Answer: A2A 'Identification' can be done in two ways - Detection and Typing. In this laboratory exercise, you will be learning the pour plate and streak plate techniques. Take a fresh agar plate and touch separate areas of the agar surface with each fingertip of your right hand. For obtaining the isolated colonies streaking method is used, the most common method of inoculating an agar plate is streaking. Dip the L-shaped glass spreader into alcohol. Heat an inoculate loop to red-hot and then allow to cool for at least 5 sec 2. Label the bottom surface of your sterile agar plates. For this isolation of only one type of microbial culture the methods that is preferable are the Streak plate method , Pour plate method and the Spread plate method Tryptic soy broth and luria bertani (most preferred media to grow the Staphylococcus aureus ) -- forms characteristic gold color colonies on media which also helps to find out the . You will be given a plate with streaked organisms on it. Make a dilution series from a sample. Fig. 4. Hold an inoculation loop in your right hand. Pour plate method • Uses the principle of mechanical separation on the surface of the agar • • Convenient and quick the surface of a nutrient agar • Colonies form on the surface of the agar • surface and in the bulk of the • Uses the principle dilution of inoculum in a large volume Inoculum added to molten . In this method, fixed amount of inoculum (generally 1 ml) from a broth/sample is placed in the center of sterile Petri dish using a sterile pipette. To ensure a countable plate a series of dilutions should be plated. 1:56 min Streak Plate Method (Figs. Procedure of Spread Plate Technique. Keep the agar dry. • Describe the different types of culture media and their possible physical state. 2. List and describe the steps of streaking for isolation Label the bottom of the plate Suspend the cells by gently shaking the tube Sterile the inoculating loop and get a sample from culture tube and sterile it. Label the plate and incubate it inverted. Reflame the loop and cool it. Look closely at it and select an area that has individual colonies. Disadvantages of Pour plate method. In the streak plate method, what is the best way to ensure that the loop is cool, after it has been flamed a. no need to cool b. If for example, an average of five bacteria is present in each ruled square, there is 5 x 20,000,000 or 10^8, bacteria per milliliter. Reflame the loop and cool it. Detection (such as through PCR assays) is confirming the presence of a suspected strain. Streak plate 12 Pour plate 13 Using a spreader 15 Spread plate 16 INCUBATION 17 IN CONCLUSION: CLEARING UP 17. ISOLATION BY STREAKING OR STREAK PLATE TECHNIQUE- This method is used most commonly to isolate pure cultures of bacteria. 8. A wrong streaking method can ruin your plate. (c) Steps in the loop dilution method and (d) the appearance of plate 3. Biology Q&A Library You are givena mix culture of S. aureus, E. coli and P. aeruginosa. Techniques in (a) and (c) use a loopful of culture, whereas (e) starts with a pre-diluted sample. Place both tubes on (into) the ice. Touch plates showing a variety of bacteria found on the fingertips. The pour plate method, spread plate method, and streak plate method are all included in the techniques. The streaking patterns shown in the figure below result in continuous dilution of the inoculum to give well separated surface colonies. Spread the sample evenly over the surface of agar . 8. Contents Different Isolation Techniques Types of Cultures Serial dilution Streak-Plate Method Pour Plate method Each time you flame and cool the loop between sectors, rotate the plate counterclockwise so you are always working in the "12:00 position" of the plate. Using the T-method (illustrated below) or quadrant method, outline the sections on the bottom of the agar plate. 1 Use a water bath at 50 °C to store bottles of molten agar.. 2 Take care not to contaminate the molten agar in the bottles with water from the water bath. Summary. To avoid contamination ensure: i that the water in the water bath is at the right depth ii that the bottles are kept upright iii that the outsides of the bottles are wiped before they are used.. 3 In an evenly spread pour plate, the base . Only as much agar as is needed for the experiment should be melted as it cannot be re-used. Four Quadrant Streak : Loosen the cap of the bottle containing the inoculum. . Date WORKSHEET 1. Same basic equipment as for conventional plate count (Chapter 3). Bacterial cells are spread over the surface of an agar plate in a continuous dilution, so that the cells will be separated from each other. Side by Side Comparison - Streak Plate vs Spread Plate in Tabular Form 6. Microbiological Laboratory Techniques. Streak plate technique. (a) Steps in a quadrant streak plate and (b) resulting isolated colonies of bacteria. The first step taken was isolating the bacterium from original broth #105 into two separate colonies using the quadrant streak method on a nutrient agar plate. 1. Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of an agar plate. These steps are refer as the 5 I's. The steps are: Inoculation, Incubation, Isolation, Inspection and Identification. 