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Streak literally means “a long, thin line”: and the streak plate method is a microbiological culture technique where a sample is spread in a petri dish in the form of a long, thin line over the surface of solid media. What is Streak Plate Method?The streak plate method is a microbiological laboratory technique of isolating pure cultures, and/or getting well-isolated colonies of bacteria from a mixed population. It is mostly used to get pure cultures of bacteria; however, yeasts can also be isolated by this method. It is one of the most commonly used aseptic techniques in microbiology to isolate and propagate bacteria. It is a mechanical isolation technique used in microbiology, commonly known as the “streaking method”. This method dilutes the bacterial load, over the surface of agar medium, successively as streaking proceeds, and ultimately only a few bacterial cells will be inoculated at the end giving well-isolated colonies in the final streaks. Thus, this method mechanically isolated the bacteria from a mixed population of either the same or different species. After inoculation, the same types of colonies are seen in the terminal streaks if the specimen contained single species, whereas, different types of colonies may be seen if the specimen contained different species. This is a very old method used in microbiology since the time of Robert Koch. This method was first devised and used by Loeffler and Gaffky in Koch’s laboratory to serially dilute bacteria over agar surface and obtain well-isolated colonies. Since that time it is used as a very important tool in bacteriology. It is a very simple and reliable aseptic technique that uses tools like cotton swabs, wooden or plastic, metal sticks and toothpicks, or inoculating loop to dilute and spread the specimen over the surface of pre-sterilized specific solid culture media. The specimen used can be either suspension or colonies from the agar surface. Well isolated colonies can be obtained from successfully performed streaking which allows describing the colony character of the organism on that specific culture media and condition. Objectives of Streak Plate Method
Principle of Streak Plate MethodThe streak plate method is based on dilution during the process of mechanical spreading of inoculum over the surface of solidified culture media in order to obtain well-isolated colonies of the sample at the terminal streaks. Sample can be either colony on solid media or suspension in broth. The sample is picked by using different tools, mostly using a sterile inoculating loop or swab. The sample is placed over a surface of sterile solid media at one edge of the petri dish and a smear is prepared. Using the tool, the smear is successively streaked over the agar medium on different patterns. As the streaking proceeds, the inoculum is gradually diluted to the point where bacterial cells are separated as individual cells or as a colony-forming unit (CFU) at a gap of a few millimeters. When these inoculated plates are incubated, the isolated bacterium or a CFU will give rise to a well-isolated colony. This will allow us to get a pure culture as well as describe the colony morphology of the organism. Types of Streak Plate MethodBased on the pattern of streaking, the streak plate method can be classified into 4 types, viz.: Quadrant Streaking, T-Streaking, Continuous Streaking, and Radiant Streaking. 1. Quadrant StreakingIt is the most commonly used and the most preferred method where four equal-sized sections of the agar plate are streaked. It is also referred to as the “four-quadrant streak” or “four sectors” or “four-way streak” method. In this method, each plate is divided into four equal sectors and each adjacent sector is streaked sequentially. The sector which is streaked first is called the first sector or the first quadrant, and it has the highest concentration of inoculum. Gradually the second, third, and fourth quadrants will have diluted inoculum. By the time the fourth quadrant is streaked, the inoculum is highly diluted giving rise to isolated colonies following the incubation. Mostly, a discontinuous fashion of streaking is followed where the loop is sterilized at the end of each quadrant prior to streaking over the next quadrant. However, if the bacterial load is too small (or highly diluted), continuous fashion can also be used. In the latter, the loop needs not be sterilized at the end of every quadrant. Although being the most popular method, it limits us to use only one specimen per plate. If we try two or more specimens in a single 10 cm plate, this method is not suitable. 