This laboratory module focuses on Littorina, trematodes, and the associated microbial communities. Field collection is scheduled for Tuesday of Week 1. Samples will be the basis for much of the lab work during Week 1.

| Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6

Littorina Data Collection Sheet



Day 1


Assess infestations using dissecting scope and phase contrast microscopy. View histology sections of littorines + trematodes


Day 2


Dissect Littorines for microbiology, preserve Littorina / trematode tissue for later RNA extraction.
- plate counts and ID using selective and non-selective media
- PCR (microbiology)


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

1. Make a serial dilution of your bacteria
    1. Label each of the cultures tubes with your 1:10 dilutions (10-1, 10-2, 10-3, 10-4,
10-5, 10-6)
    1. Start dilution series by taking 10mL of stock bacterial suspension and adding it to 90mL of broth in a tube labeled 10-1. This is your first 10-fold dilution. Mix extremely well.
    2. Next take 10mL of your 10-1 dilution tube and add to the tube labeled 10-2. This is your second dilution. Mix well.
    3. Repeat the above process until you finish the dilutions out to 10-6

  1. Obtain plates. Label each of the plates with a dilution (100 through 10-6), your name, and the date. Each student is responsible for duplicate sets of plates.

  1. Plate 0.1 mL of bacterial suspension onto the appropriate plate. If you start from the highest dilution to the lowest dilution, then you can use the same pipet.

  1. 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.

Leave on the bench right-side up for about an hour. Wrap the plates in parafilm, invert, and leave on the bench.

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)?



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.

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.

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Supplies
Sample
Inoculating loop
70% ethanol + sand
Burner
Plates
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. the plates, and you will examine the following week.

Gram Stain
    1. Select colony from your 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


DNA extraction

Qiagen DNA Stool kit Handbook



PCR (universal eubacterial primers)
Polymerase Chain Reaction involves amplifying a DNA (genomic or complementary) target using a polymerase, primers (short oligonucleotide), and dNTPs (A, C, T, Gs). In general the reaction is placed in a machine (thermocycler) where a series of temperature changes are performed [Denature (~94C), Anneal (primer specific ~50-60C), and Extention (~72C)].
For this lab you will be using Promega's GoTaq Product. Please Read!

Prepare your samples in duplicate AND make sure to include at least 2 negative controls for each primer (no template).

For a 50μl reaction volume:




27F: AGAGTTTGATCMTGGCTCAG

1492R: TACGGYTACCTTGTTACGACTT

Component
 Volume
 Final Conc.
GoTaq®Green Master Mix, 2X
 25
 1x
upstream primer, 10μM
 0.5–5.0μl
 0.1–1.0μM
downstream 10μM
 0.5–5.0μl
 0.1–1.0μM
DNA template
 1–5μl
 <250ng


Load reactions into thermocycler.

PREPARE AGAROSE GELS
1. weigh 2g of agarose and mix with 150mL 1x TAE in a 1L flask
2. microwave solution for ~ 3 minutes
3. cool solution (you should be able to touch the flask for a few seconds), then add 12uL ethidium bromide.
4. mix thoroughly by swirling, then pour into gel tray.
5. add gel combs
6. after gel is set, wrap in plastic wrap and place gel in the fridge for next week.



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Warning UV light used to visualize DNA is hazardous.




Day 3


- plate counts
- transfer colonies
- RNA Extractions to examine Host and Pathogen gene expression

List of Supplies and Equipment




Background - RNA Extraction
You will isolate RNA from whole tissue using TriReagent. TriReagent allows for separation of RNA from other cellular components, including DNA. There are three primary components of TriReagent that allow this to happen. The first is guanidine isothiocyanate which is a potent protein denaturant, the second is phenol, and the third is pH.

Guanidine isothiocyanate denatures proteins, such as the highly abundant histones that coat DNA. Even more importantly, RNases are denatured. This denaturing action allows for better access of phenol (an organic solvent) to cellular proteins and improves its ability to keep the proteins insoluble. The pH of TriReagent is acidic. The low pH keeps DNA out of solution while RNA remains soluble.

