Week 3 This week will focus on a case study/trial project to continue teaching host response methods using a week-long study in which we will expose oyster larvae to a bacterial pathogen (Vibrio tubiashii) and compare with unexposed controls at 2 pCO2s (380 ppm and 840ppm)



Bacterial Pathogens of Shellfish


Shellfish are important species for our growing marine shellfish aquaculture industry and play critical roles in our marine ecosystems, an environment that is increasingly threatened by environmental change. In the Pacific Northwest the environment has changed in a manner that has contributed to increase mortality of bivalve larvae in hatcheries and also appears to have decreased natural recruitment. Several local shellfish hatcheries, upon which nearly the entire bivalve culture industry relies, have experienced severe losses (e.g. up to 59%) over the past two years. Several factors have been attributed to this problem including temperature, ocean acidification, and re-emerging pathogens. Given the large-scale environmental change observed in our marine ecosystems and the relationship of host response and pathogen virulence with environmental conditions, it is critical to examine the problems facing bivalve larvae.

A Case Study

A local commercial oyster grower has been experiencing mortality in their larval cultures. A disease specialist working with the grower isolated a strain of bacteria from the larval oysters. He has sent us the strain of bacteria to test the pathogenicity of the isolate in the lab. We will also do some phenotypic characterization of the bacteria. In this laboratory exercise we would like to address the following questions:

  1. Could this bacterium be responsible for the mortality?
  2. Using phenotypic characterization, what type of bacteria do we have?
  3. Do environmental factors (ie: pH) influence pathogenicity?
  4. What should the oyster grower do about it?

To answer these questions, you will need to do some experiments. First, you will use Koch’s postulates to confirm the bacterial culture is capable of reproducing the disease.

Koch’s Postulates:

1. The causative agent must be present in every case of the disease and must not be present in healthy animals.

2. The pathogen must be isolated from the diseased host animal and must be grown in pure culture.

3. The same disease must be produced when microbes from the pure culture are inoculated into healthy susceptible animals.

4. The same pathogen must be recoverable once again from this artificially infected animal and must be grown in pure culture.

In this experiment you will challenge oyster larvae with the bacterial strain you were sent to assess its pathogenicity. We will conduct a LD50 (lethal dose to 50% mortality) experiment at two different pH using oyster larvae. You will monitor the larvae for signs of morbidity or mortality on the following day. You will attempt to re-isolate the bacteria from any dead larvae and then conduct some preliminary phenotypic characterization on any bacteria you recover. These characterizations should give you a tentative identification of the bacteria recovered. Finally, you will be required to write a case report, which includes background, the methods you used, results, and a discussion that will outline your recommendations on how to control the problem. It would also be interesting for you to speculate on further experiments that would help you to further characterize the bacterial strain.

Lab 1. Larval challenge using unknown bacterial strain
Serial dilutions of bacteria
Challenge larvae with serial dilutions
Return at day 1, assess larval health, and attempt to re-isolate bacteria

Lab 2. Analyze LD50 data and attempt to identify bacteria

Compare the relative pathogenicity of the challenge methods
Plate counts
Examine microscopically by wet mount and Gram stain
Antibody assay
Protease assay
Review data to date in preparation for writing your case report

LAB 1

Larval Challenge


For this exercise, the class will be split into two groups. Both groups will first begin by performing serial dilutions of the bacteria for the individual challenges.

Step 1: Serial dilutions and spread plates


Supplies

Bacterial culture
6 culture bottles
Seawater for dilutions
100mL graduated cylinder
5 T1N2 plates
2 TCBS plates
Hockey stick
Ethanol and sand bath
Parafilm

Obtain the bacteria for the challenge you will be conducting. This bacteria was grown in broth overnight. The bacteria was then washed by centrifugation and resuspended in seawater.
  1. Make a serial dilution of your bacteria (as a group)
    1. We will use 1000mL bottles to mix our dilutions (note: we will use only 150mL per tank with larvae). The bottles are filled with 675mL seawater. You will transfer 75mLs to make each dilution as outlined below.
    2. Label each of the cultures bottles with one of the following dilution labels (10-1, 10-2, 10-3, 10-4, 10-5, 10-6)
    3. Start dilution series by taking 75mL of stock bacterial suspension and adding it to the bottle labeled 10-1. This is your first 10-fold dilution. Mix extremely well.
    4. Next take 75mL of your 10-1 dilution bottle and add to the bottle labeled 10-2. This is your second dilution. Mix well.
    5. Repeat the above process until you finish the dilutions out to 10-6
  2. Obtain 5 T1N2 plates per person. Label each of the plates with a dilution (10-2 through 10-6), your name, and the date. Each student is responsible for one set of plates from the same set of dilutions (i.e. there will be 4-5 replicates of plates per group).
  3. Plate 0.1 mL of bacterial suspension onto the appropriate T1N2 plate. If you start from the highest dilution (10-6) to the lowest dilution (10-2), then you can use the same pipet.
  4. Plate 0.1 mL of bacterial suspension onto the appropriate TCBS plate. Plate only from the stock (100) and 10-1 dilutions.
  5. Dip hockey stick into ethanol sand bath and flame the hockey stick. Cool the hockey stick by touching to the media and then spread the bacterial suspension. Again starting with the highest dilution to lowest dilution on T1N2 and then continue with the TCBS plates.
  6. Leave on the bench right-side up for about an hour. Wrap the plates in parafilm, invert, and leave on the bench.

