Lab 5: Methods of Inoculation and Isolation of Pure Culture PDF

Methods of inoculation and isolation of pure culture. Lab 5 Second Stage- Pharmacy - College of Al-Zahrawi University By: Assistant Lecturer Muntadher Hameed 38 The purpose of thi...

Methods of inoculation and isolation of pure culture. Lab 5 Second Stage- Pharmacy - College of Al-Zahrawi University By: Assistant Lecturer Muntadher Hameed 38 The purpose of this experiment is to separate a mixed culture of Streptococcus aureus and Escherichia coli and obtain single, isolated colonies of each bacterial species. when working with microorganisms, it is desirable to start with single, isolated colonies to ensure you are working with a pure culture. cultures that are visible on the surface of solid media are called colonies. A colony forms on a plate when a single microbe is inoculated onto the surface of the plate and reproduces until there are enough cells to form a visible colony. since a colony theoretically forms from a single cell, a colony should then represent a pure culture. One way to obtain single, isolated colonies is using the 4-quadrant streak method (4QSM) or T streak method (TS). The quadrant streak plate method allows sequential dilution of the original microbial material over the entire surface of a fresh plate. As the original sample is diluted by streaking it over successive quadrants, the number of organisms' decreases. Usually by the third or fourth quadrant only a few organisms are transferred, and these produce single, discrete colonies. 39 We imagine that our media is divided into four quadrant & start streaking those quadrants one by one 40 MEDIA: (per student) Nutrient agar plate, Mannitol salt agar plate CULTURES: Mixed broth culture of Streptococcus aureus and Escherichia coli PROCEDURE: (Note: your instructor may present an alternative method.) 1. Divide the agar plate into 4 quadrants. 2. Place a loopful of culture onto the plate in Quadrant 1 with a sterile loop and streak the loop very gently using straight motion toward quadrant 2. 3. Sterilize loop. Go back to the edge of Quadrant 1 and extend the streaks into Quadrant2, Sterilize loop. Go back to the edge of Quadrant 2 and extend the streaks into Quadrant 3. 4. Sterilize loop. Go back to the edge of Quadrant 3 and extend the streaks into Quadrant 4, be careful not to go back into quadrant 1. 4. Tape plate closed on both sides. Make sure the plate is labeled with your name, date, and the organism(s), and incubate upside down *** (to prevent condensation from getting on to agar at 32-37 °C). Other method for cultivation of bacteria include: a. Spread plate method b. Pour plate method Spread plate method It is a microbiological laboratory technique for isolating and counting the viable microorganisms present in a liquid sample by spreading a certain volume of the sample over an appropriate solidified culture, it requires the sample to be in liquid or suspension state, this technique is used for isolating and counting the total number of viable microorganisms, it can be used for all of the culturable bacteria and fungi. 41 At first, the samples are serially diluted, then 0.1 mL of the sample is pipetted over the center of the solidified agar medium and evenly spread over the surface of the medium using bent glass rod. The plates are incubated under the optimum condition following which the numbers of colonies are counted. Following the incubation for usually 24 – 48 hours at 37°C, the viable microorganisms in the sample will grow into discrete visible colonies on the surface of the medium. 42 Pour plate method The pour plate method allows the plating of higher volumes of sample (1 mL) by dispensing a liquid inoculum onto an empty petri dish, which is then flooded with a molten medium. Pour plates allow micro-organisms to grow both on the surface and within the medium. Most of the colonies grow within the medium and are small in size and may be confluent. The few colonies that grow on the surface are of the same size and appearance as those on a streak plate. It allows the growth for more sensitive bacteria, so it is ideal for samples with lower bacterial numbers. The main difference between pour plate and spread plate is that the molten agar is poured on to the inoculum during the preparation of the pour plate whereas inoculum is spread on the surface of the solidified agar during the preparation of the spread plate. 