Was it a success?

Yes. I made progress and found a potential colony that might be the same Lactobacillus casei shirota which I inoculated the fish with. I cannot be certain, however, until I am able to perform the 16s ribosomal RNA test. If I cannot, someone else can easily conform or deny it next semester if the sample can be preserved that long.

Initially, my goal was to see if I could feed the fish Lactobacillus casei shirota with varying amounts of fructooligosaccharides and measure the concentration in their feces. I assumed (and was going to verify) that the more fructooligosaccharides I added, the higher  the concentration would be found in their fecal matter. Unfortunately, one fish didn't survive and the other won't eat the pellets I feed it (he literally spits them out). I also wanted to identify a similar bacteria already resident in the digestive tract and compare its concentration as I fed the fish the Lactobacillus casei shirota.

At the very least, I can show that the Lactobacillus casei shirota survived its digestive tract and could be isolated and identified (assuming 16s is positive). The biochemical tests I performed on fecal sample colony I isolated (g+ rod cat neg) matched 10 unique tests. However, there is a common bacteria found in fish called Erysipelothrix rhusiopathiae that shares many similar characteristics with Lactobacillus casei shirota. So many of the tests like indole, catalase etc. are the same. The morphology differs slightly under the microscope, but I'm personally not able to differentiate between the two in practice yet. Erysipelothrix rhusiopathiae is, however, supposedly set apart form Lactobacillus spp. by its positive H2S production on TSI. I only performed this test one time for each and I saw no positive signs of H2S. Also, literature states that it produces acid but -no gas- in lactose and glucose. My sample produced gas, so that's another strike against Erysipelothrix rhusiopathiae, however, it needs to be repeated and verified. Below is a photo of the lactose test of the wild sample on the left vs. the control Lactobacillus casei shirota. I'm not sure the camera could pick up the gas bubbles, but I still have these samples for verification.

Possible results in the final hour.

I got some promising results from my motility and thioglycollate tests. Both tests results from the experimental fish's resembled those of the positive lactobacillus casei which I use. I'm going to retest the glucose and lactose because although they tested positive for acid production, there was not a positive CO2 that I could see. The colonies also looked similar to my L. casei, but I'll have to stain those tomorrow. I change the tank water out twice a week (partially) and condition it once a week. I also use a large pipette to remove any debris I see (food, feces etc.). I do this equally, yet the experimental fish's tank always goes cloudy far faster than the others. Maybe the food is affecting his digestion? I also tested 3 concentrations of fructooligosaccharides in TSA with L. casei shirota. I'll use the photospectrometer tomorrow to see how significant the absorbance at 600nm (optimum wavelength my bacteria absorb theoretically) differs. I've done this once before with control versus an arbitrary amount and it was shown to increase the turbidity and absorbance. The pictures below show my Lactobacillus thioglycollate results on the right, which I'll use to compare to the potential sample from my experimental fish. The tube on the left is some other unknown bacteria that seems to be aerotolerant. Ignore that one - that was one I found previously which was a gram positive catalase positive rod.

No clear signs yet.

I've been feeding the experimental fish Lactobacilli for a few weeks now. There has been no sign of live Lactobacilli in their fecal matter. I've read that this is also the case commonly in human studies of lactobacilli consumption. The populations are better assessed directly from fluid in the intestines. I'd have to dissect the fish, which I'm opposed to unless they've already died. I could change my goal a bit and see if lactobacilli supplementation affects the concentration of the microbes I commonly find in their fecal matter. I'd have to consider the bacteria that are living within the tank water that have come from other sources however.

I did have some promising colonies today (Thursday) after looking at the morphology. There were a variety of nicely isolated colonies of varying color, elevation etc. A few resembled the Lactobacillus casei shirota I began with. I ID'd them as gram positive rods with proportions also similar to my strain. We'll see tomorrow after the lactose and other tests.

Here's an example of a gram Positive (short)rod I found (not lactobacillus). I like the honeycomb effect that is created as they chain together.

