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.

Floating fish food flakes

Many of you've probably noticed that the fish tank was partitioned into 3 areas using plastic trays. I drilled holes in them thinking this would promote circulation of O2 and keep the composition homogenous. There were a few problems. I had the bubbler in the middle on the bottom hoping it would diffuse. But, of course it just mostly went up and out like it should. This led to hypoxia in the side areas and very 'depressed' fish. Also, when the bubbler was in the middle, it would push the floating fish food flakes out to the two sides too quickly so the fish in the middle couldn't get a chance to eat. I'm letting them live together until I put them in 3 separate beakers Josh got me. That'll solve the problem. It may lead to slightly different environments - pH, ammonia etc. It should however, help isolate types of bacterial growth, so any introduced lactobacillus does not spread between fish unintentionally.

I made my MRS agar plates (encourages growth of lactobacillus while suppressing other types). It is clearly much better after one day compared with my previous lactobacillus growth on TSA which took a few days with the same species. I also tested the fish tank water in TSB and got a surprising amount of growth. Moreso than I remember getting with Encanto park water.

As for the floating fish food flakes - I need to get some floating pellets instead. I need something that I can inoculate and enrich easier (and also which floats). 

One Fish, Two Fish, Orange Fish and a slightly off-white one with dark coloring that resembles a moustache.

My fish came in this week. I've got 3 Gold Fish to work with - two have names. Most of the related research I've seen involves much larger fish like catfish. They've got a relatively large tank to live in, but no privacy. I put a tray in front of the tank to at least give them privacy from the human activity going on in the lab for now. This should extend their life and comfort. I also have a small bowl I'm going to be using to transfer then to while I attempt to collect their fecal matter. I'm hoping the excitement of moving into a new environment will encourage to cooperate and make my collection easier. I've seen some overly complex methods of collection in my research. I'll begin with simply using a net or screen hanging down to catch it. I'm also wondering if I could feed them something that would make the consistency of their fecal matter more conducive to collection.

It's interesting to note that some Lactobacillus species naturally produce bacteriocins that are harmful to bacteria like MRSA.

Growing lactobacilli in fish with varying diet.

I'm still focusing in on a specific research question, however, I have come up with a general idea. I'll be using at least two fish (probably goldfish) in the same tank. I'll either use some sort of dividing structure to keep them separated but keep the environments basically the same (water flow between). I won't have to do this if I can obtain fish which are uniquely identifiable; we'll see. I'll have two food sources, both of which will be inoculated with lactobacilli (need to choose species - there's quite a few). One food source will be mixed/enriched with a nutrient like fructooligosaccharide. Basically something that should encourage lactobacilli growth. One fish will get the food with the nutrient/bacteria and the other will get food with no nutrient and the same amount of bacteria.

Why would anyone do this? Just like as in the human gut, fish have a wide variety of bacteria. Some are beneficial, some not. The idea is to encourage dominance of beneficial bacteria in the fish so that when consumed by humans, the risk of food-borne illness is reduced. There's still going to be bacteria on the outside of the fish, but I believe (have to research more) the gut bacteria in the fish pose a larger problem. I'll be testing to see whether the nutrient enriched food yields more growth.

I inoculated a TSA plate with store bought yogurt containing probiotics and after two days this grew:

 I didn't see any other colonies other than this one. Gram Positive, catalase-negative rods. No glucose fermentation or motility results yet. But, most likely Lactobacillus casei.

On related note, supposedly goldfish aren't picky eaters. I might see if they will eat Labneh. I do, and I seem to stay healthy!

Fish and a polluted environment.

At the moment, I'm collecting and isolating different bacteria off of crickets. I've only got as far as gram staining them but, the stains look pretty interesting under the microscope. The most interesting so far are gram negative cocci which form colonies that look like flowers under the dissecting stereo-microscope (good for colony morphology). Their structure is really interesting (to me anyway) when stained and observed at 100x; they are bunched together to form pentagons (roughly) with space in the middle and the cells forming the boundaries. Maybe this is common, but I've never seen it; it seems very ordered and symmetric. I made a permanent slide of it today with cytoseal which should be ready to view tomorrow via oil immersion and maybe the video scope. I'll be sure to post some photos. FYI, if you're trying to collect bacterial samples my best results were with Peptone using 100-300 microL dilutions worked well to gather bacteria from the crickets. The direct swabs yield a useless mass of growth where it was too difficult to isolate anything.