Step 2: Oyster challenge


Serial dilutions of bacteria from step 1
Oyster larvae
T1N2 and TCBS plates
Seawater

  1. Add 150 mL of bacteria from each dilution per beaker of larvae. The final beaker volume should be 300mLs so they already contain 150mLs of larvae in seawater. We will use the stock bacterial culture (100) and the dilutions of 10-2, 10-4, 10-6.

  1. On day 1 post-challenge (Friday)
    1. Assess larval mortality via a dissecting microscope
    2. Re-isolate bacteria to fulfill Koch’s postulates
  2. Make a streak plate on TCBS and T1N2. Be sure to label group, pH, tank dilution and the date on the BOTTOM of the petri plates
Recover bacteria and make a streak plate.

It is again important to use sterile technique to avoid contamination of bacterial cultures. Everything that comes into contact with the culture is first sterilized and then sterilized again before putting it back on your bench. It is very important when working with the inoculating loop that has bacteria on it, to dip it in the 70% ethanol and sand mixture before flaming. If you do not, then the bacteria in the flame can splatter and create aerosols, which is bad sterile technique and may be harmful to you or your neighbors. As mentioned previously, cultures or contaminated materials should NEVER go down the sink or into the regular trash.

First, you will make streak plates on two types of media. A streak plate is an important for not only re-isolating the bacteria but obtaining pure cultures. The idea with a streak plate is as one progresses from the first quadrant to the second, the amount of bacteria is diluted. Eventually by quadrant 4 the bacteria is so dilute that you get single colonies. This technique is outlined below and will be demonstrated in class. We will streak on two types of agar including T1N2 and TCBS (thiosulfate citrate bile salt sucrose) agar. TCBS is a selective media for different types of bacteria of the genus Vibrio. Different species of Vibrio will have different colors on the TCBS agar.





Supplies

Larval sample
Inoculating loop
70% ethanol + sand
Burner
1 T1N2 plate
1 TCBS plate

METHODS


  1. Divide your sterile petri dish into four sections using your marking pen (always label the underside of the plate). Also label the underside of the plate with your name, date, and other information that will identify the contents
  2. Flame metal loop until red. Cool loop before use by touching an unused area of plate that is still sterile.
  3. Dip your loop into the sample. Carefully lift lid of agar plate, keeping lid over plate. Streak back and forth in 1st quadrant without going over same agar twice. Put inoculating loop into 70% ethanol and sand.
  4. Flame loop as before, touch agar and streak through 1st quadrant 2-3 times and then move to 2nd quadrant and streak without going over same area twice. Place loop in 70% ethanol + sand.
  5. Flame loop again and again streak through the 2nd quadrant and then move the streak to 3rd. Flame loop and repeat until the 4th quadrant is streaked. Place loop in 70% ethanol + sand.
  6. Flame loop BEFORE putting away or setting down so you don’t contaminate the bench or your partner. Parafilm
  7. ParafPpp the plates, and you will examine the following week.


LAB 2


Step 3: Analysis and bacterial identification

1. Count spread plates and calculate concentration of starting culture


Choose a plate with 30 to 300 colonies to count. Count the colonies and multiply by the dilution. Also remember that when you plated, you only added 0.1 mL of the bacterial suspension to the plate so you must account for that dilution (which is 1/10).

CFU/mL = # colonies * dilution * plate dilution (10)

For instance, if you had 55 colonies on the 10-5 dilution plate then your final count would equal: 55 * 105 * 10 = 5.5 x 107 cfu/mL

In your notebook, record your plate counts and calculations as well as the shape, elevation and color of the colonies (e.g. circular, raised, off-white). Does it look like your serial dilutions were precise (a ten-fold reduction on each plate)?


