Protocol for Studying Antimicrobial Activity of Natural Extracts: A Comprehensive Guide, Assignments of Microbiology

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Laboratory exercises

by:

narendra Vikram singh

(18/ibt/020)

submitted to :

dr. rekha puria

date:

th

may, 2020

Exercise1. Protocol to study antimicrobial

activity of any 2 natural extract:

 MATERIALS REQUIRED: Agar plates, broth bacterial culture, glass spreader, medicinal plants, ethanol, eppendorf, vortex, centrifuge, micropipette, incubator, etc.  PRINCIPLE: Two natural extracts are:

1. Mentha piperita: Mentha piperita against some Gram-positive and Gram-negative bacterial strains was evaluated in the present research work by agar well diffusion method. It was found that the distilled concentrations of essential oil inhibited the growth of microorganisms and the results were comparable with those of antibiotic gentamycin. Essential oils showed a wider spectrum of activity but less strong inhibition as compared to the investigated commercial antibiotic. Minimum inhibitory concentrations (MICs) for the bacterial species ranged from 0.4% to 0.7% v/v. The oil and extracts also exhibited significant antioxidant activity and the oil showed about half potency when compared to the standard BHT. These results indicated the strong antibacterial and antioxidant activities of

different extract of Acacia nilotica. In this study the pod extract shows highest antibacterial potential followed by the bark extract and leaves extract.

Exercise2. Experiment to screen antimicrobial

activity of natural extracts using plug agar

method:

 MATERIALS REQUIRED: Agar plates, broth bacterial culture, glass spreader, medicinal plants, ethanol, eppendorf, vortex, centrifuge, micropipette, incubator, etc.  PRINCIPLE: Two medicinal plants are:

1. Ficus Religiosa (pippala tree): ficus religiosa is a widely branched tree with leathery, heart-shaped, long-tipped leaves, used in the Indian system of medicine, besides which folklore medicine also claims its use in diarrhoea, diabetes, urinary disorder, burns, haemorrhoids, gastrohelcosis, skin diseases, convulsion, tuberculosis, fever, paralysis, oxidative stress, bacterial infection etc. Research carried out using different in-vitro and in-vivo techniques of biological evaluation support most of these claims. Presently there is an increasing interest worldwide in herbal medicines accompanied by increased laboratory investigation into the pharmacological properties of the bioactive ingredients and their ability to treat various diseases. Numerous drugs

have entered the international through exploration of ethnopharmacology and traditional medicine. Although scientific studies have been carried out on a large number of Indian botanicals, a considerably smaller number of marketable drugs or phytochemical entities have entered the evidence- based therapeutics. Efforts are therefore needed to establish and validate evidence regarding safety and practices of Ayurvedic medicines.

2. Calotropis Procera: Calotropis procera is a species of flowering plant in the family Apocynaceae that is native to North Africa, tropical Africa, Western Asia, South Asia, and Indochina. The green fruits contain a toxic milky sap that is extremely bitter and turns into a gluey coating which is resistant to soap. Common names for the plant include Apple of Sodom. The culture plates seeded with test organisms were allowed to solidify and punched with a sterile cork borer (7.0 mm diameter) to make open wells. The open wells were filled with 0.05 ml of the extract. The plates were incubated at 37o C for 48 hours. For the fungi, the test was carried out on SDA plates and incubated at 30o C for 72 hours. The zones of inhibition were measured and recorded.  PROCEDURE: o Prepare nutrient agar plates.

temperatures that different types of microbes grow at are as follows: Figure: Growth Rates for Different Microorganisms in Response to Temperature Since heating methods are commonly used to control microbial growth, it is important to be able to define the effectiveness of a heating method for a particular bacterial species. One way to do this is to determine the thermal death point (TDP) and the TDT (thermal death time). Thermal Death Time (TDT) temperatures that different types of microbes grow at are as Growth Rates for Different Microorganisms in Response to Temperature Since heating methods are commonly used to control microbial growth, it is important to be able to define the ness of a heating method for a particular bacterial species. One way to do this is to determine the thermal death point (TDP) and the TDT (thermal death time). Thermal Death Time (TDT) temperatures that different types of microbes grow at are as Growth Rates for Different Microorganisms in Since heating methods are commonly used to control microbial growth, it is important to be able to define the ness of a heating method for a particular bacterial species. One way to do this is to determine the thermal death

