Practical 1 & 2: Introduction of Phytopathological Techniques

Activity 1

Objectives:

1. To learn how to prepare slides from fungal culture for observation in the lab.
2. To observe and determine morphological characteristics of fungus by using compound microscope.

Methods:

1. Preparation of slide from fungal culture

1. A clean slide and cover slip are prepared. The smallest cover slip is used to reduce the area that needs to be covered up tight.
2. A drop of lactophenol(adhesive medium) is put on the slip. Only a small drop is used to cover the area below the cover slip.
3. Inoculation needle is used to transfer fungal specimen.
4. The specimen is put on the adhesive medium and the specimen is arranged properly. If bubbles are trapped in the cover slip, new slide should be made.
5. If lactophenol sips out from below the cover slip, it should be dried up before it is closed tight. Tissue paper can be used for absorption.

Demonstration on how to prepare slides.	Cultures that will be observed. Aspergillus and Bacillus cultures.
Bacillus sp. will be stained using methylene blue.	Heating up inoculation loop.
Mixing bacteria sample and water using inoculation loop.	Heating the slide to dry up excess water and make the bacteria smear stay.
Staining the bacteria smear with methylene blue.	Spreading the methylene blue on the bacteria smear.
Washing away the excess methylene blue.	Heating the slide to dry up water.
Observing the prepared slide.

1. Preparation of slide from bacteria smear

1.  A drop of water is placed on a slide.
2. Inoculation loop is heated until red and cooled before using. A colony of bacteria from culture medium is touched using inoculation loop.
3. The bacteria and water  was mixed on the slide and spread out to form a smear.
4. The slide is heated slightly by moving it above the lamp and let it cool.
5. A drop of methylene blue was put on the smear and waited for one minute. The slide was rinsed with water and dried with tissue paper.

Aspergillus sp. will be stained with Lactophenol cotton blue (LCB).	Heating up inoculation needle.
Taking specimen using inoculation needle.	Staining specimen with LCB.

Result:

Magnification	Culture
40x	Aspergillus sp. with 40x magnifications
10x	sp. with 10x magnification

Black spots and tube-like structures can be seen through the microscope for Aspergillus sp. . Fine rod shape structures can be seen through the microscope for Bacillus sp. .The structures can be seen more evidently with higher magnification.

Activity 2

Objectives:

1. To learn how to prepare culture media and how to operate a laminar air flow cabinet in a lab.
2. To learn about various culture media used in lab to cultivate microbes in the lab.

 Method:

Culture media preparation

1. 250ml of Potato Dextrose Agar (PDA) is prepared by each group.
2. Ready-to-use PDA powder is weighed according to manufacturer’s instructions and poured into conical flask.
3. 250ml of distilled water is measured then poured into the conical flask with medium and shaken to dilute the PDA powder.
4. The prepared solution is sterilized in an autoclave at 15p.s.i., 121°C for 15-20 minutes. Media needs to be sterilized to avoid contamination by destroying all microorganisms.
5. After sterilization, the medium is allowed to cool down until the flask can be hold comfortably. The medium is poured into petri dishes in the laminar hood.

Potato Dextroses Agar (PDA)	Weighing PDA agar that will be used.
PDA powder after being shaken with distilled water.	Solution placed into autoclave to be sterilized.
Closing the autoclave.	Agar solution after sterilization and cooled.
Laminar air flow cabinet is sterilized with ethanol.	Mouth cap of agar solution glass bottle is heated with an alcohol burner.
Agar solution is poured into a petri dish.	Agar solution is left to be solidified.

Discussion:

In Activity 1, it has been identified that the tube-like structure is called conidiophore and the black spot is called the conidia. The function of conidiophore is to produce spore whereas conidia is the name of the Aspergillus sp. spore. Potato Dextroses Agar (PDA) is a natural media for isolating fungi. It is a general purpose medium for yeasts and molds that can be supplemented with acid or antibiotics to inhibit bacterial growth. It is the most common media for culturing fungi.

Conclusion:

Aspergillus sp. has morphological structures such as conidiophore which function is to produce spore and also conidia which is the name of its spore.