Fish404+Lab+Notebook

**5-6-10 Gram Stain, Serum Agglitination, and Protease Testing**
SUMMARY: Continue with characterizing bacteria isolated from the hatchery. Perform phenotypic and pathogenicity testing for Vibrio sp. (since TCBS plates results indicate a Vibrio spp.)

PROCEDURES: __Colony Counts from Dilution Series__: Count the colonies on the plate with ~30-300 colonies Calculate backwards to get the original concentration of the culture we used in our challenge experiment

RESULTS: My plates were contaminated with pink and fuzzy colonies. Most of my plates were densely grown and I didn't count these. From the countable plates from the rest of the class, original concentration of the culture was approx 1 x 10^8 CFU/ml

__Gram Stain__: Place drop of water onto a clear slide Sterilize loop and touch a colony of bacteria from a plate Swirl loop in the water Let slide air dry Pass slide through flame a couple times to fix bacteria on slide Flood slide with crystal violet and let sit for 1 min Rinse with tap water Flood slide with Gram's iodine and sit for 1 min Rinse with tap water Decolorize slide with alcohol acetone solution Immediately rinse with tap water Counter stain with Saffrinin ~15 sec Let dry and view with immersion oil under 100x

RESULTS: Gram negative cells, but no real shape to the bacteria. I also stained a colony of suspected contamination and saw some large Gram positive cocci. CF mentioned these may be yeast due to size and Gram color. CS slides showed clear, Gram-negative, rod-shaped cells in one colony and Gram-positive cocci in another colony. Interesting results, but it's clear we had contamination issues on most of the plates

__Serum Agglutination__: Add 50 ul of sterile SW and 25ul of culture onto one side of a slide to use as a control On the other side, place 25ul of culture, 25ul polyclonal antibody, and 25ul of sterile SW Mix with pipette tip Incubate for 5 min View through 10x objective under Phase3 light in the scope Look for clumping and compare with control side

RESULTS: Inconclusive. There was clumping of cells on the test side of the slide, but when compared to the control side, it was approximately the same amount of clumping maybe slightly more. I can't be sure that what I saw was significantly more than the control.

__Azocasein Protease Assay__: Vortex 5 ml broth culture well Spin culture to pellets (26k rpm for 10min) Remove 100ul of supernatant and place into microcentrifuge tube Add 400ul of 1% azocasein Incubate for 30 min at 37C Add 600ul of trichloric acetic acid (TCA) to stop reaction Stick on ice for 30 min Centrifuge tube at 13000 rpm for 5 min Add 200ul of 1.8 NaOH to a new microcentrifuge tube Add 800ul of your reaction supernatant to the tube with NaOH Examine for color change

RESULTS: Positive! Bright orange color change was apparent. This sample was protease positive.

**4-29-10 Bacterial Hatchery Mystery Experiment with Cg**
SUMMARY: Mortality of C. gigas larvae has been occurring at a local hatchery. A bacterial pathogen is the suspected cause and was isolated on site. We aim to characterize the bacterium phenotypically, determine an approximate LD50 dose, reinfect a new group of C. gigas larvae and reisolate the same bacterium from this experiment. pH was also an area of concern. We will incorporate this environmental factor in our challenge experiments.

PROCEDURE: A. Serial Dilutions and phenotypic characterization The original isolate was used to grow up 3 liters of culture This culture was spun down by centrifugation (3600 rpm) at 20 min into pellet Resuspended in seawater Vortexed And placed into two 750ml aliquots (1.5L total) Serial dilutions were made up to -6 by: Adding 75ml of culture to 675ml seawater diluent to make the first dilution (10^-1) Shake rigorously Take 75ml of this dilution and place into another 675ml of seawater diluent making a 10^-2 dilution of the original culture Continue until the final dilution has been reached (10^-6) Plate out 5 dilutions(10^-6 - 10^-2): Shake each dilution rigorously Take 100ul of desired dilution and place onto separate T1N2 agar plates Using a sterilized glass hockey stick, spread the 100ul of dilution all over the plate Set aside to dry and seal with paraffin film Turn upside down and incubate for ~48 hrs at room temp Count the plates that have between 30-300 colonies and use this number to back calculate the original concentration Plate 100ul of the original stock culture and 10^-1 onto TCBS plates using the method above, spread the dilutions on the agar incubate and examine colony morphology in 24-48 hours of growth at RT