The TDT is the minimum time it takes to kill a population of microbes at a specific temperature. Figure 7.1.3: Death curves for three species at 70 Thermal Death Point (TDP) The TDP is the lowest temperature that is required to kill a population of microbes when applied for a specific time. The TDT is the minimum time it takes to kill a population of t a specific temperature. Death curves for three species at 70 Thermal Death Point (TDP) The TDP is the lowest temperature that is required to kill a population of microbes when applied for a specific time. The TDT is the minimum time it takes to kill a population of Death curves for three species at 70°C The TDP is the lowest temperature that is required to kill a population of microbes when applied for a specific time.

 The procedure of the flagella stain (wet

mount technique):

1. Grow the organism to be stained at room temperature on blood agar for 16 to 24 hours.
2. Add a small drop of water to a microscope slide.
3. Dip a sterile inoculating loop into sterile water.
4. Touch the loopful of water to the colony margin briefly (this allows motile cells to swim into the droplet of water).
5. Touch the loopful of motile cells to the drop of water on the slide. Note: Agitating the loop in the droplet of water on the slide causes the flagella to shear off the cell.
6. Cover the faintly turbid drop of water on the slide with a coverslip. A proper wet mount has barely enough liquid to fill the space under a coverslip. Small air spaces around the edge are preferable.
7. Examine the slide immediately under 40× to 50× for motile cells. If motile cells are not seen, do not proceed with the stain.
8. If motile cells are seen, leave the slide at room temperature for 5 to 10 minutes. This allows the bacterial cells time to adhere either to the glass slide or to the coverslip.
9. Gently apply 2 drops of RYU flagella stain (Remel, Lenexa, Kansas) to the edge of the coverslip. The stain will flow by capillary action and mix with the cell suspension. Small air pockets around the edge of

the wet mount are useful in aiding the capillary action.

10. After 5 to 10 minutes at room temperature, examine the cells for flagella.
11. Cells with flagella may be observed at 100× (oil) in the zone of optimum stain concentration, about halfway from the edge of the coverslip to the center of the mount.
12. Focusing the microscope on the cells attached to the coverslip rather than on the cells attached to the slide facilitates visualization of the flagella. The precipitate from the stain is primarily on the slide rather than the coverslip.

Exercise5. Characterization of microbes

through chemical analysis:

Accurate identification of bacterial isolates is essential in a clinical microbiology laboratory because the results often inform decisions about treatment that directly affect patient outcomes. For example, cases of food poisoning require accurate identification of the causative agent so that physicians can prescribe appropriate treatment. Likewise, it is important to accurately identify the causative pathogen during an outbreak of disease so that appropriate strategies can be employed to contain the epidemic.

carbon utilization and other metabolic tests. In small laboratory settings or in teaching laboratories, those assays are carried out using a limited number of test tubes. However, more modern systems, such as the one developed by Biolog, Inc., are based on panels of biochemical reactions performed simultaneously and analyzed by software. Biolog’s system identifies cells based on their ability to metabolize certain biochemicals and on their physiological properties, including pH and chemical sensitivity. It uses all major classes of biochemicals in its analysis. Identifications can be performed manually or with the semi- or fully automated instruments. Another automated system identifies microorganisms by determining the specimen’s mass spectrum and then comparing it to a database that contains known mass spectra for thousands of microorganisms. This method is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and uses disposable MALDI plates on which the microorganism is mixed with a specialized matrix reagent (Fig1). The sample/reagent mixture is irradiated with a high-intensity pulsed ultraviolet laser, resulting in the ejection of gaseous ions generated from the various chemical constituents of the microorganism. These gaseous ions are collected and accelerated through the mass spectrometer, with ions traveling at a velocity determined by their mass-to-charge

