Lab 6

Objectives

 1. To identify the fungal plant pathogen microscopically

2.  To identify the bacterial plant pathogen microscopically

Materials 

• Glass Slide

• Cover slip

• Glass Dropper Dispenser

• Inoculating Loop

• Inoculation

• Bunsen Burner

• Wooden Test tube Clamps

• Lactophenol Cotton Blue Solution

• Light Microscope

• Immersion Oil

• Tissue Paper

Procedures

A. Fungal pathogens identification

Preparing Fungal Specimen from Fresh Plant Tissue:

1. A sample was taken by gently scraping the diseased tissue with an inoculation needle.

2. The sample was carefully placed on a slide in a drop of lactophenol cotton blue dye.

3. A coverslip was placed gently with the help of a needle, ensuring it covered the specimen completely without air bubbles.

4. The slide was placed on the mechanical stage of the microscope and observed.

5. A sketch was made of what was observed in the spaces provided.

b) Preparing Fungal Specimen from Isolated Culture:

1. A sample was taken from the isolated culture with the help of an inoculation needle.

2. The sample was placed on a clean slide in a drop of lactophenol cotton blue solution.

3. A coverslip was placed gently with the help of a needle, making sure it covered the specimen completely without air bubbles.

4. The slide was placed on the mechanical stage of the microscope and observed.

5. A sketch was made of what was observed in the spaces provided.

6. A few areas of the slide were sketched with the 10X or 40X objective in place.

B. Bacterial pathogen identification

Simple Staining of Bacteria

1. A glass slide was gripped with wooden test tube clamps, and the surface of the slide was degreased with alcohol over a Bunsen burner. The slide was placed on a metal rack or staining stand with the degreased surface upwards, allowing it to cool down.

2. A small drop of water was put onto the slide (a well-degreased slide was wetted), and a small loopful of bacterial culture was mixed in the water. A thin suspension was formed. A smear was made with the needle of the inoculating loop, and the preparation was allowed to dry.

3. The preparation was fixed with heat over the Bunsen burner.

4. Methylene blue was dropped onto the fixed smear until it was fully covered, and the smear was stained for 1-2 minutes.

5. The smear was washed with tap water to remove excess methylene blue.

6. The slide was dried.

7. During microscopy, the 40x objective was used first, followed by the 100x objective lens, using immersion oil. A drawing was made of the observed microscopic field.

8. After finishing the microscopic observation, all used objective lenses were cleaned with benzene (alcohol was not used, as it could dissolve the lens adhesives).

Gram-Staining Procedure

1. A small drop of sterile water was placed on a clean microscope slide.

2. A part of a young colony was removed with a cold, sterile loop from the agar medium.

3. The bacteria were smeared onto the slide. The smear was just discernible.

4. The slide was air-dried and heat-fixed by passing it four times through a Bunsen flame, being careful not to overheat it.

5. The slide was flooded with crystal violet and left for 60 seconds.

6. It was rinsed under running water.

7. Excess water was drained off.

8. The slide was flooded with Lugol’s iodine and set aside for 60 seconds.

9. The slide was washed with 95% ethanol for 30 seconds.

10. It was rinsed with water.

11. The slide was blotted dry.

12. The slide was counterstained with safranine for 10 seconds.

13. It was rinsed with water and dried.

14. The slide was examined at 100x magnification using oil immersion.

• Gram-positive bacteria appeared dark purplish.

• Gram-negative bacteria appeared red.

Results

Discussion

Diagnosing plant diseases is a crucial step in managing crops and preventing major losses in agriculture. To make an accurate diagnosis, it’s important to identify the pathogen responsible for the disease and observe the symptoms on the affected plants. When it comes to fungal and bacterial infections, each requires a specific method for isolation and identification. For fungi, their unique structures like spores and spore-forming bodies help us determine the genus and species. Observing characteristics such as the shape, size, and color of these structures, along with the appearance of the fungus’s mycelium, helps in identifying the pathogen. To confirm the identification, experts often rely on scientific references and monographs. On the other hand, bacterial pathogens, due to their tiny size, require higher magnification for clear observation. By analyzing colony appearance, performing Gram staining, and using special media, we can begin to identify the bacteria. Bacteria are also categorized based on their biochemical and physiological properties, like the pigments they produce under UV light. Both fungal and bacterial pathogens need to be stained for a better view under a microscope. Fungi are often stained with lactophenol cotton blue, while bacteria use dyes like methylene blue or crystal violet to highlight their structures, especially the cell wall. For bacteria, using oil immersion with a 100x objective lens allows us to clearly see their small details. By combining these techniques with the right tools and knowledge, we can accurately diagnose plant diseases, leading to better strategies for managing them.

Conclusion
In conclusion, accurate diagnosis of plant diseases is essential for effective management and prevention of crop losses. By understanding the symptoms, isolating the pathogen, and utilizing microscopic examination, we can identify the causative agents, whether fungal or bacterial. Fungal identification relies on observing spore-forming structures and mycelium, while bacterial pathogens are primarily identified through colony morphology, staining techniques, and biochemical properties. Both fungi and bacteria require staining to enhance visibility under the microscope, with fungi commonly stained with lactophenol cotton blue and bacteria using methylene blue or crystal violet. The use of specialized media, such as selective agar plates, also aids in isolating pathogens and differentiating them from non-pathogenic organisms. Through these diagnostic methods, we are equipped to recognize the pathogens accurately, enabling the development of targeted disease management strategies to protect plants and ensure agricultural productivity.