PRACTICAL 1-PIPETTE WORKSHOP AND ISOLATION OF FUNGI INTRODUCTION

A pipette is tool that had been widely used in laboratories to measure and transfer to volume. Having a good pipetting technique is very important because this is one of the precursor steps when doing the experiment. A poor pipetting technique may lead to inaccurate result or wrong diagnosis in our sample. They are 3 factors that affecting the accuracy of pipetting; temperature, density and altitude. In this practical, we were showing how to practice an excellent technique of pipetting by Sartorius Company, one of the companies that manufacturing the pipette. The Kingdom Fungi plays important roles in ecological and economic aspects. Plus, many plants could not grow without symbiotic fungi or called as mycorrhizae. Fungi also involves in making drugs and food. The Kingdom Fungi cause many plant and animal diseases such as ringworm, athletes foot and also damages plant smuts, root, stem rots and many more. Therefore, it is important to isolate the certain fungi in order to identify, classify and thus manipulate them for good purpose. In this practical, we are practising to isolate the fungi plus applying the serial dilution technique. The purpose of having the serial dilution is to decrease the concentration of the bacteria. This is very important because it is irrelevant or difficult to count the colony that has too many or to less in number. In addition, a single bacterium is deposited on an agar plate, will divide to form a colony. Each bacterium represents a colony-forming unit (CFU). The acceptable range for counting and recording is between 30-300 colonies in one agar plate. The serial dilutions start with adding of 1 ml of bacteria suspension or sample to 9 ml of distilled water to makes a 10-1 dilution. Then, add 1ml of 10-1 dilution to 9 ml of distilled water to make a 10- 2 dilution. These steps are keeping repeating until you obtained the 10-3, 10-4, 10-5 and many more. This is also called a dilution factor. Various types of media can be used to isolate the fungi. They are Sabouraud Dextrose agar (SBA), Potato dextrose agar (PDA), Brain-Heart Infusion Agar (BHI), Inhibitory Mould agar (IMA), Corn Meal agar (CMA) and many more. In this practical, we are using CMA to isolate the fungi.

OBJECTIVE
1. To familiarise and applied a good pipetting technique 2. To know the process of isolating the fungi from a given sample 3. Be able to observe and identify the colony of fungi

MATERIALS

Corn meal powder, Nutrient agar powder, dried soil sample and distilled water.

PROCEDURES
1. PIPETTING TECHNIQUES All students were attending the pipetting workshop given by Sartorius Company in Lab M2

2. PREPARING OF CORN MEAL AGAR Appropriate amount of CMA were prepared and added into 500ml bottle with 300ml of distilled water. The bottle was swirled and autoclaved for 20 minute at 121C and 15psi (1 bar) The sterilised agar was poured aseptically using laminar flow into the petri dish.

3. ISOLATION OF FUNGI 1g of dried soil sample were weighed and transferred into a test tube. Then, 9 ml of distilled water were added into the test tube and mix to give 10-1 dilution of soil sample. Next, the ten-times serial dilution of the soil solution were proceeded up to 10-5 The 100l of each 10-3 to 10-5 dilution were spread onto Corn Meal agar Then, the afar plates were incubate at 25 oC for 2-5 days Lastly, the fungal colonies are observed and the numbers in cfu/g are counted.

RESULT

1. Colony count using serial dilution-spread plate technique

2. Colony morphology

Dilution Serial Colour Dilution Factor 10-3 10-4 i White 10-5 10-3 ii iii i 10-4 ii iii i 10-5 Milky orange Milky yellow White Yellow White White

Number of colonies Elevati Colony-forming Shape/Fo Margin Surface Unit Density 1 rm 2 Averageon(cfu/g) appearan 20 5 2 23 5 0 22 5