Narrowing down tank bacteria

I've been feeding my fish Lactobacillus on both flakes and pellets. However, I haven't been finding any Lactobacillus in their feces. I tested the tank pH with a calibrated meter and recorded a value of 6.5 (+/- .2pH). Various species of Lactobacillus grow best at acid pH values. The tank water is within this range. The control tank had a measured pH of approximately 7.5 - 8 (measurements fluctuated with location). I inoculated an MRS plate to test growth. If there are Lactobacillus growing within the experimental tank, I wonder what they are consuming (flake particles?).

The sample in the experimental tank were Gram negative catalase negative rods that showed positive lactose fermentation (acid production) in double strength solution. They also were positive for gas production within the lactose. This colony may be Erysipelothrix rhusiopathiae which is common in fish. Although they share some characteristics, they differ significantly in length - which I'll measure tomorrow. The Lactose fermentation was weak - which also suggests it is Erysipelothrix rhusiopathiae. The Bergey manual also suggests a glucose test to help differentiate them.

Below is the Blood Agar result which suggests Erysipelothrix rhusiopathiae. (alpha hemolysis)

Experimental fish is finicky

Last week I switched from fish flakes to floating pellet food. The two control fish eat them, and the first experimental fish also. The second experimental fish avoids them. I'm wondering if they are too large or have a different bad taste. I tried cutting them into smaller pieces, but I didn't wait around long enough to witness him eating them The pellets soak up the TSB with Lactobacillus very well compared with the flakes.

I had various growth on my MRS plates when I inoculated them with fish feces. I need to rule out the species with endospores. The remaining species won't be Lactobacillus, unless it was endogenous. I'm wondering if I should simplify my experiment as isolation is proving difficult with wild samples.

 In the mean time I made quite a bit of TSB. It's stored in our fridge and available to anyone who needs it. I learned (flip a switch and set a dial) how to use the aliquot machine to dispense it into vials.

First week of modifed feeding

This week I began feeding one of my experimental fish Lacto bacillus Casei Shirota. I'm starting with fish food flakes by coating them with growth from a MRS agar plate. I also tried coating them with TSB, but the flakes wilted quickly on contact with liquid.

I wanted to suspend the bacteria within a gel, so I cooked up some gelatin and embedded some bacteria within and on top; neither survived to the next day. Sure, they wouldn't grow on the gelatin, but I figured they'd last a bit longer. Maybe I could add some sugar to the mix. Embedding them in the gel might help deliver them into the fish effectively without loss into the water. If that doesn't work I'll try coating floating pellets which might absorb inoculated TSB.

I transferred the two control fish (I got an extra for free) into a smaller tank that has a similar environment to the other two tanks. I was advised this would be better than keeping them in the large tank. I wish I could keep them all in larger tanks so they could feel more comfortable and live longer, but I don't have that option at the moment.

The two control fish are in the leftmost tank. The large tank in the bank is out of commission. Surprisingly, it was pretty clean without a filter.

Fish food for preserving fish for food.

My star experimental fish 'Beck' died over spring break. Beck was going to be the fish that received the L. bacillus and fructooligosaccharide FOS. I noticed that the fish named 'Stache' is relatively fast and aggressive when feeding. Maybe Beck starved because of Stache. This won't matter because Beck's successor "Snowball II" will go to a separate tank. At the moment I'll be performing the experiment as follows:

Main tank:
Fish1 - Control - standard fish food
Fish2 - Stache - standard fish food


Surplus fish go here.

Secondary tank: with water and rock from main tank.
Fish3 - ET - standard fish food and L. Bacillus

Tertiary tank: with water and rock from main tank.
Snowball II - standard fish food plus enriched food with L. Bacillus and FOS.
Probably Lactobacillus casei shirota because that grew the best in my MRS plates.

I might use 'Stache' to test larger amounts of FOS if I can get another bowl/tank.
Or, I might try a different species of Lactobacillus.

All of the fish will be receiving the standard fish food. However, ET and Snowball II will be fed additional gelatin confections. I am making these by mixing gelatin with FOS and Lactobacillus and letting it cool. It can be cut into small pieces as needed. I need to keep the water in the mixture warm, but not too warm; I don't want to kill off the probiotics.