2. T-StreakingIt is another method of streaking where the agar Petri plate is divided into three sections and each section is streaked. Hence, this method is also known as the “three-sector streak” method. The media is divided into three sections by drawing a letter “T” and each adjacent section is streaked sequentially. By the time the final section is being streaked, the inoculum is diluted to the point to give rise to isolated colonies following the incubation. Mostly discontinuous fashion of streaking is followed; however, a continuous fashion can also be used in the very dilute specimen. As in quadrant streaking, it is difficult to culture two or more samples in a single 10 cm plate using this method. 3. Continuous StreakingIt is another commonly followed method where an inoculum is evenly distributed in a single continuous movement from starting point to the center of the plate. There is no need to divide the plate and sterilize the loop during the process. It is easy and quick; however, the problem is that we can use it only if the inoculum is either very diluted or we just have to propagate pure culture rather than isolate one. We can divide the 10 cm Petri plate into different sections (mostly 2 to 6), and in each section, we can streak different specimens following this method. Hence, it is used in the clinical laboratory to culture urine, sputum, pus, etc. if multiple samples have arrived at a single time. This will allow us to save media and get maximum output using a minimum resource. 4. Radiant StreakingIt is another method of streaking where the inoculum is first streaked at one edge and spread in vertical lines above the edge. Finally, the vertical lines are cross streaked diagonally. This method is suitable to propagate pure culture, and also in the case of a dilute specimen. There are other modified forms of streaking like: 5. Semi-quantitative StreakingIt is routinely followed in urine culture. It is a modified form of continuous streaking. In this method, a calibrated loop (usually a loop of 1 or 2μl) is used to streak a certain volume of the liquid specimen. A loopful of the specimen is streaked in a horizontal line in the middle of the Petri plate, and the specimen is spread all over the plate in a single continuous back and forth movement. This method allows us to approximately quantify the viable load (in a range, not an exact number) as well as get the pure culture in a single go. 6. Zigzag StreakingIt is another form of continuous streaking where a loopful of the specimen is streaked all over the plate in a zigzag pattern in a single continuous movement. It is commonly done to propagate the pure culture and culture them in large quantities. Requirements of Streak Plate MethodThis is a sterile tool used to streak the specimen over the surface of culture media. The tools used for streaking are cotton swabs, inoculating loop (both metal and plastic), toothpicks, and wooden or metal or plastic sticks/wires. The most commonly used one is inoculating loop (nichrome wire loop). (In this whole article, we will talk about inoculating loop.) Sample bacteria may be in the form of suspension, liquid broth, or colonies over solid media. The sample is picked by using an inoculating loop and transferred over the surface of fresh culture media to perform streaking. Specific culture media is used for the isolation and differentiation of suspected (or specific) bacteria. The culture medium is a solid agar medium that is pre-solidified before use.
A Bunsen burner is used to sterilize the loop and also to create a sterile zone around the flame. Besides, other chemicals, sterilizing materials, and laboratory apparatus are also required. Procedure or Protocol of Streak Plate MethodThe general procedure of the streak plate method can be summarized as:
The inoculating procedure is different according to the method of streaking, let us deal with each type: Quadrant Streaking Procedure
(if you are left-handed, hold the plate in your right hand)
(For ease, a beginner can draw two diameters intersecting each other diagonally at the back of the petri dish to divide the media into 4 equal sections)
(Be sure not to move the loop to the streaks in the first half of the first quadrant.)
(In a discontinuous fashion, the loop is sterilized after streaking each quadrant. In a continuous fashion, there is no need to flame the loop after streaking each quadrant. But, this is preferred only if the sample is very dilute.) T-Streaking Procedure
(For ease, a beginner can draw a letter “T” at the back of the petri dish to divide the media into 3 sections)
(Be sure not to move the loop to the streaks in the first half of the first quadrant.)
Continuous Streaking Procedure
Radiant Streaking Procedure
Semi-quantitative Streaking Procedure
Result Interpretation of Streak Plate Method
[Exception: in some cases where colony characters of two or more bacterial species are the same, all the colonies may look alike even if they are of a different individual.] Precautions during Streak Plate Method
Applications of Streak Plate Method
Advantages of Streak Plate Method
Limitations of Streak Plate Method
References
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