4. Loss of viability of heat-sensitive organisms coming into contact with hot agar. Streak Plate Procedure: Isolation of Bacterial Colonies Using the Quadrant Method. Have your two plates on your lab bench. After letting the first streak plate incubate at room temperature for five days there was growth on the first two streaks, but the last two had none. Sterilize loop in flame of Bunsen burner or hub of microincinerator. This is the method used by professional microbiologists and is best reserved for older students . Repeat #6, streaking the remainder of the plate. 2 Media • Classified according to three properties - Physical state - Chemical composition - Functional types . Besides using the streak plate method what other methods could you use to separate the bacteria? 1 TSA plate; 1 plate of E.coli K-12; Plate to Plate Transfers Using BSL2 Procedures. 1 Two other approaches when making a streak plate are:. Swab an area of the face (e.g., cheek or forehead) and streak a mannitol salt agar plate. • Explain and differentiate between sterilization, disinfection, and sanitization. Optional: Do your last streak with a needle and poke into the agar. Requirements: 1. Similarities Between Streak Plate and Spread Plate 5. i to lift the lid vertically (i.e. 2. What method of streak for isolation would be preferred for a sample suspected to contain a high density of cells? This is quadrant #1. Preparation for the pour plate method is time-consuming compared with the streak plate/and or spread plate technique. • Describe the general concept of aseptic techniques used in laboratory preparation and analysis. Invert the plate and store it upside down. Figure 3.10 Methods for isolating bacteria. After removal with bacteria on the loop, the quadrant streak method was used. As the pattern is traced, bacteria are rubbed off the loop . A . For accurate counts, the optimum count should be within the range of 30- 300 colonies/plate. 1 TSA plate; 1 plate of E.coli K-12; Plate to Plate Transfers Using BSL2 Procedures. Inoculate and streak another agar plate as in step 3. Microbial aerosols created from the hitting of the inoculation tip to the sides of the agar plate. 3. 5. Take an agar plate into the lavatory. Using the toothpick, make a single short streak on a blood agar plate. al and in LeBoffe and Pierce. Read the streak plate method or streaking for isolation in Tortora et. Overlap the step 1 streak 3-4 times to pull out a reduced number of bacteria, and spread them out down the side of the plate. The spread plate method of isolation is different from the streak plate method of isolation in . In the pour plate technique, an inoculum is added to melted, cooled agar. 4. The four-quadrant streak method described in the lab manual was used for this procedure. 10.6: Streaking method It was originally developed by the two bacteriologists, Loeffler and Gaffkey in the lab of Robert Koch (Father of Bacteriology). I. Streak plate method II. B. Streak Plate Technique The streak plating technique isolates individual bacterial cells (colony-forming units) on the surface of an agar plate using a wire loop. 16.13). The streak plate was then incubated at 37 degrees Celsius for 48 hours. This will make a dilution gradient across the agar plate. 9. The analysis of food products for presence of pathogenic microorganisms is one of the basic steps to control safety and quality of food. Streak Plate Procedure: Isolation of Bacterial Colonies Using the Quadrant Method. The streak plate method will require constant use of the streaking loop. List the differences in structure between bacterial flagella and eueryotic flagella 3. The colony becomes visible to the naked eye and the number of colonies on a plate can be counted. The successive streaks "thin out" the inoculum sufficiently and the micro-organisms are separated from each other. 4.8). The streak plate is only qualitative but the pour plate procedure can be used to quantitate bacteria present in a sample as in the Standard Plate Count Method (see Part III-A), The spread plate method provides a quantitative method for aerobic surface growth of cultures against which other surface growth methods such as the MF technique can be . The Streak Plate technique for the isolation of microorganism is the most practical method of obtaining discrete and well-developed colonies of the microbe in pure cultures. This is a list of molecular methods that are furthe. The inoculation of the culture is made on the agar surface by back and forth streaking with the inoculation loop over the solid agar surface. Remove the cap of the bacterial culture tube and flame the neck of the tube, and then remove a loopful of the culture using the sterilized inoculating loop 3. Once the inoculation is done, the agar or the broth containing the inoculum is kept in the incubator at a certain temperature for the required time duration. They will look like small dots on your plate. The streak-plate procedure is designed to isolate pure cultures of bacteria, or colonies, from mixed populations by simple mechanical separation. using the streak plate method (Fig. Allow the plate to dry for approximately 5 minutes. 9. Microbiologists use five basic procedures to examine and characterize microbes: Inoculation, Incubation, Isolation, Inspection (observation), and Identification—the five "I's." To culture a microorganism a small sample, the inoculum, is introduced into a culture medium usually with a platinum wire probe streaked across Click to see full answer Streak a plate of blood agar for isolation. Flame the neck of the test tube. 3. Obtain mix culture and shake gently to suspend organisms. 