1) Determination of Spores Number by using Haemacytometer Theory The hemacytometer is used for counting the fungal spores in liquid

suspension. It has a thick base and uses a special coverglass which is thick enough to stay flat under the pull of surface tension from the solution in the counting chamber. As it is difficult to distinguish between living and dead organisms unless particular stains are used to distinguish viable from non-viable cells this results in a 'total count' of the bacteria. Materials - Aspergillus flavus sp. - Sterile water - Twin 80

Method 1. The spores are suspended in the sterile water containing 0.001% Twin 80 and diluted it to get appropriate spores number in the suspension. 2. 1 ml of the spores suspension is put on haemacytometer slide and the spores under microscope is calculated.

2) Determination of glucose by using DNS Method Theory DNS reagent is used to detect the amount of glucose in the solution. This experiment involved a redox reaction between the aldehyde group of glucose and DNS, in which color develops proportionally to the concentration of the glucose present. Materials - DNS reagent - 1 N NaOH solution - Water - Glucose (sample) Methods 1. 1 ml of DNS reagent prepared earlier is added in 1ml sample and 2 drops of 1 N NaOH are added. 2. The solution is vortex for 30 seconds. 3. The mixture solution is heated in the boiling water for 5 minutes and cooled under running tap water. 4. 10 ml of distilled water is added and the absorption of wavelength of 510 nm . 5. The standard curve is prepared for different concentration of glucose 6. Dilution of supernatant that has been centrifuged and filtered is necessary.

3) Determination of organics acids by using HPLC Theory Organics acids such acetic, lactic acid and kojic acid can be detected by using high performance liquid chromatography equipped with UV detector. Highperformance liquid chromatography (sometimes referred to as high-pressure liquid chromatography), HPLC, is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture. Materials - Sample from fermentation broth - Organics acids - Sulphuric acid (mobile phase)

Method 1. The supernatant sample from fermentation broth is filtered using nylon Whatman filter paper (pore size 0.2 um and diameter 47 nm) 2. Separation of organics acids is performed by using column Biorad Amine HPX 87H cation exchange resin as stationary phase. 3. 7mM sulphuric acid is used as mobile phase at flow rate 1ml/min and control the temperature at 50 4. Detail explanation of the method is given by the demonstrator and laboratory stuff during the practical.

Results The hemacytometer is used for counting the fungal spores in liquid suspension. It has a thick base and uses a special coverglass which is thick enough to stay flat under the pull of surface tension from the solution in the counting chamber. As it is difficult to distinguish between living and dead organisms unless particular stains are used to distinguish viable from non-viable cells this results in a 'total count' of the bacteria. In case of bigger cells the number of dead (permeabilised) cells in the sample can be obtained by adding Trypan blue. Fluorescent dyes give better discrimination particularly when looking at bacteria. Firstly, the spores are suspended in sterile water which contains 0.001% of Tween 80. Tween 80 is used to prevent spores adhesion. The original suspension must be mixed thoroughly before taking the sample. This ensures the sample is representative, and not just an artifact of the particular region of the original mixture it was drawn from. An appropriate dilution of the mixture with regard to the number of cells to be counted should be used. If the sample is not diluted enough, the cells will be too crowded and difficult to count. If it is too dilute, the sample size will not be enough to make strong inferences about the concentration in the original mixture. By performing a redundant test on a second chamber, the results can be compared. If they differ greatly, the method of taking the sample may be unreliable (e.g., the original mixture is not mixed thoroughly). To count the spores, the spore suspension is diluted with sterile water. The dilution factor in this experiment is . Based on the result, the number of spores that had been count is 174.

So the number of spores in 1ml suspension is 174 spores. Next, we determined glucose concentration by using DNS method. DNS reagent is used to detect the amount of glucose in the solution. This experiment involved a redox reaction between the aldehyde group of glucose and DNS, in which color develops proportionally to the concentration of the glucose present. In this case,

glucose is the reducing agent because it reduces the DNS, while the glucose itself, specifically the aldehyde group, is oxidized. Then, the test tubes are mixed well with

vortex to produce homogeneous solutions. All the test tubes are boiled for 5 minutes to trigger the reaction. The test tubes are cooled down to room temperature to stop DNS reaction. If not, DNS reaction will keep changing the intensity of solution and give some effects on absorbance reading. After that, each test tube is added with 10 ml distilled water to undergo dilution. Spectrophotometer is used to detect the absorbance reading at 510nm. The color amount correlates with the amount of product formed during reaction. The amount of reducing sugar is determined with the use of glucose standard curve. Based on the result, the equation of the graph is y = 1.2292x. Dilution of supernatant (fermentation broth that has been centrifuged and filtered) is necessary. If the culture contains 50 g/l of glucose, the sample should be diluted at least 50 times to obtain less than 1 g/l of glucose in the sample prior to reaction with DNS reagent. High-performance liquid chromatography (sometimes referred to as high-

pressure liquid chromatography), HPLC, is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture. Some common examples are the separation and quantitation of performance enhancement drugs (e.g. steroids) in urine samples, or of vitamin D levels in serum. HPLC typically utilizes different types of stationary phases (i.e. sorbents) contained in columns, a pump that moves the mobile phase and sample components through the column, and a detector capable of providing characteristic retention times for the sample components and area counts reflecting the amount of each analyte passing through the detector. The detector may also provide additional information related to the analyte, (i.e. UV/Visspectroscopic data, if so equipped). Analyte retention time varies depending on the strength of its interactions with the stationary phase, the composition and flow rate of mobile phase used, and on the column dimensions. HPLC is a form of liquid chromatography that utilizes small size columns (typically 250 mm or shorter and 4.6 mm i.d. or smaller; packed with smaller particles), and higher mobile phase pressures compared to ordinary liquid chromatography. With HPLC, a pump (rather than gravity) provides the higher pressure required to move the mobile phase and sample components through the densely packed column. The

increased density arises from the use of smaller sorbent particles. Such particles are capable of providing better separation on columns of shorter length when compared to ordinary column chromatography.