B. LD50/pH Challenge Two pHs will be used: 7.8 and ambient seawater with pH of 8.0 Each dilution will be done in duplicate, with 2 controls for each pH level Four beakers for each dilution, 2 at low pH and 2 at ambient/"normal" pH Take 150ml of dilutions 10^-6, -4, -2 and 0 and place into beakers with 150 ml of seawater and C. gigas larvae Add 150ml of seawater to the 150 ml seawater/larvae solution for controls Let sit statically overnight In the morning stir the larvae, take 100ml of each beaker and add a 1:1000 neutral red Let sit for 6 hours Take one ml of the larvae solution and look under a dissecting scope to visulalize live vs dead Count the red larvae as "alive" Count the non-red larvae as "dead" Record the numbers for each dilution/pH and graph to determine at which dilution ~50% of larvae were dead.

C. Reisolate bacterium from challenged larvae Find a larvae that has bacterial swarming under the scope With a sterile loop touch the infected larvae Streak a quadrant of the T1N2 plate with the loop Flame sterilize the loop Drag the loop across the first quadrant once and streak the next quadrant Flame sterilize the loop Continue in this fashion until the whole plate is streaked Incubate at RT for ~24 hrs inverted

RESULTS:

Group 1:
 * 404 Larval Challenge || Start: 4/29/10 || Termination: 4/30/10 ||  ||   ||   ||
 * pH 1 = 8.0 || alive/mL || dead/mL || total/mL || T1N2 || TCBS ||
 * Control Dup 1 || 12 || 0 || 12 ||  ||   ||
 * Control Dup 2 || 107 || 0 || 107 ||  ||   ||
 * 10^0 Dup 1 || 0 || 8 || 8 || + || - ||
 * 10^0 Dup 2 ||  ||   ||   ||   ||   ||
 * 10^-2 Dup 1 || 1 || 0 || 1 ||  ||   ||
 * 10^-2 Dup 2 || 0 || 11 || 11 || + || - ||
 * 10^-4 Dup 1 || 12 || 0 || 12 ||  ||   ||
 * 10^-4 Dup 2 || 3 || 0 || 3 ||  ||   ||
 * 10^-6 Dup 1 || 10 || 0 || 10 ||  ||   ||
 * 10^-6 Dup 2 || 4 || 0 || 4 ||  ||   ||
 * pH 2 = 7.6 ||  ||   ||   ||   ||   ||
 * Control Dup 1 || 21 || 0 || 21 ||  ||   ||
 * Control Dup 2 || 53 || 4 || 57 || + || - ||
 * 10^0 Dup 1 || 0 || 3 || 3 || + || + ||
 * 10^0 Dup 2 || 0 || 17 || 17 ||  ||   ||
 * 10^-2 Dup 1 || 0 || 65 || 65 ||  ||   ||
 * 10^-2 Dup 2 || 0 || 2 || 2 || - || - ||
 * 10^-4 Dup 1 || 135 || 3 || 138 || + || - ||
 * 10^-4 Dup 2 || 47 || 2 || 49 ||  ||   ||
 * 10^-6 Dup 1 || 2 || 0 || 2 ||  ||   ||
 * 10^-6 Dup 2 || 8 || 0 || 8 ||  ||   ||
 * ||  ||   ||   || + = bacterial growth noted ||   ||
 * Seawater used for experiments: ||  ||   ||   ||
 * Low pH seawater: 7.6 pH ||  ||   ||   ||   ||
 * Ambient seawater: 8.0 pH ||  ||   ||   ||   ||
 * After bacteria was added: ||  ||   ||   ||   ||
 * Low pH: 7.8 pH at 10^-6 dilution and 7.88 pH at 10^-2 dilution ||  ||
 * Ambient seawater: not tested ||  ||   ||   ||   ||
 * After bacteria was added: ||  ||   ||   ||   ||
 * Low pH: 7.8 pH at 10^-6 dilution and 7.88 pH at 10^-2 dilution ||  ||
 * Ambient seawater: not tested ||  ||   ||   ||   ||