ce Transluce nt Opaque Opaque Transpare nt Opaque Opaque Opaque

Irregular

Filamentou s Curled Entire Filamentou s Undulate Entire Entire

= 22 103 / 0.1 = 2.2Glistening 105 Convex 1

Irregular Circular Irregular Circular Circular Circular

Convex Raised Umbona te Convex Flat Convex

Glistening Dull Dull Dull Dull Dull

ii

Creamy

Irregular

Filamentou s

Umbona te

Dull

Opaque

DISCUSSION
The serial dilution technique is undergo before cultivate the fungi in media to dilute the concentration of fungi in sample. This is very important as the best range for colony count is around 30-300. Thus, we did not want the unreliable number of colony growth in agar as it will be difficult for us to count (this is what we called as TNTC which means too numerous to count). From the result above, the total concentration of fungi in a sample soil is 2.2 x 105 cfu/g. Even though the best range is around 30 to 300, but we cannot achieve the range. The reason is may be the period of incubation is not enough for fungi to growth because certain fungus takes a long period of incubation in order to growth in media. Thus, we selected the cfu in dilution factor 10 -3 because the colony forming unit in this tube is the nearest to the range that is 22. Normally, a media that contain source of carbon, nitrogen and vitamins are favourable for fungi to growth. The carbon sources are glucose or dextrose, fructose, mannose and sucrose. The nitrogen source can be obtained from peptone, yeast extract, malt extract, amino acids, ammonium and nitrate compounds. Fungi have natural deficiencies for vitamin but other organic nutrients such as glucose are often contaminated with vitamins and this will be enough to provide vitamin for fungal growth. The Corn Meal agar is used to culture the fungi because it is best media for species identification or morphological differentiation purpose. It allows developing of sexual characteristic structure of fungi to growth. Plus, it provides a good balance of mycelial growth and sporulation. It proves when much kind of colony morphologies obtained from the soil sample. We can use Potato Dextrose agar to cultivate the fungi plus it growth many type of fungi too. But, the problem arise when excessive mycelial growth at the expense sporulation, make it difficult to identify species of bacteria as the PDA is rich with nutrient and favourable many kinds of fungi. Therefore, we use CMA that has relatively weaker medium compared to PDA. In the left hand, other microorganisms such as bacteria might be growth in the agar and thus affecting the result obtained. Therefore, the problem was corrected by lengthen the period of incubation at about 2-5 days. This is because the bacteria have to compete with fungi for nutrient resources that deplete as the days increase. As the fungi will develop the network of mycelia, therefore the efficiency to absorb the nutrient is higher than bacteria. Plus, the media was incubating at 28oC in order to inhibit the bacterial growth as the optimum temperature for bacteria growth is 37 OC. By controlling the nutrient source and temperature factor, the process of fungal identification can be done easily. They are also factor that can inhibit the growth of bacteria such as controlling the pH of media or using the antibiotic agar.

QUESTION
1. Discuss the different types of media that are usually used for the isolation of fungi.

Instead of CMA, they are other media that used to isolate fungi. Sabouraud Dextrose Agar (SAB) is the universal medium for recovery and maintenance of various kinds of fungi that usually isolate in the clinical laboratory such as Candida albicans. Potato Dextrose Agar (PDA) is media that contain potato infusion and carbon sources such as dextrose that used to culture common fungi such as Saccharomyces cerevisae and moulds such as Aspergillus niger. Brain-heart Infusion Agar (BHI) is an enriched medium that uses to isolate coliotrophs yeast such as Cryptococcus neoformans from sterile specimens (example; cerebrospinal fluid). Inhibitory Mould Agar (IMA) is nutrient media containing inorganic salts, chloramphenicol and gentamicin. It is effective media to isolate fungi and inhibits bacterial growth. Rose Bengal Chloramphenicol agar is media to isolate yeasts and molds from foods and also an effective medium to inhibit bacterial growth due to the presence of chloramphenicol, a broad-spectrum antibiotic. Malt Extract Agar is an agar to isolate zygomycetes and Yeast ExtractPhosphate Media (YEP) is media to isolate Blastomyces dermatitidis and Histoplasma capsulatum from contaminated specimens.

CONCLUSION
They are many kinds of fungi that can be found in soil environment proves that the soil contains favourable condition to varieties of microorganisms. All this can be cultivated in media in order to study the morphological of varieties fungi. They are several of media that can be used to cultivate fungi. They are Corn Meal agar, Potato Dextrose agar, Sabouraud Dextrose agar and many more. This media have the selective properties to cultivate fungi without being affected by bacteria growth.

REFERENCE Fatima, N. (2012). Preparation of culture media for fungi Retrieved 9 December, 2012, from http://www.authorstream.com/Presentation/nuzhathfatima-729125culture-media-for-fungi/ Corn Meal Agar (7350) Retrieved 9 December, 2012, from www.neogen.com/Acumedia/pdf/ProdInfo/7350_PI.pdf Micheal J. Charlie, S. C. W. G. W. G. (2001). The Fungi (2nd ed.). Great Britian: Academic Press.

PRACTICAL 2-CHARACTERISATION OF FUNGI INTRODUCTION

Just as different plants and animals have various morphologies or shapes, so do fungi. The observing and characterising of fungi in plate cultures are very important since fungi were majorly first cultured on solid media such as agar media. In addition, this technique is very important since different fungi possesses different morphology and might be used to differentiate between others in further test. In this practical, 13 types of fungi cultures were given and their morphology was observed such as size, margin, elevation, colour, appearance and opacity. The further step is simple staining. The purpose of

applying this technique is to observe whether the certain fungi have these microscopic characteristics such as hyphae, mycelium, sporocarp and others. In addition, it is very important in order to detect the pathogenic that infect animals, human and plants and to find the better solution to overcome the diseases that caused by fungi.

OBJECTIVE
1. Able to characterise the fungal morphologies in aspect of colony formed and cell 2. To familiarise with several techniques to identify the morphology of fungi such as simple stain, iodine test, cotton blue stain and cellophane tape test.

MATERIALS
Pure cultures of microorganism such as Streptomyces griseus, Staphylococcus aureus, Saccharomyces cerevisae, Schizosaccharomyces pombe and specimens of filamentous fungi. Crystal violet dye, iodine solution, cotton blue dye and cellophane tape.