2. Perform Gram stain on bacteria
    1. Select colony from T1N2 plate and suspend in a drop of water on a glass slide
    2. Let air dry
    3. Heat fix by briefly passing over a flame (if it’s too hot to touch the slide, you heated too much but don’t worry…carry on)
    4. On a rack, flood with crystal violet for 1 min
    5. Wash briefly in tap water to remove excess crystal violet
    6. Flood with Lugol’s or Gram’s iodine 1 min
    7. Wash briefly in tap water
    8. Immediately, de-colorize with alcohol-acetone solution until the stain runs past the lower edge of the section (this is very rapid: do not over de-colorize)
    9. Wash immediately in tap water
    10. If the section appears too blue repeat steps h and i
    11. Counterstain with safranin ~15 sec
    12. Blot dry, add immersion oil and view at 100x
Results
Gram positive bacteria............................... purple/dark blue/black
Filaments of nocardia and mycobacteria............ dark blue but may have red sheath
Gram negative bacteria………………….......... red
Nuclei ..................................................................red

3. Serum Agglutination Test
a. Add 50 uL of sterile seawater and 25 uL of your culture to one slide (control). What would be a good additional control here?
b. To one slide (your test slide) add 25 uL of sterile seawater and 25 uL of your culture and 25uL of the thawed polyclonal antibody.
c. Gently mix solutions on both slides with individual pipette tipes (gently suck up and down)
d. Incubate at room temperature for 5 mins
e. View at 10x or 4x on the scope to compare degree of agglutination of the cells.

4. Azocasein Protease Assay
(R. Elston protocol, Aquatechnics)

a. Vortex initial sample thoroughly for almost a minute (Vibrios are sticky and can cling to sample tube’s side during shipping)
b. Spread between 25 – 50 µL of the undiluted sample on a TCBS plate and also a 1:10 dilution of the sample on another TCBS plate (volume spread not important because you won’t be quantifying bacteria, just checking for presence or absence of vibrios and then testing for pathogenic vibrios)
c. Incubate plates at 25ºC for 24-48 hrs
d. Check plates for colony growth and pick any suspicious yellow colonies (feel free to test green ones in the beginning also, so that you have a negative control for the azocasein)
e. Grow colony in 5ml marine broth culture tube overnight

Test for protease:
a. Centrifuge culture tubes at 26,00 rpm for 10 min at 4C
b. Remove 100 μl of supernatant and add to a microcentrifuge tube (discard the rest of the pellet and culture supernatant)
c. Add 400 μl of 1% azocasein
d. Incubate for 30 min at 37°C
e. Stop the reaction by adding 600 μl of 10% trichloric acetic acid (TCA)
f. Incubate on ice for 30 min
g. Centrifuged at 13,000 rpm for 5 min
h. Add 200 μl of 1.8 N NaOH to a new microcentrifuge tube
i. Add 800 μl of the reaction supernatant to the tube with the NaOH

The supernatant will turn an extremely bright and obvious orange upon a positive result for the protease, it will remain whatever color the supernatant was (not always clear, might have slight yellowish tint) if it is negative for the protease
Azocasein Protease Assay use in detecting pathogenic Vibrio spp. Reference:

Hasegawa, H, Lind, E.J., Boin, M.A., Hase, C.C. The Extracellular Metalloprotease of Vibrio tubiashii Is a Major Virulence Factor for Pacific Oyster (Crassostrea gigas) Larvae. Applied and Environmental Microbiology, July 2008, p. 4101-4110, Vol. 74, No. 13.

Enzyme assays. V. tubiashii supernatants were assayed for proteolytic and hemolytic activity as previously described by Halpern et al. (2003) and Chan and Foster (1998), respectively. Proteolytic activity of the sterile filtered V. tubiashii supernatants was assessed by using azocasein. Briefly, 100 μl of supernatants were incubated with 400 μl of 1% azocasein for 30 minutes at 37 °C. The reaction was stopped by the addition of 600 μl of 10% trichloric acetic acid and incubated on ice for 30 minutes before being centrifuged at 13,000 rpm for 5 minutes. 800 μl of the supernatants from the centrifuged reactions were added to 200 μl of 1.8 N sodium hydroxide and the absorbances at 420 nm were measured in a Bio Rad SmartSpec Plus spectrophotometer. Hemolytic activity was determined by incubating 50 μl of 3.5% sheep blood (Colorado Serum Co.) in PBS with 450 μl of either neat supernatant or a ten fold dilution at 30 °C for one hour. The reactions were centrifuged at 4000 rpm for 10 minutes and the absorbances at 405 nm were measured