Group 2:
 * 404 Larval Challenge || Start: 4/29/10 || Termination: 4/30/10 ||  ||   ||   ||
 * pH 1 = 8.0 || alive/mL || dead/mL || total/mL || T1N2 || TCBS ||
 * Control Dup 1 || 1 || 13 || 14 || - || - ||
 * Control Dup 2 || 23 || 0 || 23 ||  ||   ||
 * 10^0 Dup 1 || 0 || 40 || 40 || + || - ||
 * 10^0 Dup 2 || 0 || 59 || 59 ||  ||   ||
 * 10^-2 Dup 1 || 0 || 50 || 50 || + || - ||
 * 10^-2 Dup 2 || 1 || 98 || 99 ||  ||   ||
 * 10^-4 Dup 1 || 6 || 16 || 22 || + || - ||
 * 10^-4 Dup 2 || 38 || 6 || 44 ||  ||   ||
 * 10^-6 Dup 1 || 32 || 0 || 32 ||  ||   ||
 * 10^-6 Dup 2 || 112 || 0 || 112 ||  ||   ||
 * pH 2 = 7.6 ||  ||   ||   ||   ||   ||
 * Control Dup 1 || 8 || 5 || 13 || + || + ||
 * Control Dup 2 || 5 || 2 || 7 || + || + ||
 * 10^0 Dup 1 || 0 || 7 || 7 || + || + ||
 * 10^0 Dup 2 || 0 || 8 || 8 ||  ||   ||
 * 10^-2 Dup 1 || 0 || 8 || 8 || + || + ||
 * 10^-2 Dup 2 || 0 || 27 || 27 ||  ||   ||
 * 10^-4 Dup 1 || 49 || 6 || 55 || + || + ||
 * 10^-4 Dup 2 || 75 || 3 || 78 ||  ||   ||
 * 10^-6 Dup 1 || 72 || 4 || 76 || + || + ||
 * 10^-6 Dup 2 || 22 || 2 || 24 ||  ||   ||
 * 10^-6 Dup 1 || 72 || 4 || 76 || + || + ||
 * 10^-6 Dup 2 || 22 || 2 || 24 ||  ||   ||

**4-22-10 Histology Scoring**
SUMMARY: Score our histology slides we made from our abalone looking for foot muscle atrophy, metaplasia, and RLOs

PROCEDURE: Examine our slides under light microscopy using 40X objective lens Find an area of interest and using that one field of vision, score our slides using this scale: Foot muscle atrophy: 0=muscle fivers comprise >90% of tissue present, 1= 76-90%, 2=50-75%, 3=<50% Digestive gland or PE WS-RLO infection: 0=absent, 1= 1-10 inclusions/200x field of view, 2= 11-100, 3= >100 Digestive gland metaplasia: 0= 0-4%, 1= 5-10%, 2= 11-25%, 3= >25%

RESULTS: Sample 10:8-8: 0 score for foot atrophy, metaplasia and RLOs. Class results:
 * Accession # || Histo Score ||
 * 10:8-1 || F=0, M=0, I=0 ||
 * 10:8-2 || F=0, M=0, I=1 ||
 * 10:8-3 || F=0, M=0, I=0 ||
 * 10:8-4 || F=0, M=0, I=0 ||
 * 10:8-5 || F=0, M=0, I=0 ||
 * 10:8-6 || F=0, M=0, I=0 ||
 * 10:8-7 || F=0, M=0, I=0 ||
 * 10:8-8 || F=0, M=0, I=0 ||
 * 10:4-4 || F=3, M=2, I=3 ||
 * 10:4-5 || F=3, M=1, I=1 ||