PROCEDURES
A. Simple stain (Bacterias test) 1. The loop is heated and allow to cool 2. A bacteria colony is picked up by the loop and thin smear of bacteria is created on the slide. 3. The slide is slightly heated to allow the bacterial smear to dry. 4. The dried bacterial smear is flooded with crystal violet for 1 minute. 5. After 1 minute, the stain is rinsed using tap water for 5 seconds. B. Iodine test (Yeasts test) 1. A loop of distilled water is placed on the slide and mixed with a loop of yeast. 2. The yeast is covered by cover slide and examined under low microscopic magnification. 3. A drop of iodine is dropped on the culture and yeasts morphologies are observed under high microscopic magnification. 4. Another yeast culture is prepared and a few drops of 2% Glucose solution are added to the culture.

5. Cell division activities are observed under high macroscopic magnification. C. Cotton blue stain (Filamentous fungi test) 1. A loop of fungis culture is picked from its plate and placed on the slide. 2. The slide is flooded with cotton blue stain. 3. The culture is covered with cover slips and observed under microscope. D. Cellophane tape test (Filamentous fungis test) 1. Upper surface of the fungi culture is sticked into cellophane tapes sticky surface. 2. Then, the cellophane tape is placed on the slide. 3. The culture is observed under low microscopic magnification. 4. Observation of hyphae and sporangia is recorded.

RESULT
1. The colony morphology

Lab el F1 F2 F3 F4 F5 F6 F7

Size

Colour

Shape/Fo rm Circular Circular Circular Circular Circular Irregular Irregular

Margin

Elevati on Convex Flat Convex Umbona te Convex

Small Small Small Big Big Big Big

White Creamy white Red Milky Green Green White

Undulate Entire Entire Entire Entire

Surface appeara nce Dull Smooth Glistenin g Glistenin g Dull Dull Rough

Density

Transluce nt Opaque Opaque Opaque Opaque Opaque Opaque

Filamento Flat us Undulate Umbona te

SE2 3 SE2 4

Big Big

Green Yellow

Irregular Irregular

Filamento Convex us Filamento Convex us

Dull Dull

Opaque Opaque

2. Cell morphology

Plate Label F1 F2

Types of microbes Bacteria Bacteria

Cell Morphology -Filament are extensively branched Have hyaline septate hyphae and conidiophores -Have grape-like structure -Large cocci -Unicellular -Rod in shape -Reproduce asexually by binary fission -Unicellular -Oval and elongated in shape -No hyphae are present -Reproduce by budding Form green brush-like cluster of conidia The conidia are round, unicellular and form an unbranching chain Multinucleated -Non septate hyphae -Have conidia -Septate hyphae -Filament - Have sickle cell, multicell macroconidia -Conidiophores, phialides, microconidia and macroconidia -Large -Filamentous -Non septate broad hyphae -Filamentous -Green brush-like structure -Have elongated and septate chains of spore

Identification Streptomyces griceus Staphylococcus aureus

F3

Yeast

Schizosaccharomyces pombe

F4

Yeast

Saccharomyces cerevisae

F5

Filamentou s fungi

Penicillium sp.

F6 F7

Filamentou s fungi Filamentou s fungi

Aspergillus sp. Fusarium sp.

SE23

Filamentou s fungi

Zygomyces sp.

SE24

Filamentou s fungi

Penicillium sp.

RESULT
In this practical, we are observing and differentiating between filamentous bacteria, filamentous fungi and yeast in aspect of colony and cell morphology. From the observation, the plate label F1 and F2 are filamentous bacteria. The plate label F3 and F4 are yeast. The plate labelled F5, F6, F7, SE23 and SE24 are filamentous fungi. The filamentous bacteria is differentiate between other two (filamentous fungi and yeast) by the way we observing it under microscope. Usually, we have to use high magnification or ocular that is 40X and 100X (with immersion oil) in order to observe the bacteria. Unlike fungi, low magnification power can be used to observe them. In addition, we can observe them by looking the size of filaments between filamentous bacteria and fungi using the same magnification power. The size of bacterial filaments is smaller than fungal filaments. The filamentous bacteria are classified as Actinomycetes, the major subdivision of the Prokaryotae. They are also transitional forms between bacteria and fungi. In bacteria form, they have prokaryotic nuclei and susceptible to antibiotics either first, second or third generation of antibiotics. Like fungi, they form filaments called hyphae that show slightly similar to the hyphal forms belong to fungi. They filamentous fungi are differing from fungi by the composition of their cell wall. The filamentous fungi or Actinomycetes do not have chitin and cellulose. In contrast, these properties are present in the cell wall of fungi. The Actinomycetes are widely found in soil and also presence in symbiotic nitrogen fixation association. Some of them are also form close association with plants by supporting the plant growth and act as biocontrol agents. Most of them are anaerobic bacteria but a few grow in anaerobic condition. Filamentous fungi or moulds and yeast are belonging to Kingdom Fungi, they are differ with Actinomycetes. Mould can be easily seen with naked eye; an aspect that made them different from the other two. Moulds have the branching structure of filaments called hyphae that multiply through vegetative reproduction. In addition, they usually presence in form of tubular hyphae that contain a large number of nuclei colonies. Yeast is differ from moulds in aspect of structure. Yeast possesses unicellular structures. Most of them reproduce asexually either budding or binary fission. The filamentous fungi (mould) and yeast can be differentiate in the several aspects such as structure, method of reproduction and appearance. From structure aspect, moulds are multicellular with tubular and filamentous hyphae structure while yeast is mostly unicellular and existing either individually or with buds on them. Moulds can reproduce sexually or asexually ( airborne spores) while yeast reproduce through budding or binary fission. In the aspect of appearance, moulds are threadlike and come in a wide variety of colours and hues while yeast appear in round or oval-shaped with dull coloured.

