Lab report 1 &2: Introduction to Phytopathological Techniques

Introduction

Phytopathology is the studies of plant diseases. In theoretical phytopathology, it focuses on the nature and causes of a disease while as for the practical phytopathology, it focuses on the methods of protection against them. In a narrowest conception of phytopathology deals with the diseases of biotic origin caused by viroids, viruses, bacteria, fungi and parasitic plants. While in a broader sense they are studied Plant Pathology and diseases caused by nematodes and protozoa, resp. disorders caused by abiotic factors.

Objectives

1. To be able to know the techniques of preparation of slides under the microscope.

2. To know the types of culture media that can be used to for slide preparation.

3. To identify the methods of sterilization to prevent contamination from the surrounding when preparing slides.

Materials and Appertatus
Method:

Activity 1: Preparing Slide from Fungal Culture (Aspergillus sp.)

1. The needles are dipped in spirit to sterilize the needle from other unwanted microorganism. 

Figure 1.0

2. The inoculation needle is then heated up until it turns red using the spirit lamp to ensure further sterilization process.

Figure 1.2

3. A clean slide was prepared with a drop of Lactophenol cotton blue (LCB) which act as a staining agent.

Figure 1.3

4. After the inoculation needles is fully sterilized, the needle is used to transfer the Aspergillus sp. (fungal specimen).

Figure 1.4

5. The fungal specimen is then put on the drop of Lactophonel cotton blue and arranged properly.
6. A cover slip is gently placed on the sample.

Figure 1.5

7.The slide is then slowly placed above the spirit lamp to remove trapped buddles, concentration and also moisture from the slide sample.
8. Next, the slide is than put on a microscope to examine the Aspergillus sp. (Fungal).

Figure 1.6

Figure 1.7

Result and Discussion

Activity 1: Preparing Slide from Fungal Culture (Aspergillus sp.)

The Aspergillus sp. is observed through the microscope as shown in Figure 1.0, Figure 1.1 and Figure 1.2.

Figure 1.8

Figure 1.9

Figure 1.10

There was three fungal culture slide that was observed to ensure the best slide preparation for fungal culture. Based on Figure 1.0, the Aspergillus sp. from the slide that was prepared wasn’t able to observed the specimen fully which only the spores were seen through the microscope whereas in Figure 1.1, the specimen was able to observe the filamentous fungi such as the spores and the conidiophore. As for Figure 1.2, the specimen was able to be observed the filamentous fungi clear which the conidiophore, vesicle and also the spore of the specimen. 

Precaution steps

1. The materials used in the procedure of preparing the slides from fungal culture must be sterilized and disinfected fully to ensure that the result will only be focused on the fungi specimen rather than being contaminated by other environmental factors. The procedures of dipping the inoculation needle in spirit in one of the procedures of sterilization and disinfection (Figure 1.1). The spirit acts as agent to kill microorganism that if found on the inoculation needle.

2. Another precaution is to heat up the inoculation needles over the spirit lamp for further sterilization as shown in Figure 1.2. This procedure is called burning which the inoculation needle is heating up to the point it becomes red hot to kill microorganism and also to dry the needle from the spirit.

3. The precaution to prevent contamination from the environmental surrounding is to always ensure that the fungal culture is keep closed before and after transferring the specimen onto the slide. While taking the specimen, only a small opening must be made to transfer the fungal specimen must be done (Figure 1.12). Other than that, the procedures must be done quickly to prevent contamination from the environmental surroundings.

Figure 1.2

Activity 2: Culture agar media

1. The 250ml PDA was prepared by each group.
2. The ready-to-use PDA powder was weighted according to manufacturer’s instruction and put into a conical flask.
3. The 250ml of distilled water was measured, poured into the conical flask with medium and shook well to dilute the PDA powder.
4. The prepared solution was sterilized in an autoclave at 15 p.s.i, 120 ^oC for 20 minutes.
5. The medium was left to cool down after sterilization until the flask can be hold comfortably. The medium was poured into Petri dishes in the laminar hood.

Result and discussion 

Activity 2: Preparing culture media (Potato Dextrose Agar, PDA)

 

                                                                          Figure 2.0    

                                                                                           

 Figure 2.1

After the Potato Dextrose Agar (PDA) is completely sterilized and left cool until can be hold comfortably (Figure 2.0), the solution was distributed into several Petri dishes within the laminar hood (Figure 2.1).

PDA is a common and widely used medium to cultivate fungi or isolate fungi. Generally, it has been used as medium for culturing yeasts or molds and can be used with acid or antibiotics to inhibit bacterial growth. PDA is composed of dehydrated Potato Infusion and Dextrose that encourage luxuriant fungal growth. In addition, the agar is function as the solidifying agent. On the other hand, the other example of medium to culture or isolate fungi including, Corn Meal Agar (CMA), V-8 Juice Agar (VJA) and Malt Extract Agar (MEA). An example of medium for isolate bacteria is Nutrient Agar (NA).

Precaution steps

Figure 2.2

1.   The well mixed solution or medium need to be sterilized to avoid contamination by destroying all microorganism and bacteria. The sterilize method used during the lab practical is by using autoclave machine (Figure 2.2). Always make sure that the water level in the autoclave is sufficient and not too less or exceed the maximum level. Insufficient of water will overheat the machine. In addition, do not open the autoclave when there are pressures in to autoclave.

 

Figure 2.3

The cooled solution needed to be distribute into Petri dish within the laminar hood. The function of laminar hood or cabinet is to prevent contamination of semiconductor wafers, biological samples, or any particle sensitive materials. These hoods protect the samples from contamination by using the circulated air within the enclosed bench and drawn through a High efficiency particulate air (HEPA) filter. Generally, there are two type of laminar hood namely Horizontal Laminar Flow Hoods and Vertical Laminar Flow Hoods. They are differentiated by the airflow patterns for different purposes.

3.      

Figure 2.4

Before distribute the medium into Petri dishes, the bottle mouth should be sterile by heat to destroy any microorganism and bacteria. Contamination will waste time and money, and confuse the result.

 

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