Microbiology Practical (6) Inoculation of Culture Media Lab 6 (AUW) PDF

# **Microbiology practical (6)** ## Inoculation of culture media Before all check the medium for visual contamination or any change When inoculating, an aseptic technique must be used. This will: - Prevent contamination of cultures and specimens. - Prevent infection of the laboratory workers and...

# **Microbiology practical (6)** ## Inoculation of culture media Before all check the medium for visual contamination or any change When inoculating, an aseptic technique must be used. This will: - Prevent contamination of cultures and specimens. - Prevent infection of the laboratory workers and the environment. ### Aseptic techniques: - Using Bunsen burner flame sterilizes the wire loop, straight wire and metal forceps before use. - Flame the neck of specimen bottles, culture bottles, and tubes after removing and before replacing caps, bungs, or plugs. - When inoculating does not let the tops or the caps and tubes touch the unsterile surface. This can be done by holding the top or the cap in the hand. - Always use racks for holding tubes and bottles containing specimens or culture media. - Make slide preparation from specimens after inoculating the culture media. - Decontaminate the workbench before starting the day's work and after finishing. - Use a safety cabinet when working with hazardous pathogens. - Wear protective clothing, wash the hands after handling infected material, and never mouth pipette, eat drink, or smoke in the lab. ### Inoculation of media in Petri dishes: The technique used to inoculate media in Petri must provide single colonies for identification. It must also show whether the culture pure or mixed. - Dry the surface of the culture medium, by remove the lid of the plate and place this face upward on an indicator shelf. - Invert the base containing the medium and let it rest at an angle on the lid usually 30-40 mins incubation at 35-37°c, it is sufficient time to dry the surface of an agar plate. ### Inoculating technique: - Use a sterile loop or swab of the specimen, apply the inoculums to a small area of the plate. - Flame sterilized the loop, when cool spread the inoculums. ### Inoculation of slopes: To inoculate a slope use a sterile straight wire to streak the inoculums in a zigzag pattern. To inoculate a slope and butt medium use a sterile straight wire to stab into the butt first and then use the same wire to streak the slope in a zigzag pattern. **Figure 1.** Showing the steps of aseptic techniques: (a) Flaming a wire loop. (b) Inoculating a petri dish. (c) Flaming before putting back in a test tube. (d) Flaming before placing a new culture. ### Inoculation of stab media (deep): Use sterile straight wire to inoculate the stab medium. Stab through the center of the medium, taking care to withdraw the wire along the line of inoculums without making further stab lines. ### Inoculation of fluid media: Use sterile wire loop, straight wire or Pasteur pipette depending on whether the medium is a colony, a fluid culture, or specimen inoculate the fluid media. ### Labeling of inoculated media: Using a grease pencil, or marker pen, label with the date, and the patient's number, label the base of the culture plate. The slope labeled on the underside of the medium. A stab culture should be labeled above the level of the agar. When a plate anaerobically incubated it should be marked as (An O2) or when in carbon dioxide atmosphere it should be marked (CO2). ### Incubation of inoculated media: Incubate inoculated media as soon as possible, a delay in the incubation can affect the viability of the pathogens especially anaerobes. It can also increase the risk of plates contamination with small insects and dust. Un inoculated and inoculated media should be protected from sunlight. The temperature, humidity, and gaseous atmosphere should be suitable for the requirement of the microorganism metabolism. ### Temperature of incubation: The temperature at which the microorganism grows best is referred as the optimum one, temperature below which growth stops (not necessary resulting in death) is called minimum temperature, and that above which growth stop and death occurs is called maximum temperature. ### Humidity: An atmosphere too dry can affect the growth and viability of the microorganism. ### Gaseous atmosphere: Depending on its atmospheric requirements an organism can be described as: - An obligatory (strict) aerobe: Requires free oxygen to survive. - A microaerophilic organism: Growth best in the presence of only a trace of free oxygen. - An obligatory anaerobe: Survive only in the absence of oxygen. - A facultative anaerobe: Can live with or without free oxygen. - A carboxyphilic organism: Requires an atmosphere that contains carbon dioxide. ### Culturing of anaerobes: An anaerobic atmosphere is essential for the growth of strict anaerobe; anaerobe incubation also helps to differentiate pathogens and to isolate facultative anaerobe from specimens containing commensal. An anaerobic condition can be obtained suitable to the microbiology labs as: - Commercially produced sachets containing oxygen-removing chemicals. - Copper coated steel; wool to remove oxygen. - Reducing agents in culture media. **Figure 2.** Showing steps for streaking a petri dish: (A) Flame the loop. (B) Transfer inoculum to the plate. (C) Spread the loop across the plate. (D) The four stages for streaking method. (E) Shows the final colonies after incubation. **Figure 3.** Showing how to inoculate a slant with a straight wire: - **Top:** Inoculation of an agar slope (slant). - **Bottom:** Inoculation of a butt and slope. Use a straight wire to inoculate the butt first. **Figure 4.** Showing how to inoculate a stab media: - **Top left:** Inoculation of a slope (slant). - **Top right:** Inoculation of a butt and slope. Use a straight wire to inoculate the butt first and then the slope. - **Bottom:** Inoculation of a deep (stab).