CONCLUSION

The morphological study of colony and cell for filamentous bacteria, filamentous fungi and yeast are important in order to classifying them into appropriate and correct group. In addition, they are several aspects that can be used to differentiate the three of them. They correct classification is very important because the information of them can be used in future for the purpose of specific or critical study.

REFERENCE Micheal J. Charlie, S. C. W. G. W. G. (2001). The Fungi (2nd ed.). Great Britian: Academic Press. Actimycetes & Actinomycetales. (2012). Retrieved 9 December, 2012, from http://www.gopetsamerica.com/bio/bacteria/actinomycetes.aspx Davidson, C. (2000). Actinomycetes & filamentous fungi Retrieved 8 December, 2012, from http://www.bio.net/bionet/mm/microbio/2000February/017761.html Shukla, I. C. (2010). Mold vs Yeast Retrieved 8 December, 2012, from http://www.buzzle.com/articles/mold-vs-yeast.html Chandoliya, C. S. (2009). Fungi Biological Divesity (1st ed.). New Delhi, India: Cyber Tech Publication.

PRACTICAL 3

: FUNGAL INHIBITION AND ANTIBIOTIC PRODUCTION : MAKING TEMPE

INTRODUCTION
The testing and identifying certain chemicals that has the ability to inhibit the fungal activity is important to overcome the disease and problem cause by pathogenic fungi. Different fungi species have different chemicals that have ability to inhibit their activity as each species has different morphology and characteristics. In this practical, we are using potassium dichromate and nystatin to inhibit different fungal species. Nystatin was the first polyene macrolide antibiotic founded by New York State Public Health Laboratories. It functions on disrupting the plasma membrane function on fungi. It is used topically against dermatophytic fungi and orally against fungal infections in the gut. In 1928, Sir Alexander Fleming founded that Penicillium notatum have the capability to kill Gram-positive bacteria. The compound then named as penicillin and was the first member of beta lactam class (four atoms cyclic amide) of antibiotics to be discovered. We are using to types of bacteria; Escherichia coli and Staphylococcus aureus in order to test the capability of different type of fungi to produce antibiotic. Tempe is a solid fermented soy bean product that is widely consumed in Indonesia. In recent years, there is considerable interest in Western such as USA in developing tempeh as an alternative protein source. The fungi used in making tempeh are Rhizopus oligosporus and takes at about 2 days to complete the process.

OBJECTIVE
1. To study the effectiveness of certain chemicals (antifungal) in inhibiting the growth of certain fungi. 2. To study the effectiveness of certain chemicals (antibiotics) produced by certain fungi in inhibiting the growth of certain bacteria. 3. To produce tempe from fungi Rhizopus oligosporus

MATERIALS
Fungal inhibition

Nystatin, Potassium dichromate, isolated fungi from previous practical (Penicilium sp., Aspergillus sp., Fusarium sp.), Nutrient Agar, microbial disk. Antibiotic production E.coli culture broth, P. aeruginosa culture broth, S. aureus culture broth, isolated fungi from previous practical (Penicilium sp., Aspergillus sp., Fusarium sp.), Novobiocin disk, Nutrien Agar. Making tempeh Soy beans, tap water, inoculum (sporulating culture of R. oligosporus), banana leaves, newspaper.

STEP AND PROCEDURES

A. Antifungal test 1. Each of the filter paper (act as empty microbial disk) is diffused with 100 l of 0.05mg concentration of Nystatin and Potassium dichromate respectively. 2. Some area of the isolated fungi ( Penicilium sp.), which is prepared from previous practical or already in the lab, is plugged out using the sterile straw including its bottom agar. It is placed in the middle of the Nutrient Agar. 3. The Nystatin and Potassium dichromate disk are placed on the left and right side of the fungi respectively. 4. The agar plates are incubated at 25oC for 2-5 days. 5. The presence of zone of inhibition around both disks is observed. The diameter of the zone is measured. 6. Procedures 1-5 are repeated for fungi Fusarium sp. and Aspergillus sp.

B. Antibiotic production 1.A sterile cotton swab is prepared and its cotton is dipped into E.coli culture broth. 2.A lawn of E.coli culture is performed on the agar. 3.The plate is divided into four regions for placing of the Novobiocin disk, Penicilium sp., Aspergillus sp. and Fusarium sp. plug respectively on the agar. 4. The agar plates are incubated at 25oC for 2-5 days.

5. The presence of zone of inhibition around fungal plug is observed. The diameter of the zone is measured. 6. Procedures 1-5 are repeated for agar plates with a lawn of P. aeruginosa and S. aureus.

