Methods and Materials A. Experimental Set up and Sampling.

The set up of the study will be located at Institute of Aquaculture Multi-Species Hatchery. 18 concrete tanks with proper organized aeration will be used as containers for the biofloc system. One ton (25 ppt) of seawater filled up each tank. Three concrete tanks represent triplicates in each of the five (probiotic) treatments and a control. Sampling is done every week for 13 times and is scheduled every Friday. A preassessment for the present microbes before the probiotic applicationwill be conducted. The Figure 1 shows the experimental set up.



Probiotic 1 Triplicates

Black Tiger Shrimp
Penaeus monodon

Probiotic 2 Triplicates

Probiotic 3 Triplicates

Legends: Control Probiotic 1Probiotic 2Probiotic 3Probiotic 4Probiotic 5Probiotic 6-

Probiotic 4 Triplicates

Probiotic 5 Triplicates

Figure 1. The Experimental Set up

and the smear is covered with iodine.0g 1 liter . the purple dye is washed off.negative bacteria appear dark violet or purple. The specimen is now ready for the microscopic examination. the group will identify microbes shapes. a mordant. Next. But before staining. while all bacteria that colored pink are the gram. fixing microorganisms to the slide entailed drying and flaming. The smear is washed again. A heat. both grampositive and gram. When the iodine is washed off. The solution will wash off the purple from the cells of some species but not from the others.positive bacteria.B. Composition of Nutrient Agar Constituents Peptone (partially digested protein) Beef Extract Sodium Chloride Agar Water Amount 5. In most staining preparations. Nutrient Agar The nutrient agar is used in this study. The alcohol is rinsed off. blotted dry. They might be presented as rod. Gram Staining. ii. C. a basic red dye. The composition of the Nutrient Agar shown in Table 1 was described in the book of Tortora.acetone solution (decolorizing agent). All bacteria that colored dark violet characterizes gram. Characterization of Microbes Staining. then the slide is blotted with absorbent paper for the excess water on the slide. Quantification of Microbes a.0g 15.negative bacteria. the microorganisms must be first fixed to the microscope slide by spreading them over the surface of the slide through a thin film (a smear) containing the microorganisms. The smear is being exposed to the air for drying and to the Bunsen burner for flaming. After a short time. Stain is applied and then washed off by distilled water. Table 1.0g 3.positive and gram.0g 8. i. Safranin is used for counter staining. and examined microscopically. or tubular in form. spiral. For this study.fixed smear is covered with a basic purple dye. the group will used Gram Staining for the characterization of gram. 2005. the slide is wash with alcohol or an alcohol. and the slide is then stained with Safranin. Shapes. Stained preparation is a basic procedure for the initial observation of microorganisms.negative bacteria. Under light microscope. usually crystal violet.

tenth the numbers of microbial cells as the preceding tube. Each colony will be counted and each count per plate will be multiplied to the reciprocal of the dilution of sample to determine the number of bacteria per milliliter. 8th Edition.L. Serial dilutions ensure a countable plate for the study. . Pearson Education South Asia PTE. LDT. This is done by diluting the original inoculum in a series of dilution tubes. Inc.b. Funke B.way ANOVA to analyze the data on the amount of bacteria from each tank treated with five different probiotics and from the control. solidified agar medium.. Plating samples is done in the last diluted tube from 10-5 dilution series. Serial dilutions. MICROBIOLOGY: An Introduction Component. (2005)..1 mm inoculum from 10-5 dilution tube is added to the surface. Statistical Analysis. Sterile water is used in the dilution series. The inoculum is then spread uniformly over the surface of the medium with a specially shaped. In this study. The group will use One. Spread plate. A 0.R. Plate count and Serial dilutions i. & Case C. Philippines. Reference: Tortora G.. Colonies grow on the surface of the medium only. ii. Each succeeding dilution tube will have only one. the group will inoculate the original inoculum up to 10-5 dilutions. D.J. sterilized glass rod.

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