After homogenizing/lysing your tissue in TriReagent, chloroform (another organic solvent) will be added to your sample to allow for separation of the phenol and insoluble cellular components (DNA, proteins) from soluble cellular components (RNA). This will result in three distinct layers: the organic phase (the bottom portion), the interphase (layer of cell debris) and the aqueous phase (the top portion). The aqueous phase (the RNA) can then be easily isolated.

The RNA can be precipitated and washed to remove residual phenol and salt carryover. Then the RNA can be resuspended in a suitable solution and quantitated.

RNA is quantitated using a spectrophotometer and measuring the absorbance of your RNA sample at 260nm (A260). The concentration of your sample is calculated with the following equation:
[RNA] = 40ug/mL x A260 x Dilution Factor

In addition to the A260, absorbance at 230nm and 280nm should be taken. The ratio of A260 to each of these absorbances can be used to assess the purity of your RNA. Various substances will absorb at 230nm, which will indicate carryover of phenol, ethanol or high salt in your sample. Proteins generally absorb light at 280nm. For clean RNA, A260/A280 should range between 1.8-2.0. The A260/A230 should range between 1.5-2.0 for clean RNA.

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IMPORTANT NOTES

1. Wear clean gloves - For your own safety as well as the integrity of your RNA samples, you must wear gloves throughout this week's lab. Phenol and chloroform are nasty, cuastic chemicals, so gloves are necessary when handling anything that comes in contact with either reagent. Additionally, RNases are constantly secreted from your skin and can easily enter, and subsequently degrade, your RNA sample.

2. Phenol/Chloroform Handling and Disposal -
A. Handling - You must wear gloves, safety glasses and lab coats at all times! These chemicals have potential to do damage to clothing and exposed body parts. TriReagent may be used on the benchtop, but be aware that it is caustic, very volatile and has a very strong odor. Chloroform must only be used in a fume hood! It is extremely caustic, volatile, and inhalation of fumes can be dangerous.
B. Disposal - All tips/tubes/gloves that come in contact with phenol/chloroform must be disposed of in the "Solid Phenol/Chloroform Waste" container found in the fume hood. None of this type of waste should be discarded in regular trash! Any liquids that have phenol/chloroform must be disposed of in the "Liquid Phenol/Chloroform Waste" container found in the fume hood. None of this type of waste should be disposed of down the drain or in the regular trash!

3. RNA Handling - Due to the prevalence of RNases, gloves should be worn at all times when handling your samples. Samples should also be stored on ice at all times (to reduce the activity of any contaminating RNases remaining in your sample), unless otherwise noted.

4. Razor Blades Handling and Disposal -
A. Handling - Obviously, these are extremely sharp. Use them with extreme caution. Pay careful attention to what you are cutting. Only cut tissue that is on a flat, stable surface. Do NOT attempt to cut anything with a razor blade while holding the object in your hand!
B. Disposal - Razor blades MUST be disposed of in the available "Sharps" container! The "Sharps" container is bright red and easily visible. If you cannot find it, ask the TA. Under no circumstances are razor blades to be disposed of in the regular trash!