C. Making tempe

1. Soy beans is soaked overnight in tap water and allowed to drain. 2. The beans are dehulled by hand. 3. Soy beans are placed in glass beaker and an excess of water is added. 4. The beans are brought to boil and are boiled for 5 minutes. 5. The beans are drained thoroughly / put into new beaker. 6. The beans are allowed to cool by stirring continuously with spatula. 7. Inoculum (sporulating culture of R. oligosporus) is added and the whole mass is mixed thoroughly. 8. The inoculated beans is placed onto banana leaves and wrapped with newspaper. 9. The wrapped inoculated beans are incubated in the dark at 30 oC. 10.Tempe is examined and the fermentation is assessed after 24 hours. 11.The tempe is readied after 48 hours of incubation and can be stored at 10oC for up to a week or deep frozen. The product is a firm white cake.

RESULT
A. The antifungal test Type of antifungal used Nystatin Potassium dichromate Zone of Inhibition (in mm) Fusarium sp. Penicillium sp. No inhibition No inhibition No inhibition No inhibition

Rhizopus oligosporus No inhibition No inhibition

B. Antibiotic production Type of fungus Zone of inhibition (in mm)

used for antibiotic activity NOVOBIOCIN (control) Aspergillus sp. Penicillium sp. Fusarium sp. C. Tempe Period of incubation 2 days (48 hours)

Escherichia coli No inhibition No inhibition No inhibition No inhibition

Staphylococcus aureus 26.0 11.0 15.0 10.0

Pseudomonas aeruginosa No inhibition No inhibition No inhibition No inhibition

Obseravation Soybean are clumped with white substance covering them The colour of soybean is constant (light brown) Slight odour produced

7 days

The intensity of white colour increased The colour of soybean turns brownish Strong odour produced

DISCUSSION
Two types of antifungal are used in this practical to observe and determine the effectiveness of antifungal to inhibit the fungal growth; nystatin and potassium dichromate. The result is no inhibition zone formed on Penicillium sp., Fusarium sp. and Aspergillus sp. agar either using nystatin or potassium dichromate. Nystatin is an antifungal agent in class polyenes. It is the first polyene macrolide antibiotic found. It is used tropically against dermatophytic fungi and orally against fungal infections in the gut. Nystatin disrupts the plasma membrane of fungi in such way that potassium ions are lost from the cell and protons will eventually enter the cell. This condition leads to the acidification environment in cell and thus, causes the cell to die. It works to different species of Candida, Aspergillus, Histoplasma, Coccidioedes, Cryptococcus and Saccharomyces. Potassium dichromate is generally used to remove fungi in water especially for water treatment and keep the healthy environment for ornamental fish. It is relatively mild chemical oxidants, soluble, non-toxic, do not decompose and do not produce developmental abnormalities. Theoretically, nystatin and potassium dichromate should create the zone of inhibition toward three type of fungi used. But, the result is vice versa. They are possibilities that being derived upon this false result. First, the fungi might create or have the resistance towards these two antifungal as nystatin is the first generation antifungal found. Nowadays, many fungi have the ability to create their resistance towards many antifungal. This is because long period of expose toward same antifungal give them time to

adapt with it and thus, resist the antifungal. Second possibility that arise is the concentration of antifungal used. For nystatin, the minimum inhibitory concentration for fungi is in the range 1.56 to 6.25 g/ml. The provided concentration in this practical is 0.05g/ml. Therefore, the concentration provided is not in the range that being state. Third possibility is the way we undergo the process of making the antifungal disk. The heating of antifungal disk may affect the concentration and the effectiveness of the antifungal itself. For the test of antibiotic production from different fungus, only bacteria lawn of Staphylococcus aureus show the zone of inhibition towards 3 type of fungi used; Aspergillus sp., Penicillium sp. and Fusarium sp. in addition, we are using Novobiocin antibiotic that act as a control antibiotic. Novobiocin is an aminoglycoside antibiotic that works by binding to the bacterial 30S ribosomal subunit that lead to the vital of the bacteria due to inability of the bacteria to synthesize protein. The Novobiocin is produced by Streptomyces nivens and usually used to treat infections caused by gram-positive bacteria. The fungi Aspergillus sp. have the ability to produce antibiotic 3-chloro-5, 2-dihydroxy-3, 7, 8-trimethoxy flavone. This antibiotic had been found from the fungi Aspergillus candidus under an aerobic condition. This antibiotic has the bactericidal and fungicidal properties. Penicillin, a very general antibiotic in world is produced by fungi Penicillium sp. Penicillin are categorised in -lactam antibiotics and used to treat bacterial infections mainly caused by the Gram-positive organisms. Penicillin works by inhibit the formation of peptidoglycan cross-link in the bacterial cell wall. It did not give the major effect in degrading the bacteria but eventually the bacteria will die due to the osmotic pressure process. This process is called cytolysis. The third fungi, Fusarium sp. produce an antibody that have C 22H31NO4 chemical formula. This antibiotic appear white in colour with 65 oC to 66oC degree of melting point. It inhibited some gram-positive bacteria such as Bacillus subtilis, Mycobacterium phlei and Staphylococcus aureus. Based on the result, there are no inhibition zone formed on bacterial lawn Escherichia coli and Pseudomonas aeruginosa. This is because these two bacteria had already create the resistant towards antibiotics produced by Aspergillus sp., Penicillium sp., and Fusarium sp. Plus, they have also create resistant toward Novobiocin. In contrast to bacterial lawn Staphylococcus aureus, all of the fungi show the reaction toward the bacteria by and form the zone of inhibition. Penicillium sp. show the largest zone of inhibition; 15mm followed by Aspergillus sp.; 11mm. Fusarium sp. have the lowest zone of inhibition that is 10mm. It can be conclude that Penicillium sp. is the best antibiotic production to against bacteria Staphylococcus aureus even though the penicillin is the classic antibiotic. In this practical, we are also being familiarised with the making of tempeh. The fungi Rhizopus oligosporus are used and it takes at about 2 days for

