yeast suspensions of less than about 1 million cells per mL are not visibly turbid. Above this threshold density, the suspension is cloudy.
When you adjust the number of cells in a suspension until just barely visible, you obtain a suspension of known density (approximately 1 106 cells/ml). When you have a suspension that contains approximately 1 106 cells/ml, you will dilute it to get the right concentration for plating. You will make the dilutions in known steps so you can calculate the number of cells in each dilution tube. This procedure helps you plate a countable number of colonies.
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