TIU Faculty of Science Medical Analysis Department General

TIU - Faculty of Science Medical Analysis Department General Microbiology Heshu J. Ahmed/Assist. Lecturer _______________________ Pure Culture techniques – Stage /1 st Semester Heshu. jalal@tiu. edu. iq https: //tiu. edu. iq/ 2020 - 2021

• WHAT IS A COLONY? • Different bacteria give rise to colonies that may be quite distinct to the bacterial species that created it. Therefore, a useful preliminary step in identifying bacteria is to examine a characteristic called colonial morphology • Colony morphology: is defined as the appearance of the colonies on an agar plate or slant. Ideally, these determinations should be made by looking at a single colony.

The following three characteristics are readily apparent whether you’re looking at a single bacterial colony or more dense growth, without the aid of any type of magnifying device. Texture: describes how the surface of the colony appears: smooth, glistening, mucoid, slimy, dry, powdery, flaky etc. Transparency—colonies may be transparent (you can see through them), translucent (light passes through them), or opaque (solid-appearing). Color or Pigmentation—many bacteria produce intracellular pigments which cause their colonies to appear a distinct color, such as yellow, pink, purple or red.

Two different types of bacterial colonies on an agar plate.

• Pure culture: is a culture that contains a single known species or type of Microorganism. • mixed culture contains two or more different bacteria. • If a bacterial culture is left in the same media for too long, the cells use up the available nutrients, excrete toxic metabolites, and eventually the entire population will die. • Thus bacterial cultures must be periodically transferred, or sub-cultured, to new media to keep the bacterial population growing. Aim: To identify the bacterial pathogens.

• Mixed culture need to be separate.

Determining pure colony: • The colonies should all look the same • they should have the same cultural characteristics. • In a mixed sample, individual colonies show different color. • To verify you have a pure sample use the sterile needle to isolate and grow on a new streak plate to make sure you see only one culture.

What is a contaminant? • A contaminant is defined as a microorganism that is supposed to be introduced into the culture during either specimen collection or processing and that is not pathogenic for the patient.

• The most commonly used method in the laboratory for isolating microbes is: - The streak plate and to a lesser extent - The pour plate. • Both methods rely on dilution of bacterial cells in a sample to the point at which a single cell can divide giving rise to a single pure colony.

Streak Plate technique (Quadrant Streak, loop dilution)

Types of streaking

Pour plate technique • one loopful from the mixed culture is transferred between 3 tubes contain nutrient agar(wormed to 50 C degree). • then poured to plates and allowed to solidify and then incubated. melted nutrient agar

Lab Exercise Streak plate method What is required for pure culture? • Sterile apparatus • Aseptic technique • Appropriate media

Step 1: Do the initial inoculation then FLAME the loop, let cool about 15 seconds.

Step 2: Use the sterile cooled loop and draw over the agar surface in the first section, flame and cool.

Step 3: Use the sterile cooled loop and draw of the agar surface in the second section. Note that you overlap with the first section a few times. Flame the loop and allow to cool.

Step 4. Use the sterile cooled loop and draw of the agar surface in the third section. Again overlap with the second section only. Now flame loop.

Step 5. place the plate in incubator.

Isolated Colonies