completion. This is to ensure that the fungi are fully attached and hold the soy bean together. In other words, the beans are knitted together by a mat of white mycelia produce by the fungi.

QUESTION
1. Name two examples of antifungal medications. Why are some fungal diseases difficult to treat? The examples of antifungal medications are imidazole (class Azoles) and Terbinafine (class Allylamines). Fungi are eukaryotic organisms like human and therefore it is difficult for us to target them without give any harm to human body. The nature of fungi also is one of the reasons on why to treat some fungal diseases. Usually, the nature of fungal infections occurs in old, sick or immunosuppressed persons. Thus, it is difficult for the body to fight the infection as the immune system become weaker even though antimicrobial treatment being given.

CONCLUSION
They are many types of antifungal used to treat the growth of fungi especially the pathogenic fungi. Other than that, the fungi have the ability to produce the antibiotic that gives benefit to human in order to fight bacterial infection. Fungi also benefit in production of food such as the making of tempeh.

REFERENCE

Micheal J. Charlie, S. C. W. G. W. G. (2001). The Fungi (2nd ed.). Great Britian: Academic Press.

Kavanagh, K. (2011). Fungi-Biology and Applications (2nd ed.). New Delhi, India: John Wiley & Sons Limited Publication. Nystatin preparation. Retrieved 8 December, 2012, from www.sigmaaldrich.com/etc/medialib/docs/.../2/.../n1638pis.pdf Balhorn. (1982). Fish egg fungicide Retrieved 8 December, 2012, from http://www.patentgenius.com/patent/4331660.html Active ingredient: Novobiocin - Brands, Medical Use, Clinical Data (2006). Retrieved 7 December, 2012, from http://www.druglib.com/activeingredient/novobiocin/ Richards, M., Munden & Jeffery Edward. (1972). ANTIBIOTIC PRODUCTION USING A STRAIN OF ASPERGILLUS CANDIDUS Retrieved 8 December, 2012, from http://www.freepatentsonline.com/3632477.html H. R. Burmeister, G. A. B., R. F. Vesonder & C. W. Hesseltine. (1974). Antibiotic Produced by Fusarium equiseti NRRL 5537 Retrieved 8 December, 2012, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC429026/ Penicillin. (2012). Retrieved 7 December, 2012, from http://en.wikipedia.org/wiki/Penicillin Fungal disease vs bacterial disease. (2007). from http://www.biologyonline.org/biology-forum/about12237.html?hilit=Pear Tempeh. (2012). Retrieved 7 December, 2012, from http://en.wikipedia.org/wiki/Tempeh

PRACTICAL 4-MYCORRHIZA AND LICHEN INTRODUCTION

Most plants are able to grow well in certain area because of the presence of specialized fungi in the soil which form close associations with their roots. The

association is called as mycorrhizae. The mycorrhizae can increase the uptake of plant nutrients such as phosphorus and nitrogen even though the nutrient state is low in certain soil. They are two mechanisms that used by the fungi to assist the uptake of nutrient by plants from dilute concentrations. First, the hyphae extending into the soil from the root and search for the nutrient and then being absorbed by the root through active transport. Second, the fungi act as a reservoir of nutrient that can be used for future utilization. They are several different types of mycorrhiza such as Vesicular-arbuscular mycorrhizas (VAM), Orchidaceous mycorrhizas, Ericaceous mycorrhizas and ectomycorrhizas. Lichens are one type of symbiotic relationship species. Like fungi forming mycorrhiza and endophytes, lichen fungi depend for their carbon and energy supply on photosynthetic organisms. Lichens are unicellular filamentous green algae and in the remainder they are cyanobacteria or a combination of these and green algae. Lichens can live on exposed surfaces such as rock wherever there is light and water.

OBJECTIVE
1. Be able to observe and identify the mycorrhiza that present in plat, orchid and grass roots 2. Be able to identify and differentiate three different growth form of lichen in surroundings

MATERIAL
Grass roots, orchid roots and banana plant roots. Potassium hydroxide, tryphan blue/lactophenol blue dye.

PROCEDURE
Mycorrhizae 1. Roots are cut into 1 cm pieces and boiled for 5 minutes in 10% KOH. 2. The roots are rinsed with tap water. 3. The roots are stained by boiling with 0.05% tryphan blue/ lactophenol blue for 5 minutes and rinsed with water. 4. The roots are sliced into thin sections, placed onto a clean slide and examined under microscope. Lichen 1. Students are divided into groups and walked around laboratory buildings and surroundings to search for lichen. 2. The type of lichen found is examined and drew.

