**2.4. Bacterial challenge**

diluted with dilution factor of 10−4. 2 mL was taken from each diluted sample and dispensed into two Petri dishes (1 mL to each). The first dish received plate count agar (PCA, LAB M, LAB149) for total viable count (TVC), while the second Petri dish received MacConkey agar (LAB M, LAB002) for total enterobactereceae count (TEB) using the pure plate count method [11–12]. Each dilution was overlaid, respectively, with PCA and MacConkey that have been

**Table 1.** Ingredients and chemical composition (%; on dry matter basis) of experimental diets containing graded levels

Note: DCP = Dicalcium phosphate; LA = *Lactobacillus acidophilus.*\*Premixes = HI-MIX®AQUA (Fish) each 1 kg contains; vitamin A, 4000,000 International Unit (IU); vitamin D3, 8,00,000 IU; vitamin E, 40, 000 IU; vitamin K3, 1600 mg; vitamin B1, 4000 mg; vitamin B2, 3000 mg; vitamin B6, 3800 mg; vitamin B12, 3 mcg; Nicotinic acid 18,000 mg; Pantothenic acid, 8000 mg; Folic acid, 800 mg; Biotin, 100 mcg; Choline chloride 120,000 mg; Iron, 8000 mg; Copper, 800 mg; Manganese, 6000 mg; Zinc, 20,000 mg; Iodine, 400 mg; Selenium, 40 mg; Vitamin C C(coated), 60,000 mg;

Inositol, 10,000 mg; Colbat, 150 mg; Lysine, 10,000 mg; Methionine, 10,000 mg; Antioxidant, 25,000 mg.

The dishes were then gently swirled to mix the bacteria with the liquid agar. The mixtures were allowed to harden. When the mixture was hardened, the individual cells were fixed in place and incubated (Newlife Laboratory Incubator NL-9052-1) for 24 h at 37°C to allow distinguished colonies to form. The colonies formed were counted using Wincom Colony Counter (16 W, 220 V ± 10%, 50 Hz). The experiments were replicated three times. The TVC

cooled to 50°C. At this temperature, agar is still in liquid form [12].

**Ingredients (%)** *Lactobacillus acidophilus* **inclusion levels cfu/mL**

Groundnut cake 25.00 25.00 25.00 25.00 25.00 25.00 Soya meal 24.00 24.00 24.00 24.00 24.00 24.00 Fish meal 22.15 22.15 22.15 22.15 22.15 22.15 DCP 1.00 1.00 1.00 1.00 1.00 1.00 Salt 1.00 1.00 1.00 1.00 1.00 1.00 Flour 0.89 0.89 0.89 0.89 0.89 0.89 Vegetable oil 0.50 0.50 0.50 0.50 0.50 0.50 Maize 24.96 24.96 24.96 24.96 24.96 24.96 Premix\* 0.50 0.50 0.50 0.50 0.50 0.50 LA 0 101 102 103 104 105 TOTAL 100.00 100.00 100.00 100.00 100.00 100.00

Moisture 9.16 9.16 9.16 9.16 9.16 9.16 Crude protein 40.00 40.04 40.10 40.11 40.12 40.12 Ether extract 9.63 9.63 9.54 9.63 9.66 9.65 Total ash 8.27 8.27 8.27 8.27 8.27 8.27 Carbohydrates 20.17 20.13 20.16 20.06 20.02 20.03 Crude fibre 12.77 12.77 12.77 12.77 12.77 12.77

and TEB were expressed in Log10CFU/g [12].

**Proximate composition**

96 Aquaculture - Plants and Invertebrates

of *Lactobacillus acidophilus*.

To evaluate prawn resistance to disease infection, a challenge test using the pathogenic bacteria, *Pseudomonas aeruginosa* and *Aeromonas hydrophila* was performed following 84 days feeding trial. Prior to the challenge test, a preliminary experiment was performed to determine the LD50 (lethal dose) of the pathogenic bacteria. For the challenge test, 10 prawn were selected from each replicate tank and transferred into another tank filled with dechlorinated freshwater. *Pseudomonas aeruginosa* was grown nutrient agar broth and *Aeromonas hydrophila* on PBS broth for 24 h at 35°C in an incubator in Microbiology Laboratory. Bacterial cells were then centrifuged at 3000× g for 30 min to form pellets. The pellets were re-suspended in 1 mL of 0.1% peptone water and after which 100 mL of the suspension were diluted at 4:10 with water from experimental tank. The prawn were exposed to bacterial solution of both *Pseudomonas aeruginosa* (1 × 107 CFU/mL) and *Aeromonas hydrophila* (5 × 105 CFU/mL) for 30 min and returned into experimental set up [13, 14]. The fish were fasted for 24 before infection and feeding with experimental diets resumed 12 h later. All prawn groups were kept under observation for 14 days to record any abnormal clinical signs and the daily mortality.
