Chapter III

Presentation, Analysis and Interpretation of Data

This chapter represents the design of the study. It discusses the procedure materials and the subject of the study. The statistical treatment and the manner in which the data analyses and interpretation were presented in this chapter.

3. 1 Research Design The research design is shown in Figure 1. The independent variables were the different treatments namely: the control, the 1 Molar, 2 Molar and 3 Molar sodium chloride solutions prepared culture media. Adjusted culture media was prepared such that Treatment One, control contains no sodium chloride, Treatment Two was concentrated with 1 Molar sodium chloride solution, Treatment Three was concentrated with 2 Molar sodium Chloride, then Treatment Four was concentrated with 3 Molar sodium chloride solution. Sterile circular fertile paper was soaked in an Aspergillus niger embedded solution, prepared by flooding with sterile water and streaking a three weeks old test tube cultured Aspergillus niger and poured in a watch glass for petri dish culture. Four sterile perti plates were prepared and pour into it 15 mL of adjusted culture media corresponding to its respective treatment. Three Aspergillus niger embedded soaked filter paper were placed on the top of the solidified culture media of each petri plates in triangular formation. The dependent variable was the zone of inhibition of Aspergillus niger and the corresponding sporelation.

3. 2 Procedural Flow Chart

Aspergillus niger

R1
T1 R2 R3 R1 R2 R3 R1 R2 R3

Growth of Aspergillus niger (Zone of Inhibition)

T2

Sporelation of Aspergillus niger

T3

Analysis of Data

R1 T4 R2 R3

Analysis of Variance

Inhibition of Growth and Sporelation of Aspergillus niger Using Sodium Chloride Legend T1 = Distilled Water Culture Media T2 = 50 ml culture media + 2.9g NaCl = 1 Molar T3 = 50 ml culture media + 5.8g NaCl = 2Molar T4 = 50 ml culture media + 8.8g NaCl = 3 Molar 3. 3 Materials and Equipment

This sub-section presents the materials and laboratory equipment used in the conduct of the experimentation. These were categorized based on preparation of the culture media, paper disc, measurement of zone of inhibition, preparation of slide for spore microscopic examination, and data collection 3.3.1 Sabouraund Agar Sabouraud agar can be purchased from a variety of commercial sources, either as the original recipe (Sabouraud agar, modified), or in a slightly altered version termed Sabouraud agar. Per liter of medium: Peptone, 10 g Glucose, 40 g Agar, 15 g Combine all ingredients in ~900 ml of deioinized water. Adjust to pH 5.6 with hydrochloric acid and adjust final volume to 1 liter. Autoclave 20 minutes at 121C, 15 lb/in2. Cool to ~45 to 50C and pour into petri dishes or tubes for slants. 3.3.2 Adjusted Sabouraund Agar To make Sabouraund Agar more selective amoxicillin was added which inbihit gram-negative bacteria, and chlorampenicol which inhibits grampositive and gram-negative bacteria. To adjust the Molarity of the culture media, the following processes were followed: a. Distilled Water Culture Media or the pure sabouraund agar. b. For One Molar sabouraunf agar, To 50 mL pure sabouraund agar a 2.9 g NaCl was dissolved c. For Two Molar sabouraund agar, To 50 mL pure sabouraund agar a 5.8 g NaCl wa dissolved d. For Three Molar sabouraund agar, To 50 mL pure sabouraund agar an 8.8 g NaCl was dissolved. This was placed in different containers with corresponding labels. The 12 pieces of filtrate paper were placed and equally distributed in different

treatments. Test tube cultured Aspergillus niger was flooded with 5 mL sterile water and carefully scrapped using inoculating loop not destroy spores. Pour the sterile water with Aspergillus niger into a watch glass. Then the filtrate papers were soaked for about 5 minutes in the watch glass with Aspergillus niger then it was placed in a triangular location in each petri-dish. All cultures were incubated for 6 days in dark at 30 degrees Celsius. Treatments were replicated thrice. Observations were recorded after 6 days for the extent of radial growth of the fungal colony as well as the central sporulation zone on solid medium. Daily observations were also recorded for the initiation of sporulation in all fungal cultures. Data were subjected to one way analysis of variance (ANOVA) and multiple comparisons for difference in mean values for treatment and control were made by Bonferroni - Holm Test at P(<_)0.05, XL Stat Created by Daniel's XL Toolbox version 4.01 (http://xltoolbox.sf.net).