43 44 Action of dyes and antibiotics; Enzymes assays for some specific microbial enzymes. Lab 6 Second Stage- Pharmacy - College of Al-Zahrawi University By: Assistant Lecturer Muntadher Hameed 45 Introduction Dyes are used in microbiology for their inhibitory and differential properties ex: the use of carbol fuchsin in differentiating between acid-fast & non-acid-fast bacteria. In addition, some dyes (like crystal violet & safranine) have also antibacterial activity in some dermatologic infection especially against multidrug resistance bacteria like Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) but such use (the use of dyes as antibiotics) will remain limited and inconvenient due to their possible toxicity on our body. In recent years, many bacterial species have developed an ability to resist antibiotics by different mechanisms. One of these mechanisms is the release of special enzymes that will interfere with the antibacterial structure rendering it inactive. An example of such enzyme is the bata lactamase enzyme that will break open the beta-lactam ring present in penicillin and cephalosporine, thus inactivating them. These enzymes are released by many bacterial families especially by gram negative bacteria. Many techniques (quan ta ve and qualita ve) have been developed to test bacterial resistance to antibiotics. Such techniques include agar dilu on (the gold standard), broth macrodilu on and microdilu on, and strips with an an bio c gradient. 46 agar dilu on method is a method by which different concentra on of an bio c is used on a gar full of bacteria to determine the minimum inhibitory concentra on of that an bio c on bacteria. 47 broth dilu on method is a technique in which containers holding iden cal volumes of broth with an microbial solu on in increasing concentra ons are inoculated with a known number of bacteria. Qualitative technique: Kirby – Bauer method is a method of determination of antibiotic sensitivity of the bacteria by disc diffusion method. In this method, a standard suspension of bacteria to be tested are inoculated on the surface of Mueller Hinton agar plates. Filter paper discs containing specific concentration of antimicrobial agents are pressed on to the surface and incubated at 35°C overnight (18-24 hr.). After incubation, the zone of inhibition of growth of bacteria around each disc is measured and the susceptibility is determined. Preparation of suspension of bacteria: Approximately, 4-5 well isolated colonies of the bacterial strain to be tested are inoculated into 5 ml of peptone water, and is incubated at 37 °C for 3-4 hours. The turbidity of the suspension is adjusted to match 0.5 McFarland standards*. If the density is more it is diluted with sterile saline. The comparison is made against a white back ground with a contrasting black line. *(The McFarland 0.5 standard has particular application in the preparation of bacterial inocula for performing antimicrobial susceptibility testing. Turbidity standards are prepared by mixing chemicals that precipitate to form a solution of reproducible turbidity). 48 PROCEDURE 1. After standardization of bacterial suspension, immerse a sterile cotton swab in it and rotate the swab several times with firm pressure on the inside wall of the tube to remove excess fluid. 2. Prepare a Mueller Hinton agar (MHA) plate (pH 7.2-7.4) with a depth of 4 mm. 3. Inoculate the dried surface of the MHA agar plate by streaking the swab three times over the entire agar surface. It is streaked in three directions by rotating the plate 60° after each streak. 4. Place the appropriate antimicrobial impregnated discs on the surface of the agar using sterile forceps. 5. Gently press each disc onto the agar to provide uniform contact. Do not move the disc once it has contacted the agar because some of the antibiotics diffuse almost immediately. Discs must be placed in such a way that they are at least 20 mm from one another. 6. Invert the plates and incubate at 35 °C -37°C for 16-18 hr. 49 McFarland standard comparison with bacterial broth Infront of white and black background Antibiotic disc 50 RESULTS AND INTERPRETATION Each antibiotics produces a specific zone size for each bacteria tested. Depending on the zone size, the bacteria are classified as follows: Sensitive (S): Infection treatable with normal dosage of the antibiotic. Intermediate (I): Infection may respond to therapy with higher dosage Resistant (R): Unlikely to respond to the antibiotic at the usual dosage. Antibiotics: It is a substance that inhibits the growth or causes death of organisms in low concentrations. Bacteriostatic drug: Certain antimicrobial agents inhibit the growth by preventing the multiplication of organisms. They do not cause death. These are called bacteriostatic agents. e.g., tetracycline. Bactericidal drug: The drugs that cause irreversible damage to bacteria resulting in death are called bactericidal drugs. e.g., penicillin 51 52 53 Mueller Hinton agar with bacteria 54