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.

Control

Triplicates

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

B. Characterization of Microbes Staining. Stained preparation is a basic procedure for the initial observation of microorganisms. But before staining, 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. In most staining preparations, fixing microorganisms to the slide entailed drying and flaming. Stain is applied and then washed off by distilled water; then the slide is blotted with absorbent paper for the excess water on the slide. The specimen is now ready for the microscopic examination. For this study, the group will used Gram Staining for the characterization of gram- positive and gram- negative bacteria. i. Gram Staining. A heat- fixed smear is covered with a basic purple dye, usually crystal violet. After a short time, the purple dye is washed off, and the smear is covered with iodine, a mordant. When the iodine is washed off, both grampositive and gram- negative bacteria appear dark violet or purple. Next, the slide is wash with alcohol or an alcohol- acetone solution (decolorizing agent). The solution will wash off the purple from the cells of some species but not from the others. The alcohol is rinsed off, and the slide is then stained with Safranin, a basic red dye. Safranin is used for counter staining. The smear is washed again, blotted dry, and examined microscopically. All bacteria that colored dark violet characterizes gram- positive bacteria, while all bacteria that colored pink are the gram- negative bacteria. ii. Shapes. Under light microscope, the group will identify microbes shapes. They might be presented as rod, spiral, or tubular in form. C. Quantification of Microbes a. Nutrient Agar The nutrient agar is used in this study. The composition of the Nutrient Agar shown in Table 1 was described in the book of Tortora, 2005.

Table 1. Composition of Nutrient Agar

Constituents Peptone (partially digested protein) Beef Extract Sodium Chloride Agar Water Amount 5.0g 3.0g 8.0g 15.0g 1 liter

b. Plate count and Serial dilutions i. Serial dilutions. Serial dilutions ensure a countable plate for the study. This is done by diluting the original inoculum in a series of dilution tubes. Each succeeding dilution tube will have only one- tenth the numbers of microbial cells as the preceding tube. In this study, the group will inoculate the original inoculum up to 10-5 dilutions. Sterile water is used in the dilution series. Plating samples is done in the last diluted tube from 10-5 dilution series. ii. Spread plate. A 0.1 mm inoculum from 10-5 dilution tube is added to the surface, solidified agar medium. The inoculum is then spread uniformly over the surface of the medium with a specially shaped, sterilized glass rod. Colonies grow on the surface of the medium only. 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.

D. Statistical Analysis. The group will use One- way ANOVA to analyze the data on the amount of bacteria from each tank treated with five different probiotics and from the control.

Reference: Tortora G.J., Funke B.R., & Case C.L.. (2005). MICROBIOLOGY: An Introduction Component. 8th Edition. Pearson Education South Asia PTE. LDT, Inc. Philippines.