2. A . How would the pH of the stain affect the staining of bacteria? Both of these techniques depend upon the physical/spatial isolation of a single bacterial cell on/in a solid medium. Have your two plates on your lab bench. BASIC PRACTICAL MICROBIOLOGY A MANUAL Compiled by John Grainger, . Gram's staining method 24 Using the swab, streak the Mueller-Hinton agar plate to form a bacterial lawn. Pour plate; Streak plate. Significance of Streak Plate Method. Standard Plate Count (SPC) By far the most widely used method for determining the number of viable colony forming units (CFU) in a food. Being able to visibly differentiate bacteria based on the appearance of their colonies is a crude, but essential first step in isolating the different types of bacteria in the sample. 6. Methylene blue can be prepared as a basic stain or an acidic stain. Single colonies are comprised of millions of cells growing in a cluster on or within an agar plate (Figure 1). step - give a preliminary ID for an infectious organism Selective! ii to place the lid on the working surface, lift out the base, invert it and inoculate the upwards-facing agar surface. Inoculation of a plate culture uses a streaking technique to make a streak plate. Repeat this rotation 3 times. The most common method of isolation is the streak plate, in which a sterile loop is inserted into a sample and streaked onto a plate in a pattern, to obtain individual colonies Colony: A group of descendants of an original cell. Label first, streak second. To obtain uniform growth, streak the plate with the swab in one direction, rotate the plate 90° and streak the plate again in that direction. Make sure the agar plates are free of droplets of condensed moisture. Method used for inoculating the solid media depends upon the purpose of inoculation- whether to have isolated colonies or to know the bacterial load of the sample (quantitative analysis). There was only one colony that was apparent. Use a sterile inoculation loop to pick up a single colony of bacteria from your starter plate. Proper use of a bunsen burner, inoculation loop, and aseptic technique are shown. Once again, the idea is to obtain isolated . Embedded colonies are much smaller than those which happen to be on the surface. 3. more of the agar. Isolation of bacteria is . Streak inoculum to obtain isolated colonies. Remove the cap of the bacterial culture tube aand flame rhe nexk of the tube snd them remove a loopful of the cukture using the sterilized inoculating loop 3. The isolation method most commonly used to get pure cultures is the streak plate technique. Go To Results of Streak Plate Lab Procedures 25 . List the basic steps of the streak plate method in correct order from start to finish 1. Plate Culture. Close, invert, and incubate the plate at 35°C. These are the real thing. 10.6: Streaking method use a marker to divide each plate into sectors: e.g. to the plate method) . Then, using your inoculating loop, streak out the toothpick streaks as illustrated below. 2. Turn the plate 90°, overlap the previous area 1-2 times, and streak into the next quadrant as in step 4. The streak plate is a qualitative isolation method; quadrant streaking is mostly done to obtain pure colonies. Once the agar reaches this temperature, it is ready to pour. Fig. Flame the loop and cool it in the agar. The plate count method or spread plate relies on bacteria growing a colony on a nutrient medium. Isolation of bacteria is a primary method to separate different groups of microorganisms. A sterile inoculating loop is dipped into a mixed culture that contains more than one type of microbe and is streaked in a pattern over the surface of the nutrient medium. Watch the following movie which demonstrates the technique of "streaking a plate" and then practice the technique on a piece of paper with a pencil and then using an empty petri dish. insert the cooled loop into the broth culture and sterile by passing through flame. Look closely at it and select an area that has individual colonies. (e) Spread plate and (f) its result. Remove the cap/ cotton wool plug of the test tube with the little finger of your right hand. Streak each sector carefully, staying within the sector margins to minimize the possibility of cross contamination. Incubate plates 48 h at 35-37°C. that is streak plating on two . Clearly labeling your plates to identify the microorganism only takes a few seconds and will help you avoid a big headache later. See page 84 of the Difco/BBL Manual. Single colonies are comprised of millions of cells growing in a cluster on or within an agar plate (Figure 1). Incubate the plate at 37°C for 48 hours. It also used to avoid having to pipette very small volumes (1-10 µl) to make a dilution of a solution. 2. three sectors for three bacterial test species. 5. Follow these best practices to get your streaking technique down to a science. Above: Streak plate of . Step 4. Touch the loop to an uninoculated area of the plate c. Touch the loop to your finger to determine the temperature d. 4. Overlap the step 2 streak 3-4 times and spread over the surface. Flame the loop. Incubate the MOX at 35 ± 2°C for 26 ± 2 h. Flame the loop. Pour plate method is usually the method of choice for counting the number of colony-forming bacteria present in a liquid specimen. Method had been performed, the idea is to obtain isolated are much smaller than those happen... 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