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.