Prokaryotic Pathophysiology
MARAAN, Lonesa Mae V. |2012-08040|ENG10 G-4R
Isolation, Characterization, and Identification of Prokaryotes from Public and Private Hotspring Resorts in Pansol, Laguna
METHODOLOGY
A. Materials
a. Prokaryotic samples from swimming pools of public and private resorts in sterile amber bottles
b. Glassware and Apparatus
1. Insulation Box or Thermos
2. Filtration set-up
3. Incubator
4. Dilution bottles
5. Test tubes
6. Screw cap tubes
7. Petri plates
8. Wire loop
9. Alcohol lamp
10. Microscope
c. Chemicals/ Reagents
1. Nutrient Agar
2. Nutrient Broth
3. Gram staining set
4. 3 % KOH
5. Endospore Staining set
6. Thioglycollate broth
d. Bergey’s Manual of Systematic Bacteriology Volume 1 and 2
B. Methods
a. Collection of Samples
Prokaryotic samples from different water depths (i.e.5 in. and 36 in. from the surface and 10 in. from the bottom) will be collected from swimming pools in private and public hot spring resorts in Pansol, Calamba, Laguna. Then, the collected samples will be placed into sterile amber
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The set-up will be properly labeled with assigned dilution and name of samples. First, ten ml volume samples will be transferred into a dilution bottle which contains 90 ml diluent, nutrient broth (NB) in particular; second, one ml from the previously made dilution will be transferred into a screw cap tube with 9 mL NB; and third, another one ml from the second dilution will be transferred into a different screw cap tube with 9 ml NB. Samples of 0.1 ml obtained from the three dilutions will be plated into nutrient agar (NA) plates. The plates will be then incubated for 24 hours at temperature ranging from 55 oC to 70 oC. When the maximum time of incubation is reached, colonies will be observed and then selected from the NA plates. The seleceted colonies will be picked up with sterile wire loop and will be relocated into another set of NA plates, as well as into NA slants, via streak plating technique (Aguda,
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