RNA ISOLATION PROTOCOL
1. Turn on heating block to 55C. Also turn on spectrophotometer.
2. Add 500uL of TriReagent to a 1.5mL snap cap tube. Store on ice.
3. Cut a piece of frozen tissue weighing between 50-100mg and add to tube containing TriReagent.
4. Carefully homogenize the tissue using a disposable pestle.
5. Add an additional 500uL of TriReagent to the tube and close the tube.
6. Vortex vigorously for 15s.
7. Incubate tube at room temperature (RT) for 5 mins.
8. In the fume hood, add 200uL of chloroform to your sample and close the tube.
NOTE: Due to the high volatility of chloroform, pipetting needs to be done carefully and quickly. Have your tube open and close to the container of chloroform before drawing and chloroform into your pipette tip.
9. Vortex vigorously for 30s. You are vortexing correctly if the solution becomes a milky emulsion.
10. Incubate tube at RT for 5 mins.
11. Spin tube in refrigerated microfuge for 15 mins. @ max speed.
12. Gently remove tube from microfuge. Be sure not to disturb the tube.
13. Slowly and carefully transfer most of the aqueous phase (the top, clear portion) to a fresh microfuge tube. Do NOT transfer ANY of the interphase (the white, cell debris between the aqueous and organic phase).
14. Close the tube containing the organic and interphase and properly dispose of the liquid inside the tube as well as the tube itself at the end of the lab.
15. Add 500uL isopropanol to the new tube containing your RNA and close the tube.
16. Mix by inverting the tube numerous times until the solution appears uniform. Pay particular attention to the appearance of the solution along the edge of the tube. If mixed properly, it should no longer appear viscous/"lumpy".
17. Incubate at RT for 10 mins.
18. Spin in refrigerated microfuge at max speed for 8 mins.
19. A small, white pellet (RNA and salts) should be present. If not, do not fret. Continue with procedure.
20. Remove supernatant.
21. Add 1mL of 75% EtOH to pellet. Close tube and vortex briefly to dislodge pellet from the side of the tube. If the pellet does not become dislodged, that is OK.
22. Spin in refrigerated microfuge at 7500g for 5mins.
23. Carefully remove supernatant. Pellet may be very loose. Make sure not to remove pellet!
24. Briefly spin tube (~15s) to pool residual EtOH.
25. Using a small bore pipette tip (P20 or P200 tips), remove remaining EtOH.
26. Leave tube open and allow pellet to dry at RT for no more than 5mins.
27. Resuspend pellet in 100uL of 0.1%DEPC-H2O by pipetting up and down until pellet is dissolved.
28. Incubated tube at 55C for 5mins. to help solubilize RNA.
29. Remove tube from heat, flick a few times to mix and place sample on ice. This will be your stock RNA sample.
30. Quantitate RNA yield using spectrophotometer (we will be using the Nanodrop).

RNA QUANTIFICATION
NOTE: Always keep your RNA samples on ice!
1. Pipette 2µL of 0.1%DEPC-H20 onto the Nanodrop pedistal and lower the arm.
2. Click "Blank", to zero the instrument
NOTE: steps 1 and 2 only need to be done once for the whole class.
3. Pipette 2µL of your RNA sample onto the Nanodrop pedestal and lower the arm
4. Click "Measure". Record your A260 absorbance, RNA concentration (ng/µL), A260/280 ratio and A260/320 ratio.
NOTE: The Nanodrop uses the Beer-Lambert Law to calculate RNA concentration for you. See Lab 1 notes on RNA extraction for more information on the calculation and how to evaluate RNA purity using A260/280 and A260/A320 ratios.
5. Raise the arm and wipe off you sample with a Kim Wiple
6. Clearly label your stock RNA sample with the word "RNA", source organism/tissue, your initials, today's date and the concentration in ug/uL.
7. Give your samples to Mac for storage at -80C.

Day 4


- Complete RNA Extractions
- Innoculate API strips
- Vibrio tubiashii assays
- Reverse Transcription
- Quantitative PCR



Vibrio tubiashii assays

Serum Agglutination Test
a. Add 50 uL of sterile seawater and 25 uL of your culture to one slide (control).
b. To another 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 tips (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.

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.





REVERSE TRANSCRIPTION PROTOCOL
1. Mix your stock RNA sample by inverting tube several times.
2. Transfer 25ug of your RNA (.25ug of mRNA) to a fresh PCR tube. Bring the volume up to 5uL with PCR water. If necessary, spin tube briefly to pool liquid.
3. Incubate tube at 75C for 5mins in thermal cycler.
4. Transfer tube IMMEDIATELY to ice and incubate for at least 5mins.
5. Make Master Mix (MM)

PER RXN
4 ul 5x Buffer (AMV RT Buffer)
8 ul dNTPs (10 mM total)
1 ul AMV RTranscriptase
1 ul Oligo dT Primer
1 ul RNase free water
Total = 15 ul

  1. Add MM to tube with diluted RNA in it (total volume now 20 ul)
  2. Vortex
  3. Spot spin
  4. Incubate at RT for 10 min
  5. Incubate at 37C for 1 hr in thermocycler
  6. Heat inactivate @ 95C for 3 min
  7. Spot spin
  8. Leave cDNA on ice or store at –20C



Putative primers for Littorina qPCR



Background Information
Lec16 Realtime PCR
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For gene expression analysis
Arbitrary expression value =10^(-(0.3012*Ct)+11.434)

Day 5

- read API strips, Continue QPCR


Day 6

- read 48 hr API strips