ratio (m/z), thus, reaching the detector at different times. A plot of detector signal versus m/z yields a mass spectrum for the organism that is uniquely related to its biochemical composition. Comparison of the mass spectrum to a library of reference spectra obtained from identical analyses of known microorganisms permits identification of the unknown microbe. Fig1: MALDI-TOF methods are now routinely used for diagnostic procedures in clinical microbiology laboratories. This technology is able to rapidly identify some microorganisms that cannot be readily identified by more traditional methods. (credit “MALDI plate photo”: modification of work by Chen Q, Liu T, Chen G; credit “graphs”: modification of work by Bailes J, Vidal L, Ivanov DA, Soloviev M). Microbes can also be identified by measuring their unique lipid profiles. As we have learned, fatty acids of lipids can vary in chain length, presence or absence of double bonds, and number of double bonds, hydroxyl groups, branches,

“culture”: modification of work by the Centers for Disease Control and Prevention; credit “graph”: modification of work by Zhang P. and Liu P.) A related method for microorganism identification is called phospholipid-derived fatty acids (PLFA) analysis. Membranes are mostly composed of phospholipids, which can be saponified (hydrolyzed with alkali) to release the fatty acids. The resulting fatty acid mixture is then subjected to FAME analysis, and the measured lipid profiles can be compared with those of known microorganisms to identify the unknown microorganism. Bacterial identification can also be based on the proteins produced under specific growth conditions within the human body. These types of identification procedures are called proteomic analysis. To perform proteomic analysis, proteins from the pathogen are first separated by high- pressure liquid chromatography (HPLC), and the collected fractions are then digested to yield smaller peptide fragments. These peptides are identified by mass spectrometry and compared with those of known microorganisms to identify the unknown microorganism in the original specimen. Microorganisms can also be identified by the carbohydrates attached to proteins (glycoproteins) in the plasma

membrane or cell wall. Antibodies and other carbohydrate- binding proteins can attach to specific carbohydrates on cell surfaces, causing the cells to clump together. Serological tests (e.g., the Lancefield groups tests, which are used for identification of Streptococcus species) are performed to detect the unique carbohydrates located on the surface of the cell.

Exercise6. Draw cycle of endospore formation

Endospore formation is usually triggered by a lack of nutrients, and usually occurs in gram-positive bacteria. In endospore formation, the bacterium divides within its cell wall, and one side then engulfs the other. Endospores enable bacteria to lie dormant for extended periods, even centuries.

The outer membrane of the Gram-negative cell (lipopolysaccharide layer) is lost from the cell, leaving the peptidoglycan layer exposed. Gram-negative cells have thin layers of peptidoglycan, one to three layers deep with a slightly different structure than the peptidoglycan of gram- positive cells. With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell. The highly cross-linked and multi-layered peptidoglycan of the gram-positive cell is dehydrated by the addition of ethanol. The multi-layered nature of the peptidoglycan along with the dehydration from the ethanol treatment traps the large CV-I complexes within the cell. After decolorization, the gram-positive cell remains purple in color, whereas the gram-negative cell loses the purple color and is only revealed when the counterstain, the positively charged dye safranin, is added. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Q2: How will you prepare 5 fold serial dilution upto 5 fold with final volume of 200ul?

Q3: Name media is used for growth of bacteria in lab?? Write its composition. Ans: Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Blood agar, chocolate agar, Loeffler's serum slope etc are few of the enriched media. Blood agar is prepared by adding 5-10% (by volume) blood to a blood agar base. Chocolate agar is also known as heated blood agar or lysed blood agar. COMPOSITIONS: LB Broth and LB Agar Most referenced bacteria media Terrific Broth Nutritionally rich bacteria media for E. coli stains. M9 Minimal Commonly used bacterial media MagicalMedia Medium Customized bacterial media for protein expression ImMedia Medium Pre-mixed, pre-sterilized bacteria strains Other Media Microbial media for outgrowth media (invitrogenTM^ Select Agar Broth)