3. The type of lichen found is classified and differentiated between the types crustose, foliose and fruticose.

RESULT
1. The mycorrhiza diagram (plant roots, grass roots and orchid roots)

2. The lichen diagram

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/ /

DISCUSSION
Mycorrhiza is a symbiotic or mutualistic association that form between the roots of most plant species and fungi. It derived from Greek term; myco=fungus and rhiza=root. At about 95% of all plant species have these kinds of associations. The presence of mycorrhiza is very important in order to support the plant growth. They associate with plant in aspects of sugar-water or mineral exchange, resist to disease and drought, colonise the barren soil and resist to toxic. They are several different types of mycorrhiza such as Vesicular-arbuscular mycorrhizas (VAM), Orchidaceous mycorrhizas, Ericaceous mycorrhizas and ectomycorrhizas. Lichen is a mutually beneficial partnership between fungi and autotrophic unicellular algae. In other word, it is symbiotic life form between mycobiont and photobiont green alga. They are three main growth form of lichen. It can be differentiated by the shape of thallus, arrangement of cortex and attachment to substratum. The three main growth forms are crustose, foliose and fruticose. Crustose has crust-like appearance with more or less prostrate. It attached over entire lower surface and cannot be removed intact from substratum. It always lacks a lower cortex. Crustose has no lobes and rhizines (rootlike attachment structures). Foliose derive from the Greek word; foli=leafy. It has flattened low and lobed appearance. It attached over part of lower surfaces and there may be rootlike rhizines. The foliose thallus composed of branched, leaflike lobes and have a lower cortex. The colour of foliose varies from pale green, yellowish green, dark green and many more depend on species. Fruticose has bushy, strap-like or hair like appearance. It attached to substratum at a single point or not at all. The thallus are roughly cylindrical in shape. Fruticose anchored at the base to the substrate with their hollow stalks thallus. Fruticose mainly can be found hanging from tree branches.

QUESTION
1. Why are mycorrhizae important to plants? Mycorrhiza are important in increasing the plant nutrient supply. They extending the volume of soil accessible to plants. In addition, mycorrhiza acquiring nutrient forms that would not normally be available to plants. Some Ericaceous mycorrhizas and ericoid fungi have the capacity to breakdown phenolic compounds in soils which can interfere with nutrient uptake. Furthermore, the root colonisation by Ericaceous mycorrhizas and Vesicular-arbuscular mycorrhizas can provide protection from parasitic fungi and nematodes.

2. What are the habitats of lichens? Lichen has wide range of habitat. They can leave on rock, soil, bark and wood. The characteristics of lichen that can tolerate wide range of temperature and tolerate of alternating wet-dry conditions made them to be one of the successful organism on Earth. In addition, they will in dormant form if the environment is dry and very sensitive to air pollution.

CONCLUSION

Mycorrhiza is the symbiotic interaction between fungi and plant roots. It gives a lot of benefit especially in supporting plant growth. Lichen is the mutualistic interaction between alga and fungi. It has widespread habitat and plays an important role as the air pollution indicator, involve in nitrogen fixation and as the animal food source.

REFERENCE

Micheal J. Charlie, S. C. W. G. W. G. (2001). The Fungi (2nd ed.). Great Britian: Academic Press. Chandoliya, C. S. (2009). Fungi Biological Divesity (1st ed.). New Delhi, India: Cyber Tech Publication. Mycorrhiza. (2012). Retrieved 8 December, 2012, from http://en.wikipedia.org/wiki/Mycorrhiza Armstrong, W. P. (2004). Fruticose Lichens Retrieved 8 December, 2012, from http://waynesword.palomar.edu/pljan98c.htm

PRACTICAL 5- ALGAE AND PROTOZOA INTRODUCTION

Algae refer to a large and diverse assemblage of eukaryotic organisms that contain chlorophyll and carry out oxygenic photosynthesis. Algae are either unicellular or colonial (occurring as aggregates of cells). Most algae are green in colour but there are also algae that appear brown or red because of their pigments such as carotenoids that mask the colour of chlorophyll. Protozoa are unicellular eukaryotic microorganisms that lack cell walls, generally colourless and motile. Protozoa feed by pinocytosis and phagocytosis. Major groups of protozoa include flagellates, amoebas, ciliates and sporozoans.

OBJECTIVES
1. Be able to observe and identify the algae in given water sample 2. Be able to observe and identify the protozoa in given water sample

MATERIAL
A water sample from different location; Taman Jaya lake, Varsity lake, 12 th Residential college lake and one of the river in Seremban, Negeri Sembilan.

PROCEDURES
1. A drop of water sample is placed on a clean slide and protozoa and algae are observed. 2. A tiny amount of 10% methyl cellulose is added to the water sample using a toothpick, covered with coverslip and observed protozoa and algae under microscope.

RESULT

Scenedesmus sp.

Cercomonas sp.

Paramecium sp.

Diatom sp.

Phacus sp.

Cylindriospermium sp.