**4-22-10 Agarose Gel and cPCR**
SUMMARY: make a 1.5% agarose gel to visualize cPCR. We are looking for ~160bp fragment to identify WS in our sample. PROCEDURE: Measure out 15g of agarose powder Add 100ml of TBE (1X) to the powder Microwave for ~1.5 mins Add 10ul of Ethidium Bromide and swirl the mixture Pour into gel mold and let solidify for ~20min After gel is solidified, add 1X TBE to the gel box, enough to cover the gel Pipet 7ul of 100bp molecular weight ladder to the left well Pipet 7 ul of each PCR product into the following wells Run gel for 45 min at 115v After done cooking, remove gel from box and visualize bands under UV light

RESULTS: qPCR Results: 10:8-8: 2.14E+07 10:4-4: 3.27E+04

cPCR Results: Positive for both samples

**4-15-10 DNA Extraction, PCR and qPCR**
SUMMARY: DNA extraction using Qiagen Stool Kit, PCR and qPCR will be performed to help detect and quantify the presence of withering syndrome (__Candidatus Xenohaliotis californiensis__) in our abalone tissues taken at necropsy.

Samples: 10:8-8 and 10:4-4

PROCEDURE: DNA extraction: Label tubes Remove tissue from cryovial with sterile forceps, weigh (g), and record Cut tissue into small pieces and place into 2.0 ml microcentrifuge tube Add a total of 1.4 mL of Buffer ASL using this protocol: - Add 700ul of Buffer ASL - Vortex for 1 min - Add another 700ul of Buffer ASL - Vortex for 1 min Place 1.4 ml of lysate into a new 2ml microcentrifuge tube Heat at 70C for 5 min Vortex for 15 sec Centrifuge at full speed for 1 min Take 1.2ml of supernatant and place into new 2 ml microcentrifuge tube (discard pellet) Add 1 inhibitEX tablet to the supernatant and quickly vortex for 1 min or until tablet is dissolved Incubate for 1 min at RT Centrifuge for 3 min at full speed Take all supernatant and place into new microcentrifuge tube (discard pellet) Centrifuge at full speed for 3 min Place 15 ul of Proteinase K in to new microcentrifuge tube Place 200 ul of supernatant into the Proteinase K Add 200ul Buffer AL Vortex for 15 sec Incubate at 70C for 10 min Remove samples from water bath and centrifuge briefly Add 200ul of 95% EtOH to sample and pulse vortex to mix for 15 sec Centrifuge briefly Place mixture into QIAamp spin column (~610ul) Close cap and centrifuge at 8000rpm for 1 min Place spin column in new collection tube Carefully open the spin column and add 500ul of Buffer AW1 Close and centrifuge for 1 min at 8000rpm Discard collection tube with buffer and put spin column in new collection tube Add 500 ul of Buffer AW2 Centrifuge for 3 min at full speed Re-centrifuge if there is still buffer in the column Place spin column into new microcentrifuge tube and add 100ul of Buffer AE Incubate at RT for 5 min Centrifuge for 1 min at 8000rpm Remove spin column and throw it away Close tube and make sure this is labeled thoroughly. Take 50ul of DNA and put into another microcentrifuge tube. (one for cPCR and one for qPCR) Store in -20C until ready to use.

Conventional and Quantitative PCR protocol: Calculate the volumes needed for PCR reactions Recipes here: [|PCR Recipes][|qPCR recipe] Wipe down bench with 10% bleach solution Add reagents to a clean microcentrifuge tube (except DNA template) Mix reagents by finger pipetting Label PCR products with sample number and primers Label two other tubes for negative and positive controls Label PCR tubes Add 18ul of master mix to each PCR tube Add 23ul of master mix to each qPCR tube Add 2 ul of water to negative controls Close tubes Add 2 ul DNA template into each PCR tube Add 2 ul of positive control to positive control tubes Flick to mix and briefly spin down

Place cPCR samples in thermocycler set to these parameters: [|thermo cycles]

Define parameters on qPCR program and start qPCR.

RESULTS: Note: tissue chucks were still visible in the tube during the initial maceration and I used a sterile pipet tip to help break the tissue down. But the supernatant was brownish in color and I continued with the rest of the protocol as usual.

