Total Plate Count (TPC):
- Advantage And Disadvantage Of The Serial Dilution Agar Plate Procedure Form
- Advantage And Disadvantage Of The Serial Dilution Agar Plate Procedure Chart
- Advantage And Disadvantage Of The Serial Dilution Agar Plate Procedure Pdf
- What Are Some Advantages And Disadvantages Of The Serial Dilution Agar Plate Technique
The advantages to serial dilution-agar plate procedure is it has countable viable cells to count. As for the disadvantage of it, the techniques used which are spread and pour plates might not always have a single colony that represents the progeny of a single cell. If 0.1 mL of a 10-6 dilution plate contains 56 colonies, the number of cells per millimetre of the original culture is 5.6 x 10 8. Mueller–Hinton agar is used in this method. Serial dilution of the antibiotic are made in agar and poured onto Petri dishes. Dilutions are made in distilled water and added to the agar that has been melted and cooled to not more than 60°C. One control plate is inoculated without antibiotics. What is the major disadvantage of microbial counts performed by methods other than the serial dilution-agar plate procedure? The total count includes dead cells as well as living cells.
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.
ADVERTISEMENTS:
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.
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 How to paint space marines book pdf.
2. Spread plate technique Ttm57sl drivers for mac.
ADVERTISEMENTS:
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:
In this technique 0.1 ml of the bacteria suspension is dropped onto a prepared agar plate. 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. Pdmworks 2015 keygen ssq exe.
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:
ADVERTISEMENTS:
https://drivers-gerber-infinity-45-s1.peatix.com. 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. Dell security device driver pack windows 7 64 bit. 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. 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’.
ADVERTISEMENTS:
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.
Advantage And Disadvantage Of The Serial Dilution Agar Plate Procedure Form
![Chart Chart](https://www.coursehero.com/thumb/b6/b1/b6b14850f90d0bc56cdaf2d5c16c0c9b8f7c39a6_180.jpg)
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.
Advantage And Disadvantage Of The Serial Dilution Agar Plate Procedure Chart
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.
Advantage And Disadvantage Of The Serial Dilution Agar Plate Procedure Pdf
5. If no colony is observed in any dilution taken, it is represented as: Estimated <1 x lowest dilution. https://gugufiber216.weebly.com/fugees-the-score-320-rar.html.
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.
Related Articles:
- Performed to determine the minimum inhibitory concentration (MIC) of an antimicrobial agent.
- MIC is defined as the lowest concentration of an antimicrobial agent that inhibits the growth of organisms.
Estimation of the MIC is useful to:
■ Regulate the therapeutic dose of the antibiotic accurately in the treatment of many life-threatening situations, such as bacterial endocarditis.
■ Test antimicrobial sensitivity patterns of slow-growing bacteria, such as M. tuberculosis.
![Serial Serial](https://www.coursehero.com/thumb/f8/5e/f85e0b13fac17238375a02ed5718c6b5eaa112f7_180.jpg)
What Are Some Advantages And Disadvantages Of The Serial Dilution Agar Plate Technique
Following methods are carried out to determine the MIC
Broth dilution method
Agar dilution method
Epsilometer test (E-test)
- Quantitative method for determining the MIC of an antimicrobial agent that inhibits the growth of organisms in vitro.
- In this method, the antimicrobial agent is serially diluted in Mueller–Hinton broth by doubling dilution in tubes and then a standard suspension of the broth culture of test organism is added to each of the antibiotic dilutions and control tube.
- This is mixed gently and incubated at 37°C for 16–18 hours.
- An organism of known susceptibility is included as a control.
- The MIC is recorded by noting the lowest concentration of the drug at which there is no visible growth as demonstrated by the lack of turbidity in the tube.
The main advantage of this method is that this is a simple procedure for testing a small number of isolates. The added advantage is that using the same tube, the minimum bactericidal concentration (MBC) of the bacteria can be determined.
- The MBC is determined by subculturing from each tube, showing no growth on a nutrient agar without any antibiotics.
- Subcultures are made from each tube showing no growth into the nutrient agar plates without any antibiotics.
- The plates are examined for growth, if any, after incubation overnight at 37°C.
- The tube containing the lowest concentration of the drug that fails to show any growth on subculture plate is considered as the MBC of the antibiotic for that strain.
Broth microdilution is done using microtiter plates and is considered the “gold standard.”
- Quantitative method for determining the MIC of antimicrobial agent against the test organism.
- Mueller–Hinton agar is used in this method.
- Serial dilution of the antibiotic are made in agar and poured onto Petri dishes.
- Dilutions are made in distilled water and added to the agar that has been melted and cooled to not more than 60°C.
- One control plate is inoculated without antibiotics.
- Organism to be tested is inoculated and incubated overnight at 37°C.
- Plates are examined for presence or absence of growth of the bacteria.
- The concentration at which bacterial growth is completely inhibited is considered as the MIC of the antibiotic.
- The organisms are reported sensitive, intermediate, or resistant by comparing the test MIC values with that given in CLSI guidelines.
The main advantage of the method is that a number of organisms can be tested simultaneously on each plate containing an antibiotic solution.
- Based on the principle of disc diffusion, is an automated system for measuring MIC of a bacterial isolate. In this method, an absorbent plastic strip with a continuous gradient of antibiotic is immobilized on one side.
- MIC interpretative scale corresponding to 15 twofold MIC dilutions is used on the other side.
- The strip is placed on the agar plate inoculated with the test organism with the MIC scale facing toward the opening side of the plate.
- An elliptical zone of growth inhibition is seen around the strip after incubation at 37°C overnight.
- The MIC is read from the scale at the intersection of the zone with the strip.
- The end point is always read at complete inhibition of all growth including hazes and isolated colonies.
E test is a very useful test for easy interpretation of the MIC of an antibiotic.