A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

The overall goal of the following experiment is to introduce a miniaturized algal bioassay aimed at rapidly screening algal cultures to identify microbial symbiance and or pathogens that might influence the health or physiology of the algal culture. This is achieved by first making control mixtures of algae and bacteria with media and making experimental algal bacteria co-culture at various concentrations. The next step is to prepare 48 well plates with aliquots of the mixtures for a robust replicable and rapid screen.

Next, the plates are incubated and monitored using pulse amplitude modulated fluorometry, which takes measurements that infer the photosynthetic health of the system. The results of this time course experiment reveal a pathogenic, symbiotic, or neutral effect of the bacteria on the algae. Additional small volume screens can also be applied during the time course experiment.

The main advantage of this technique over conventional methods that employ large volumes for algal bioassays is that the miniaturized bioassay allows for small volumes, which allows for increased replication. This method was originally developed to provide insight into an alga response to a bacterial pathogen or symbiant, but it can also be applied to answering questions about the effect of a chemical bioactive molecule or nutrient on analogous health and physiology. Initially, individuals new to this method might struggle with aliquot and algal samples into the miniaturized micro teeter plate format because this method requires careful aseptic technique and good experimental planning in order to succeed.

Time the experiment. So bacteria grown to stationary phase and algal cells grown to early log phase already at the same time. It is crucial to have a good understanding of the bacterial and algal strains before starting an experiment.

It's important to know when early log phase is, as well as the initial concentrations for the bacterial and lgal strains, and the time necessary for dark adaptation. When using Pam To prepare the bacteria, wash any biofilm present on the glass test tube down into the bacterial culture, then take one milliliter of the culture and a septically. Transfer it to a micro centrifuge tube.

Centrifuge the micro centrifuge tube for a minute. At 14, 000 gs, remove the supernatant without disturbing the palate and resuspend the palate in one milliliter of sterile algo media such as L one, vortex the tube to get a homogeneous suspension and then wash the bacterial cells once more by centrifuging, removing the supernatant and resus suspending the pellet in sterile media. Washing with lgal media thoroughly removes the bacterial media detritus and small molecules, which is critical to downstream steps.

After these two washes, dilute the bacterial cells in almedia using a serial dilution so that they are 100 times more concentrated than needed for a one-to-one culture with the lgal cells. Add a volume of this 100 X culture to sterile almedia such that the final concentration of bacterial cells is one x or equal to that of the lgal cells. This culture is referred to as the one x bacterial stock.

For the next step, have three labeled autoclave conical flasks at the ready. Now transfer the calculated control volume of the one x bacterial stock to the bacterial control flask and add an equal volume of sterile almedia. Next, transfer the calculated volume of one X bacterial stock to the co-culture flask.

Now use a wide mouth pipette to mix the early log algal culture. Then with a 10 milliliter pipette, transfer the control volume to the algal control flask, followed by an equal volume of algal media. Finally, transfer the calculated volume of algal culture to the co-culture flask.

First, divide a sterile 48 well microtiter plate is indicated. Keep the labels above the outer wells containing the diluent or non-photo synthetic samples. Load the appropriate wells with one milliliter of PBS to minimize edge effects and evaporation.

Then add one milliliter of properly diluted bacterial control culture to the bacterial control wells. Remember to mix the flask before taking an aliquot. Next, add one milliliter of algal control culture, bacterial control culture, and algal bacterial co-culture to the appropriate wells.

Again, try to only take aliquots from a uniform suspension. Now seal the plate with paraform and transfer it to a diurnal incubator. Arrange all the plates in the same orientation to get uniform light exposure and be sure that the middle of the dark cycle arrives at the same time during the workday.

Next, prepare the contamination test plates. Transfer 20 microliter aliquots of algal media and algal control to 1.5%auger plates made with a non-selective medium like marine broth. 2 2 1 6.

Incubate the auger plate at 30 degrees Celsius for 24 to 72 hours depending on the bacteria if any unexpected growth appears. Do not use data from the micro microtiter plate. Keep the remaining algal control and co-culture samples in the dark diurnal incubator until ready to take the day.Zero.

PAM measurements. Perform this measurement during the middle of the algal culture's dark cycle. To begin, zero.

The water pam machine with sterile algal media in a clean vete to measure the day zero samples pipette 300 microliters of algal control culture or co-culture into a clean vete containing 2.7 milliliters of sterile lgal media and mix the Q vet contents with a wide mouth pipette. Load the Q vet into the water Pam, cover the sample and let it dark. Adapt for three minutes.

This time can vary depending on algal strain, but should not be more than 20 minutes. Then take a reading by pressing F zero. If either the F zero or FM reading is above 3, 900, dilute the sample by 50%with sterile algal media and repeat the reading after another three minute dark adaptation period.

Repeat this process until a reading below 3, 900 is obtained. Then multiply up the value as required. With the F zero value set.

Take a saturating pulse reading every 90 seconds by pressing the SAT button. The time interval can vary with algal strain. To take measurements from a microtiter plate, prepare six sample tubes with 2.7 milliliters of algal media and transfer them into the proper diurnal incubator For 30 minutes to equilibrate to the desired temperature, remove the microtiter plate ensuring that the wells remain in the dark by wrapping the plate with foil and leaving them covered as much as possible.

Using wide mouth tips slowly mix 300 microliter aliquots from wells B2B three, B four, C two C3, and C four. Load the aliquots to the six sample tubes and take the PAM water readings as before using the described protocol. Algal fluorescence was measured by water Pam when cultured alone compared to when co cultured with bacteria over 10 days in the co cultures, there was a dramatic decline in the health of photo system two, the primary electronic between five and 10 days.

The maximum fluorescence reading was taken directly after a saturating LED pulse. In this example, lgal cells with healthy chlorophyll have a high fm, but the chlorophyll of the lgal cells in co-culture are negatively impacted by the bacteria later in the time course Once mastered. This technique can be done in as little as 20 minutes per three well set for continuous sampling over a time course experiment.

While executing this procedure, it's important to remember that different algal strains have different diurnal cycles, and that experimental measurements should always be taken at the midpoint of the dark cycle for each algal strain.