Microbiology Lab: Cultivation of Bacteria PDF

University of Sharjah College of Health Sciences- MLS Department Microbiology Lab Cultivation of Bacteria  Aseptic transfer of bacteria and inoculation  Plate streaking  Culture and subculture technique...

University of Sharjah College of Health Sciences- MLS Department Microbiology Lab Cultivation of Bacteria  Aseptic transfer of bacteria and inoculation  Plate streaking  Culture and subculture techniques  Bacterial colonies description Material updated by Instructor: Dana Salahat, MSc. Objectives  Learn aseptic transfer technique.  To take cultures from selected areas of the environment to identify sources of contaminating microorganisms.  To isolate a pure culture from a specimen containing mixed flora  To obtain isolated colonies from streaked plate cultures and grow them as a pure subculture  To describe colonies morphology.  Aseptic Technique Main Purposes:  To ensure no contaminating organisms are introduced into culture materials during handling or inoculation.  To ensure that the organisms that are being handled do not contaminate the handler or others who may be present.  No contamination remains after you have worked with cultures.  The aseptic technique does reduce the likelihood of contamination. The general procedure for the aseptic technique includes:  Work Area Disinfection  Loops and Needles  Working with Culture Tubes Bacterial incinerator  Working with Agar Plates  Final Flaming of the Loop or Needle  Final Disinfection of the Work Area Bunsen Burner Definitions  Inoculation: Introduction of microbes into culture medium  Inoculum: Suspension of microorganisms  Colony: macroscopically visible collection of millions of bacteria originating from a single bacterial cell. Procedure for removing organisms from a broth culture with an inoculating loop. For Glass Tubes Removing organisms from a broth culture with an inoculating loop Inoculation Procedure If using a broth culture Sterile broth Inoculation: Transfer the inoculum to a sterile medium (solid or broth) Procedure for inoculating a nutrient agar slant from an agar plate Inoculation of nutrient broth from two different sources (plate culture and liquid culture)  Streaking Technique to Obtain Pure Culture  Good spacing between colonies on the plate is critical so that a single pure colony can be aseptically isolated from quadrant 4 and used for further testing and study. This will ensure that you are not working with a mixed or contaminated culture. Protocol of Streak Plate Method Different streak patterns that can be used to obtain isolated colonies Mixed Culture vs. Pure Culture Subculturing: to isolate a pure colony from a mixed culture  Culturing microorganisms from the environment Procedure: 1. Moisten the swab with sterile water  press it against the inner wall of the tube to drain off excess fluid. 2. Wipe the surface of the desired place  by rubbing and rotating it over an area. (Avoid letting the swab dry completely) 3. Inoculate an agar plate with the swab by rotating it over a small area near one edge. 4. Use a wire loop (inoculating loop) to streak out the plate to obtain isolated colonies. 5. Close, invert, and label the base of the plate with the sample location. 6. Place the plate in an incubator at 37° C overnight. 7. Examine the plate on the following day and record your observation. Bacterial Colony Morphology  Colony morphology is a method that scientists use to describe the characteristics of an individual colony of bacteria growing on agar in a Petri dish  It can be used to help to identify them.  Appearance of bacterial colonies  Size  Color  Overall shape  Elevation  Appearance of the edge of the colony Blood Agar MSA Klebsiella Catalán-Nájera, J. C., Garza-Ramos, U., & Barrios- Camacho, H. (2017). Hypervirulence and hypermucoviscosity: Two different but complementary Klebsiella spp. phenotypes? Virulence, 8(7), 1111–1123. https://doi.org/10.1080/21505594.2017.1317412