aspergillus niger and sodium chloride chapter iii
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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
T1
T3
T2
T4
R3
R2
R2
R3
R2
R1
R3
R2
R1
R3
R1
R1
Legend
T1 = Distilled Water Culture Media T2 = 50 ml culture media + 2.9g NaCl = 1 MolarT3 = 50 ml culture media + 5.8g NaCl = 2MolarT4 = 50 ml culture media + 8.8g NaCl = 3 Molar
Growth of Aspergillus niger
(Zone of Inhibition)
Sporelation of Aspergillus niger
Analysis of Data
Analysis of Variance
Inhibition of Growth and Sporelation of Aspergillus niger Using Sodium Chloride
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 gGlucose, 40 gAgar, 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 121°C, 15 lb/in2. Cool to ~45 to 50°C 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 gram-positive 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 121°C, 15 lb/in2. Cool to ~45 to 50°C 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 gram-positive 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
Treatments
Replication
Total Mean
Percentage
of Inhibition1 2 3
Water 1.60 1.50 1.90 5.00 1.67
1 Molar 1.50 1.40 1.50 4.40 1.47 11.98%
2 Molar 1.20 1.10 1.00 3.30 1.10 34.13%
3 Molar 0.50 0.50 0.50 1.50 0.50 70.06%
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.