LAB 7 Streak plate technique.pdf

LAB 7 STREAK PLATE TECHNIQUE Asst. Prof. Dr.Saharut Wongkaewkhiaw The specimen obtained from the patient contained a large number of microbes that comprise the norm...

LAB 7 STREAK PLATE TECHNIQUE Asst. Prof. Dr.Saharut Wongkaewkhiaw The specimen obtained from the patient contained a large number of microbes that comprise the normal flora and pathogenic microorganisms. Colonies of the pathogenic species must be picked out of the mixed culture and grown in isolated pure culture. The microbiologist can then proceed to identify the isolated organism by examining its biochemical and immunological properties. Pure culture technique is critical to successful, accurate identification of microorganisms. The most common method to isolate individual cells and produce a pure culture is to prepare a streak plate. This method is a means to separate the microbial population physically on agar plate. Upon incubation, colonies will arise, and single cells (Figure 7.1) will be isolated from clinical samples.. Figure 7.1 A single colony of Escherichia coli (a green metallic sheen) on Eosin Methylene Blue (EMB) 20626104 General Microbiology for Dental Sciences 1  The streak plate technique is a fundamental method used in microbiology for isolating and identifying bacterial colonies from a mixed culture (Figure 7.2). This technique involves spreading a small sample of a bacterial mixture onto the surface of an agar plate in a way that results in individual bacterial cells being separated from each other. Figure 7.2 Mixed culture: streak plate isolation of M. luteus (bright yellow) and E. coli (greyish white). The importance of the streak plate technique in microbiology cannot be overstated, and here are several key reasons why it is essential: - Isolation of pure cultures: The primary purpose of the streak plate technique is to isolate individual bacterial colonies from a mixed culture. By streaking the bacteria in a specific pattern, it is possible to dilute the sample progressively, leading to the isolation of individual colonies on the agar plate. These isolated colonies are essential for further study, characterization, and identification of bacteria. - Identification and characterization: Microbiologists use isolated colonies obtained from streak plates to study and identify different bacterial species. This technique allows for the observation of colony morphology, color, size, and other characteristics, which can provide initial clues about the identity of the microorganisms present. - Quantification: The streak plate technique can be used to estimate the concentration of bacteria in a liquid sample. Researchers can also determine the viable bacterial cell in the original sample. 20626104 General Microbiology for Dental Sciences 2  - Purity assessment: Streak plates are used to assess the purity of a culture. A pure culture contains only one type of microorganism, whereas a mixed culture contains multiple types. By streaking bacteria onto an agar plate, microbiologists can visually inspect the colonies for uniformity. If a single colony morphology dominates the plate, it indicates that the culture may be pure. However, if multiple colony types are present, further purification may be necessary. - Preservation and storage: Isolated colonies obtained from streak plates can be preserved for future use. They can be stored in culture collections, such as agar slants or cryopreservation, for research, quality control, or diagnostic purposes. - Diagnostic applications: In clinical microbiology, streak plate techniques are used to diagnose infections. By isolating and identifying the pathogenic bacteria present in a patient's sample, healthcare professionals can determine the appropriate treatment and management strategies. In summary, the streak plate technique is a critical tool in microbiology that facilitates the isolation, identification, and quantification of bacteria. It is essential for various applications, from diagnosing infections to advancing our understanding of microbial biology. Figure 7.3 A single colony of Klebsiella pneumoniae (pinkish-red) on MacConkey (MAC) agar When primary isolation plates have been properly streaked, individual colonies can be picked up on an inoculating loop or straight wire and inoculated to fresh agar or broth media. 20626104 General Microbiology for Dental Sciences 3 These new pure cultures of isolated organisms are called subcultures. If they are indeed pure and do not contain mixtures of different species, they can be identified in stepwise procedures, as you will see in later exercises. Pure subcultures are also required for several experiment such as biochemical test and antimicrobial susceptibility testing. OBJECTIVE 1. To practice the streak plate technique for bacterial isolation 2. To isolate pure cultures from a specimen containing mixed bacteria 3. To understand the importance of pure culture for clinical microbiology laboratory MATERIALS - 24-hour broth culture of K. pneumoniae - 24-hour broth culture of E. coli - 24-hour mixed broth culture of K. pneumoniae and E. coli - Eosin Methylene Blue (EMB) - MacConkey (MAC) agar - Nutrient agar (NA) - Inoculating Loop PROCEDURES Streak Plate Technique 1. With a marking pen or pencil, label the bottom of agar plate with your name and the date. Be certain to write on only a small section of the plate (preferably near an edge) or you will not be able to examine the colonies that grow. 2. Make certain the contents of the broth culture tube are evenly mixed. 20626104 General Microbiology for Dental Sciences 4 3. Sterilize the loop and let it cool in the air. Figure 7.4 Diagram of plate streaking technique. The goal is to thin the numbers of bacteria growing in each successive area of the plate as it is rotated and streaked so that isolated colonies will appear in sections D and E. 4. Place a loopful of broth culture on the surface of the labeled NA, near but not touching the edge. With the loop pressed lightly against the agar surface, streak the inoculum back and forth over approximately one-eighth the area of the plate; do not dig up the agar (Figure 7.4, area A). Sterilize the loop again and let it cool. 5. Rotate the open plate in your left hand so that you can streak a series of four lines back and forth, each passing through the inoculum and extending across one side of the plate (Figure 7.4, area B). Sterilize the loop again and let it cool. 7. Rotate the plate and streak another series of four lines, each crossing the end of the last four streaks and extending across the adjacent side of the plate (Figure 7.4, area C). Sterilize the loop again and let it cool. 8. Rotate the plate and repeat this parallel streaking once more (Figure. 7.4, area D). 9. Finally, make a few streaks in the untouched center of the plate (Figure. 7.4, area E). Do not touch the original inoculum. 10. Incubate the plate at 37°C for 24 h. 20626104 General Microbiology for Dental Sciences 5 RESULT 1. Examine the incubated agar plate carefully. Compare your streaking with that illustrated in Figure 7.4 and discussed. Single Culture ………………………………………………………………………………………….. ………………………………………………………………………………………….. ………………………………………………………………………………………….. ………………………………………………………………………………………….. ………………………………………………………………………………………….. ………………………………………………………………………………………….. ………………………………………………………………………………………….. ………………………………………………………………………………………….. 20626104 General Microbiology for Dental Sciences 6 Mixed Culture ………………………………………………………………………………………….. ………………………………………………………………………………………….. ………………………………………………………………………………………….. ………………………………………………………………………………………….. ………………………………………………………………………………………….. ………………………………………………………………………………………….. ………………………………………………………………………………………….. ………………………………………………………………………………………….. 2. Questions - Why is obtaining a pure culture or single bacterial colony from a patient specimen crucial for diagnostics? - What experiments can be conducted after obtaining a pure isolated bacterial culture? Please provide at least two examples. 20626104 General Microbiology for Dental Sciences 7 REFERENCE 1. Morello, J. A., Granato, P. A., & Mizer, H. E. (2004). Laboratory manual and workbook in microbiology. McGraw-Hill Science, Engineering & Mathematics. 2. Sandle, T. (2015). Pharmaceutical microbiology: essentials for quality assurance and quality control. Woodhead Publishing. 20626104 General Microbiology for Dental Sciences 8