NEXT STEPS: Make 1% agarose gel to visualize PCR bands. Interpret PCR and qPCR results

**4-8-10 Abalone Necropsy**
SUMMARY: Perform abalone necropsy and gather tissues for histology to help diagnose Rickettsia-like organisms (RLOs) or other disease-causing agents that may be present in our animal. Three tissues will be taken for PCR and histology: post esophagus (PE), digestive gland (DG) and foot muscle. We will also place PE tissue on a glass slide for fluorescent dye detection of RLOs with SYBR green by light microscopy.

PROCEDURE: PPE: gloves, lab coat, goggles Weigh animal (g) Measure shell using calipers Record on data sheet

Perform gross examination: Look for any swollen or inflamed tissue on the animal Note any shrinkage or discoloration of foot muscle or metaplasia in PE/DG area

Perform necropsy: Flame sterilize all SS instruments by dipping in EtOH and passing through flame Remove animal from shell by using spatula-like tool to scrap the adductor muscle from the shell starting from the mantle edge. Weigh and record shell weight Orient the animal with caudal end to the left, internal organs up Locate the PE area of the animal Make first cut on the PE/DG separation (there should be a line of separation between the two tissue types) Make a second cut about 2-3mm to the right of the first cut. Lay the PE side down in the cassette Take another section the same way directly to the right of the cut you just made Cut this in half and place some PE tissue onto a glass slide and half into the cryovial labeled "PE/DG" Cut the DG out for easier handling Cut a mid-section (same width as previous sections) DG Place in cassette posterior side down Take another piece of DG and place in cryovial labeled "DG" Cut through the middle of foot muscle and take a 2-3mm section for histology Place section in cassette Cut another piece of foot muscle and place into cryovial for PCR Place sponge on top of tissues inside the cassette and close the lid Place labeled cassette into Davidson's fixative for 24hr After 24hrs, pour off fixative and store in 70% ethanol for storage Dispose of animal and parts into biohazard waste Bleach down bench and clean up

Fluorescent Dye Detection of RLOs: Place glass slide with PE on bench and heat gently with hair dryer for 15-20 mins to heat fix tissue Circle the tissue with PAP pen Flood the tissue with SYBR green dye (1:1000 with DI water) Incubate for 1 min at room temp Place coverslip onto tissue View under microscope Look for fluorescent oval bodies

RESULTS: Abalone Accession Number: 10:8-8 Total Lgth: 54.11 mm Total Wgt: 21.8 Shell Wgt: 5.85 Total Body Wgt: 15.95

Gross Examination: The animal is very active. No discoloration or atrophy of the foot muscle is apparent. The animal is normal in appearance and behavior. It keeps looking at me.

Necropsy: Internal organs appeared normal. No significant findings.

Tissues taken for histology and PCR: Post Esophagus Digestive Gland Foot Muscle

Fluorescent Dye Detection of RLOs Result: Score: 0 (absent) Tissue: Most of the PE was gone after sampling for histo and PCR, most of the tissue on the slide appeared to be DG. No RLOs or any other significant organisms were seen in my PE slide.

Class Results:[|RLOs][|Unknown Org]

**4-1-10 Intro to Histology**
SUMMARY: General introduction to histology. Tour of histology instruments and procedures for paraffin preparations of tissues and mounting of tissue sections to glass slides.

PROCEDURE: Perform gross examination before necropsy Look over necropsy protocol Take necessary measurements of animal Cut small sections for histology (tissue section sizes may vary according to protocol/animal/organ) Place tissues of interest in cassette Chemically fix tissue with formalin or glutaraldehyde (again, may vary) for a specified time Process for histology: Ascending gradient of EtOH Hydrophobic xylene used to clear EtOH Paraffin impregnation of cassette Sectioning performed with microtome Deparaffination process Descending gradient of EtOH to rehydrate tissue Mount section on slide Stain Coverslip

RESULTS We did not go through this process in lab, but will perform necropsies next lab section.