Microbiological techniques inoculation & growth monitoring.pptx

Microbiological techniques inoculation & growth monitoring.pptx

1. Microbiological technique: Inoculation & Growth monitoring Dr Showkat Ahmad Wani

2. What is Inoculation?

3. Tools and Materials Used Inoculating loop/needle – for picking up and transferring microbes. Bunsen burner – for sterilizing the loop and maintaining aseptic conditions. Culture media – agar plates, slants, or broth tubes containing nutrients. Petri plates and test tubes – sterile containers for growing microbes. Incubator – provides optimal temperature (e.g., 37°C for bacteria).

4. Steps of Inoculation Sterilize the inoculating loop in the flame until red hot. Cool the loop for a few seconds to avoid killing the microbes. Pick up the inoculum from the source culture. Transfer it gently to the fresh sterile medium (agar or broth). Flame the mouths of the tubes before and after inoculation. Close and label the tubes or plates properly. Incubate them at suitable temperature and time. 👉 Purpose: To obtain pure, uncontaminated, and healthy microbial growth.

5. Types of Inoculation Techniques There are several ways to inoculate depending on the purpose of the experiment. A. Streak Plate Method Used to isolate pure colonies from a mixed culture. The loop is streaked across the surface of agar in a zigzag or quadrant pattern. Each streak thins out the sample, leading to single, isolated colonies . 🧠 Example: Isolation of E. coli from a mixed bacterial sample.

6. B. Spread Plate Method A measured volume (usually 0.1 mL) of diluted microbial suspension is spread evenly on the agar surface using a sterile glass spreader (hockey stick). Suitable for counting colonies (viable count). 🧠 Example: Counting bacteria in drinking water samples.

7. C. Pour Plate Method In this technique, molten agar (at about 45°C) is mixed with the inoculum and then poured into a Petri dish. Colonies grow both on the surface and inside the agar. Useful for counting microorganisms and detecting anaerobes (those that grow without oxygen).

8. D. Stab or Deep Inoculation

9. E. Slant Inoculation The surface of a slanted agar tube is streaked with an inoculum using a loop. Commonly used for maintaining stock cultures or for biochemical testing . 🧠 Example: E. coli grown on nutrient agar slant for storage.

10. F. Broth Inoculation The loop carrying microbes is dipped into liquid nutrient broth . Growth appears as turbidity (cloudiness) or sometimes as pellicle or sediment . 🧠 Example: Testing antibiotic sensitivity in broth media.

11. G. Replica Plating

12. Monitoring Microbial Growth Once inoculated, we need to monitor the growth of microorganisms to study their behavior and response to different conditions. A. Visual Observation Turbidity in liquid broth → indicates growth. Colonies on agar → visible clumps of cells. B. Quantitative Methods Direct Microscopic Count – counting under microscope using a counting chamber. Viable Plate Count – counting colonies formed on agar plates. Turbidimetric (Spectrophotometric) Method – measures cloudiness (optical density). Dry Weight Measurement – measures mass of dried cells.

13. The Bacterial Growth Curve Microbes in a closed system (like broth) pass through four stages:

14. Safety and Aseptic Practices Work near a flame or in a laminar air flow cabinet . Wash hands before and after. Disinfect the work area before and after inoculation. Dispose of cultures properly (autoclaving or disinfectant).

15. Summary Table Technique Medium Type Purpose Streak plate Solid agar Isolate pure colonies Spread plate Solid agar Count viable cells Pour plate Molten agar Count and study anaerobes Slant Agar slant Maintain stock culture Stab (deep) Agar deep Study motility, oxygen use Broth Liquid Observe turbidity and growth Replica plating Agar plate Mutant/strain screening

16. Inoculation and growth monitoring form the foundation of microbiological work . By mastering these techniques, we can safely culture microorganisms, observe their behavior, and use them in medicine, food, agriculture, and biotechnology .

17. thank you