3.4 Experimentation and General Procedure This section showed the step-by-step process in the conduct of the experiment. 3.4.1 Procurement of Aspergillus niger Aspergillus niger culture was given by a researcher-teacher of Notre Dame of Dadiangas University for free. However, morphological examination was done in the laboratory of Hope Christian School prior to experimentation to ensure purity of the culture. 3.4.2 Preparation of the Culture Media Sabouraud agar can be purchased from a variety of commercial sources, either as the original recipe (Sabouraud agar, modified), or in a slightly altered version termed Sabouraud agar. Per liter of medium: Peptone, 10 g, Glucose, 40 grams and Agar, 15 grams. Combine all ingredients in ~900 ml of deioinized water. Adjust to pH 5.6 with hydrochloric acid and adjust final volume to 1 liter.

Autoclave 20 minutes at 121C, 15 lb/in2. Cool to ~45 to 50C and pour into petri dishes or tubes for slants. 3.4.3 Adjusted Sabouraund Agar To make Sabouraund Agar more selective amoxicillin was added which inbihit gram-negative bacteria, and chlorampenicol which inhibits grampositive and gram-negative bacteria. To adjust the Molarity of the culture media, the following processes were followed: a. Distilled Water Culture Media or the pure sabouraund agar. b. For One Molar sabouraunf agar, To 50 mL pure sabouraund agar a 2.9 grams NaCl was dissolved c. For Two Molar sabouraund agar, To 50 mL pure sabouraund agar a 5.8 grams NaCl wa dissolved d. For Three Molar sabouraund agar, To 50 mL pure sabouraund agar an 8.8 grams NaCl was dissolved.

3.4.4. Inoculation of Aspergillus niger in respective petri-plates Fifteen mL. of adjusted culture media was poured in four petri plates, let it stand for minutes to allow solidification. Then to petri dish for Treatment 1 containing pure sabouraund agar was placed Aspergillus niger impregnated circular filter paper in triangular location. Then to petri dish for Treatment 2 containing one molar sabouraund agar was likewise planted with Aspergillus niger impregnated circular filter paper in triangular formation. Then to petri dish for Treatment 3 containing two molar sabouraund agar was likewise planted with Aspergillus niger impregnated circular filter paper in triangular formation and, Then to petri dish for Treatment 4 containing three molar sabouraund agar was likewise planted with Aspergillus niger impregnated circular filter paper in triangular formation. In an inverted position, the petri dishes were with the cultures were incubated for 6 days in dark at 30 degrees Celsius. Treatments

were replicated thrice. Observations were recorded after 6 days for the extent of radial growth of the fungal colony as well as the central sporulation zone on solid medium. Daily observations were also recorded for the initiation of sporulation in all fungal cultures. Data were subjected to one way analysis of variance (ANOVA) and multiple comparisons for difference in mean values for treatment and control were made by Bonferroni - Holm Test at P(<_)0.05, XL Stat Created by Daniel's XL Toolbox version 4.01 (http://xltoolbox.sf.net). 3.5 Data Presentation and Analysis 3.5.1 Growth Inhibition of Aspergillus niger This sub-section presents the data gathered during the course of the experimentation. Along with the data collected is the corresponding analysis of the data collected with a short explanation on the daily basis until six days. Day One: Table 1. Growth Inhibition of Aspergillus niger in Different Concentration of Sodium Chloride for First Day of Experimentation Replication Treatments Water 1 Molar 2 Molar 3 Molar 1 1.60 1.50 1.20 0.50 2 1.50 1.40 1.10 0.50 3 1.90 1.50 1.00 0.50 Total 5.00 4.40 3.30 1.50 Mean 1.67 1.47 1.10 0.50 11.98% 34.13% 70.06% Percentage of Inhibition

Table

1 showed the growth inhibition of Aspergillus niger using different

concentration of sodium chloride during the first day of experimentation. It is evident that the three molar concentrations showed 0.50 cm radial fungal growth the least growth of Agpergillus niger at a rate of 70% inhibition compared to water the negative control. It is

also observed that as the molar concentration of the culture media increases the percentage of inhibition on the growth of Aspergillus niger likewise increases.