Isolation and Identification of Bacteria (Frerichs and Millar (1983, 1993))

To study the possibility of hepatopancreatic necrosis syndrome infection in shrimp, a series of infection experiments were conducted. Healthy shrimp breeds were tested for infection through rearing together with diseased shrimp, feeding food mixed with hepatopancreas collected from diseased shrimp, soaking shrimp in an environment with hepatopancreas collected from diseased shrimp, and injecting hepatopancreas extract from diseased shrimp. The experiments were conducted at different concentrations of infection and different times. The results showed that infection occurred in experimental shrimp, but the level of infection was not high, it seems that the level of infection depends on environmental conditions. Shrimp with hepatopancreatic necrosis syndrome, when transferred to clean water environment, had reduced mortality rates and were able to recover.

In May 2013, a research team led by Professor Donald Lightner of the University of Arizona (USA) identified a strain of Vibrio parahaemolyticus bacteria that, when infected with a virus called a phage, will produce an extremely toxic toxin. strong, similar to the phenomenon of cholera in humans, is the causative agent of acute hepatic necrosis disease (AHNPD).

Chapter 2: SUBJECTS, CONTENT AND RESEARCH METHODS

2.1 Research object and content

2.1.1 Research subjects

Black tiger shrimp and White leg shrimp

2.1.2 Research content

- Determine the presence of bacteria on shrimp with AHPND

2.2 Time and location of research

2.2.1 Research time

Implemented from April 2013 to April 2014

2.2.2 Research location

- Sample collection location: Nghe An and Nam Dinh

- Location of disease sample analysis: Research Center for Environmental Warning Monitoring and Prevention of Aquatic Diseases in the Northern Region - Aquaculture Research Institute 1.

2.3. Materials and research methods

2.3.1 Determine AHPND

- Clinical signs (Department of Veterinary Medicine, 2012):

o Acute hepatopancreatic syndrome causes death in black tiger shrimp and white leg shrimp

15-45 days old period after stocking.

o Sick shrimp show signs such as stopping eating, swimming slowly, thin shell, pale shrimp color, and the hepatopancreas appears swollen, limp, and atrophied.

- Histopathological techniques (Lightner, 1996)

The steps are sequenced according to the following diagram:

Sample:

Fix with Davison solution (12-72 hours) Transfer to 70 0 C alcohol for preservation

Sample processing

Cut the sample 0.2-0.5cm into the cassette. Process with an automatic sample processor

Cast and cut samples

Model casting: Pour paraffin and create mold Model cutting: microtom cutting machine

Tint​

Hematocyline-Mayer and Eosin.

Read the results

Observe the microscope

Step1: Take sample

+ For Post shrimp

Soak the entire shrimp directly in the fixing solution with a sample/fixing solution ratio of 1/10 for 12-24 hours.

+ For larger shrimp

Use scissors to cut and separate the armor layer at the top of the chest to remove the internal organs. For small sized organs, just use scissors and pints

Separate it and put it in a bottle containing Davidson's fixative solution. For large organs, use a syringe to inject the fixative solution into the tissue of that organ and then put it in Davidson's fixative solution. In order for the fixing solution to penetrate well into the tissues, the ratio between the volume of shrimp and the fixing solution should be 1/10. Keep the sample in the fixative solution for 12-72 hours.

All collected samples must be labeled with complete sample information. After being kept in the fixative solution for 12-72 hours (depending on the sample), they will be transferred to a 70-degree alcohol solution for preservation.

Step 2: Sample processing:

+ Prepare the sample for processing: Use a pint to remove the sample from the bottle containing the sample preservation solution, then use a sharp knife to cut the sample into pieces of 0.2 - 0.5cm in size and put them in the cassette.

+ Sample processing process: processed by automatic sample processing machine (put the cassette containing the sample into the sample basket and put it into the automatic processing machine).

Step 3: Cast and cut the sample:

+ Model casting: Use the machine to pour molten paraffin into the mold that already has the model, then place the mold on the cooling system. After a few minutes, molds containing the sample can be created. Use a paring knife and cut the sample into isosceles trapezoidal or rectangular blocks.

+ Cut the sample: Attach the cassette with the peeled sample to the microtom cutting machine, cut slices with a thickness of 5µm. Put the slices in moist water (40-50 o C) (you can add egg whites) for about 1-2 minutes so that the slices relax and do not wrinkle. Use a clean slide to remove the slices from the water, place them on the dryer at a temperature of 45-60 o C for 1-4 hours.

Step 4: Dye

After drying, samples will be placed in dye baskets and stained. We use traditional staining methods with Hematocyline-Mayer and Eosin dyes.

Step 5: Read the results:

Based on the following signs to determine AHPND (Department of Animal Health, 2012)

o Acute hepatopancreatic degeneration;

o Lack of isomitotic mitotic activity of cells derived from embryonic tissue (E, Embryonalzellen cells);

o Dysfunction of central hepatopancreatic cells: B secretory cells (Basenzellen), F fibrous cells (Fibrillenzellen), R reserve cells (Restzellen);

o Hepatopancreatic cells have abnormally large nuclei and cell sloughing.

o In the final stage, blood cells aggregate between the hepatopancreatic ducts and become infected

2.3.2 Isolation and identification of bacteria (Frerichs and Millar (1983, 1993))

c as follows:

Vietnam – Master's thesis in Agricultural Sciences

Check colony morphology characteristics Culture on basic medium (Nu + ) Check colony morphology (after 24 hours)

Culturing pure bacteria

Culture transferred to TCBS medium

Summary of steps

Academy of Agriculture

Page 30

Staining pure bacteria:

Gram stain 10-40-100 microscope

Test biochemical reactions

By API Kit 20E

Step 1: Check colony morphological characteristics: After taking samples of sick shrimp, culture them on basic medium (Nutrient Agar), at a temperature of 28 - 30 0 C, after 24 hours check the morphological characteristics. Colony morphology (colony size, colony morphology, colony color).

Step 2: Cultivate pure bacteria: After checking the characteristics and morphology of the colonies, mark loose colonies that grow on the inoculation line, use a sterile culture rod to take the marked colonies, and transfer to TCBS selective media plate and leave the sample at a temperature of 28 - 30 0 C.

Step 3: Staining pure bacteria: Take pure colonies, spread thinly on a slide with 1 drop of 2% physiological saline, heat over an alcohol lamp flame to immobilize bacteria and perform gram staining and bacterial examination. Using a microscope from a 10 - 40 - 100 objective lens to observe the shape and color of bacteria.

Step 4: Test biochemical reactions with API Kit 20E (BioMerieux, France):

Table 2.1: How to perform and read biochemical reactions

Tests

Maybe you are interested!

• Isolation and Identification of Ammonia Metabolizing Bacteria (Aob)
• Study on the ability to cause acute hepatopancreatic necrosis disease of some species of vibrio bacteria in brackish water shrimp in some northern provinces - 1
• Some Research Results in the World and in the Country on Xanthomonas Axonopodis Pv. Citri Bacteria and Ulcer Disease Caused by Xanthomonas Axonopodis Pv. Citri Bacteria on
• Study on nitrogen-metabolizing bacteria in the bottom of the lobster farming area Panulirus sp. for aquaculture - 2
• Survey on antibiotic resistance of acne-causing bacteria Probionibacterium acnes in Can Tho city - 1