DISCUSSION
Algae have no formal taxonomic standing. Like protozoa, it is also indicate a polyphyletic, noncohesive and artificial assemblage of oxygen-evolving, photosynthetic organism. Algae did not have roots, stems, leaves and well defined vascular tissues. In addition, they did not form embryos and their reproductive structures consist of cells that are potentially fertile and lack sterile cells covering or protecting them. Algae are usually occurring in dissimilar forms such as microscopic single cell, macroscopic multicellular loose or filmy conglomerations.

Protozoa is an informal collective term used in referring to a polyphyletic assemblage of animal-like protists. Protozoa includes more than 92000 species inhabit the marine, freshwater and terrestrial soil environment. At about 25% of the total species recorded participate in symbiotic associations and majority of them are pathogenic that infect vertebrates and invertebrates and some of them parasite to plants. In the marine protozoan, the osmoregulation in water is done by the contractile vacuoles that pump hyposmotic urine from the cytoplasm back into the environment. In many protozoans, the contractile vacuole is a conspicuous feature of the cell.

QUESTION
1. How do protozoa move? Protozoa move by using the cilia, flagella and pseudopods. As example, amoebas move by extending their parts of cell (pseudopods).

2. What is algae fuel? How can we produce it? Algae fuel is a biofuel which is derived from algae. Algae have the ability to capture the energy of sunlight and can be transform into transportation fuel. To produce the algae in the large scale (commercially), first we have to culture the algae on agar plate. Then, after we had seen the green colonies formed on agar, we can grow the algae in a small flask. After that, the algae are being taken to the algae farm in order to grow them in a large scale. By this, we can isolate the algae fat (also called as lipid) and the lipid has quite similar properties to the diesel fuel.

CONCLUSION
Protozoa and algae are the collective term to describe a polyphyletic organism. Protozoa refer to the animal-like protists while algae refer to photosynthetic organism. The algae have the good benefit in future for producing the biofuel that can be a source of new fuel.

REFERENCE Protozoans Retrieved 9 December, 2012, from http://www.cbv.ns.ca/mchs/diversity/ProtozoansPage1.html The Microbe world. Protozoa Retrieved 9December, 2012, from http://www.edu.pe.ca/southernkings/microprotozoa.htm Algae fuel. (2012). Retrieved 9 December, 2012, from http://en.wikipedia.org/wiki/Algae_fuel Gualtieri, L. B. P. (2006). Algae-Anatomy, Biochemistry and Biotechnology (1st ed.). NY, USA: Taylor & Francis Group. PRACTICAL 6- DEMONSTRATION ON VITEK 2 (MICROBIAL ID SYSTEM) INTRODUCTION
In the last practical, we are being demonstrated by the BiOLOG TM Company about how to use the Biolog Gen III Microbial Identification System. It is one of the advanced identification system that can identify around 2686 species of microbes and claim to be the highest identification devices in the world. The species that identify includes 1350 species from aerobic bacteria, 361 species from anaerobic bacteria, 267 species from yeast and 708 species from filamentous fungi.

OBJECTIVE
1. To familiarise using the Biolog GENIII Microbial Identification System

MATERIALS
Pure culture and the Biolog GENIII Microbial Identification Technology system

PROCEDURES
1. The bacteria culture (was prepared before) is picked and released into the Inoculation Fluid using sterile cotton swab.

2. Then, by using the same sterile cotton swab, the wall of the Inoculation Fluid tube was being rubbed in order to crush any cell that clumps on the wall. 3. Next, the density of the fluid was checked using Biolog Turbidimeter in order to get the suitable cell density (around 93-98%) 4. After that, the inoculate fluid is transferred into pipette reservoir before transferred into the GEN III Microplate (100l for each well). 5. The plate then incubated in an incubator for overnight in 33 oC temperature.

6. Lastly, the incubated plate was read using Biology Microstation Reader and the result obtained was interpreted.

RESULT
As it is a demonstration practical, we did not obtain any result.

Biolog GENIII Microbial Identification System

DISCUSSION
The BiOLOGTM GENIII Microbial Identification System has many advantages. There are no gram stain needed, no pre-tests and no follow-on tests have to be carry out in order to identify the bacteria. In addition, it uses only one ID panel for both Gram-positive and Gram-negative aerobic bacteria. It also used a minimum sample and the setup only takes about one minute. Plus, it only uses one standard sample suspension (100l into each well) and one incubation temperature (33oC). The plate used provides 94 types of different tests which include 71 carbon source and 23 chemical sensitivity tests. The BiOLOGTM GENIII Microbial Identification System come with 4 different plates; FF MicroPlateTM (fungi identification test panel), EcoPlate TM (microbial community analysis), YT MicroPlateTM (yeast identification test panel and AN MicroPlate TM (anaerobe identification test panel).

CONCLUSION

The microbial identification process become easier because the technology in worldwide had improved in order to make the human works become easier and precise. One of the technologies is The BiOLOG TM GENIII Microbial Identification System.

REFERENCE BiOLOG GENIII Microbial Identification System. (2012). Retrieved 12 December, 2012, from http://www.biolog.com/products/?product=Microbial%20ID %20%2F%20Characterization

Biolog GENIII Microbial Identification System