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Lab Manual / Exp. 8
Experiment 8 : Determination of antimicrobial activity
Objective
:
Some microorganisms produce antimicrobial compounds and these compounds are detected based on their ability to inhibit the growth of other microorganisms microorganisms To perform methods those are normally used to determine the antimicrobial activity of microorganism. microorganism.
Overview :
Antimicrobial compounds are chemicals acting against bacteria. They include disinfectants which show a low selectivity and are only used for surface decontamination of non-biological material and chemotherapeutics and antibiotics which have low or minimal activity against higher eukaryotes. In contrast to chemotherapeutics which by definition d efinition are compounds which have been chemically synthesized in a laboratory, antibiotics are substances which are produced by certain taxonomic groups of microorganisms and therefore do occur naturally. This phenomenon was first observed by Alexander Fleming (1928) who had obtained his now famous Penicillium contamination on a staphylococci plate which he correctly recognized as caused by the synthesis of an antibacterial compound by the fungus. He however never purified the active ingredient and also failed to recognize its therapeutic potential. This was left to a research group in Oxford consisting of Howard Florey and Ernest Chain who demonstrated the selectivity of Penicillins in 1940. After their emigration to the US during the war they then developed the large scale fermentation processes in order to satisfy the growing demand caused by the high casualties suffered during the second WW. ndh/dbt/July 2010
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The late 1940s and early 1950s then saw the discovery of other antibacterial drugs such as Streptomycin, Chloramphenicol, Neomycin and Tetracyclin. These compounds were now called antibiotics because they were of biological origin as opposed to the chemotherapeutics of the early 20th century; these compounds can act either bacteriocidal or bacteriostatic. Measuring antimicrobial activity Determining the antimicrobial activity of a compound is often essential for its effective use. For example, to fight a bacterial infection a series of antibiotics and antimicrobials will be tested to determine which should be prescribed as a treatment. In a food processing plant the concentration of disinfectant to use for cleaning equipment can be determined experimentally to maximize killing power, yet minimize the amount of antimicrobial used. A good antimicrobial assay will achieve two purposes, it first verifies that the compound actually has the desired antimicrobial activity and second it indicates the concentration of the antimicrobial that will be needed to inhibit the target organism. To determine the activity of an antimicrobial compound against microorganisms, it is tested under carefully controlled conditions. The effectiveness of a compound is dependent upon a number of conditions such as the nature of the target microbes, concentration of the microbes, composition of the environment, time in contact with the compound, temperature, pH and amount of aeration. A standard assay often used to gauge the effectiveness of an antimicrobial is the minimum inhibitory concentration (MIC) test. The MIC is the lowest concentration of a compound that still inhibits its growth. The MIC of a given compound for a certain bacterial species is determined using a series of test tubes containing medium in which the microbe will normally grow, but where each tube contains progressively lower concentrations of the test compound. Each tube is inoculated with the microbe and after incubation the tubes in which growth does not occur are noted. The lowest concentration of the antimicrobial compound that prevents growth defines the MIC. Since many parameters affect MIC, it is only valid for the specific conditions tested. If it is desired to compare the effectiveness of different antimicrobials on a certain strain or to examine the resistance of a number of microbes to a single agent, test conditions need to be rigorously standardized. In some situations it is important to determine the minimum concentration at which a compound is lethal to a microorganism and this is defined as the minimal lethal concentration (MLC). Antimicrobials that are cidal will normally kill a microorganism at two to four times their inhibitory concentration, while a static agent will require a much higher concentration and may not ever be lethal.
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Another commonly used assay to assess the potency of an agent is the agar diffusion method. A microbial culture is spread evenly on the top of an agar plate containing medium that will support its growth. Disks impregnated with antimicrobial compounds are then placed onto the agar and the plate incubated at an appropriate temperature for that microbe. During incubation the antimicrobial compound diffuses away from the disk and into the agar creating a concentration gradient that is highest near the disk and decreases as one moves away from the disk. If a microbe is inhibited by the agent, it will be unable to grow near the disk, which we see as a zone of clearing in the lawn of growth. Farther away from the disk, where the concentration of the antimicrobial compound is much lower, growth will be evident. The size of the zone of clearing around the disk is an indication of the potency of the antimicrobial for the tested microbe. Using the agar diffusion method it is possible to test a number of different compounds simultaneously on the same agar plate. Unlike the previous method, this one does not readily provide the MIC, since we cannot easily know the concentration at different places in the agar. The agar diffusion method is most useful in determining the approximate antibiotic sensitivity of a microbe.
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Lab Manual / Exp. 8
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Lab Manual / Exp. 8
Glassware / apparatus and chemicals for 2 student : 1.
A bottle of cell suspension or cell broth of
Escherichia coli
and Bacillus
subtilis
2. 3 Petri dishes contain sterile nutrient agar 3. Sterile nutrient broth to be diluted with 1 % (w/v) of chloramphenicol 4. 2 pieces of Whatman filter paper which is sterilized prior to use (6mm disc) 5. 1 % (w/v) of chloramphenicol 6. A bottle containing 200 ml of sterile distilled water 7. 10 sterile Universal bottles 8. Sterile forceps 9. Hockey stick 10. 70% ethanol solution
Experimental procedures
A. Disc agar diffusion technique 1. Place the agar plates right side up and divide one of the plates into two sections by scoring the underside of the plate with a marker pen. 2. Label each section on the plate with the name of organism to be inoculated (Escherichia coli and Bacillus subtilis ). – To see the inhibition growth between the two microbes. 3. Label the other two plates with the name of organism to be inoculated (one plate for Escherichia coli and one plate for Bacillus subtilis ).- to see the antimicrobial activity of the microorganism 4. Using the cultures from Escherichia coli and Bacillus subtilis , perform the spread plate method as described in Experiment 2 on the plate which has been divide into two sections (each organism on each section as being labeled respectively). These will be used as the test organisms. 5. Perform the spread plate method as well on the other two plates (each organism on each plate as being labeled respectively). 6. Prepare the filter paper disc of diameter 6 mm using the 2-layer Whatman filter papers using the paper hole puncher. Sterile the paper discs by autoclaving at 121°C for 15 min. 7. Hold the filter paper disc using a pair of sterile forceps and dip the paper in the 1% chloramphenicol solution. Similarly, using the filter paper, dip in the distilled water which will be used as control. 8. Place the disc on the surface of the agar containing the cultures of E.coli and B. subtilis. Gently press each disc down with the wooden end of a cotton
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swab or sterile forceps to ensure that the discs adhere to the surface of the agar. Do not press the discs into the agar. 9. Incubate the plates at 35°C for 24 h and observe the growth inhibition zone.
B. Minimum inhibitory concentration, MIC (Tube dilution technique) 1. Prepare a serial double dilution of the 1 % chloramphenicol using the nutrient broth. The final concentration of the antibiotic will now be 0.5, 0.25, 0.125, 0.0625%. 2. Prepare the antibiotic in 2 sets, one for Escherichia coli and the other for Bacillus subtilis.
3. To each tube of known concentration of the antibiotic, add 0.1 ml of the cell suspension of the test organisms and vortex them gently. 4. A nutrient broth without any addition of the antibiotic is used as control 5. Incubate all the tubes at 37°C for 24 h. 6. After 24 h, observe the turbidity, and check the minimum concentration of the antibiotic that prevents the growth of the test organisms.
Reports.
1. Determine the area of growth inhibition of the test organisms by the agar diffusion technique. 2. Determine the MIC value of chloramphenicol for Escherichia coli and Bacillus subtilis.
3. Discuss the effectiveness of the methods used.
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