I was speaking with one of our instructors today about the focus of my research this semester and he suggested perhaps working with either fish and a polluted (with one specific pollutant) environment vs unpolluted. If we can't get fish in the lab, I'll try crickets with polluted water sources or maybe even plants. One possible pollutant could be 4-Methylcyclohexanemethanol - which was in the news recently from West Virginia as a side effect of coal processing. It's not entirely understood how this affects humans. I'll do some reading how it might affect anything else. A fun side note - we just learned how to name organic compounds like this in o-chem, which isn't as bad as it looks. I could also continue working from 182 with eutrophication. Maybe I could test how it affects bacterial growth in organisms with in the waters. 

I also identified my unknown bacteria today: Micrococcus luteus. I did an extra motility test because although it wasn't needed, I read they are highly motile and wanted to see this. Also, I'm going to try to extract dna from the sample and see if it matches. Below are photos of my sample of Micrococcus luteus. on isolation TSA, lawn TSA, MSA (negative for manitol fermentation) plate, and a pre-iodine starch plate (was negative).

Lactose Intolerant crickets aided by probiotics?

I was thinking of various ideas relating to crickets and their microbiomes. Specifically, in their digestive tract. Many people are lactose intolerant, so I'm assuming crickets haven't evolved an efficient way to break down dairy products. But what if they ingested bacteria that could? Could the bacteria survive within the cricket? Would the crickets that were fed diary and had the beneficial bacteria thrive more than those without the bacteria that ate dairy? Also, how would the crickets that never dairy at all do compared with the ones that did? I'm not sure if this is a practical experiment yet, but we'll see.

I also noticed in reading some anecdotal reports that bearded dragons could be fed yogurt and not have any ill-effects. I wonder if any creature that doesn’t normally have enzymes to digest lactose and related proteins could acquire certain bacteria that could and live relatively well. Lifespan and activity level might be good variables that would be affected by their diary diet.

At the moment, I’m simply trying to find a good way to collect bacteria samples from a very small cricket without creating a huge mess. I’ve literally streaked the plate with a cricket, embedded a cricket into agar, and finally used peptone to separate the cricket into a solution that was distributed onto a TSA plate.

 

Fundamentals of fundamental particles

This month's Scientific American has an article suggesting that a proton's radius might not be known to an accuracy which was previously assumed. In all our chemistry and physics books we have all sorts of values for all sorts of masses, radii etc. Each to a seemingly razor (a very tiny one) sharp accuracy. This recent article suggests that while the group of scientists went in thinking they were improving the accuracy of the proton radius, they obtain much different results.

They claim that the odds of it being a mistake are quite slim.
One interesting part of info in this article says that electrons have a non zero probability of existing -inside- the proton because of their wave nature. It would be useful, then, to know the radius of a proton so that you can determine the effects inner electrons have when their are within or very close to the proton. The researchers used particles called muons to measure the radius by bombarding it and measuring the wavelengths of virtual (I don't understand virtual particles) photons emitted. They used muons because they seem to spend more time within the proton according to their wave function and make it easier to measure the proton radius. They used very specific energy levels that should exist for electrons in certain orbitals. The author describes changing the 'atom's' energy state from 2s to 2p, but we were taught that it was the electron that moved to that state. Maybe it's just phrasing.

The wave function view of an atom is thought to better describe and predict behavior of small particles.

Taken from: http://faculty.wcas.northwestern.edu

Anyhow, it's neat to see there are still very fundamental properties being discovered. I think discoveries like this are great for moral in the sciences because they show the scientific method and verification works. Many of us could discover new useful and interesting biological interactions in our studies and work.

Another recent fundamental discovery involved creating stem cells by adding acids to normal cells in mice. Whether we directly contribute to new discoveries or simply verify existing theories, it's all useful to our society. This semester I hope to work with any type of genetic analysis so that I can explore the large field of DNA analysis, evolution and how it ties in to disease. Also of interest to me is human microbiome and it's relation to health.


This article can be viewed through ebsco at our library in the February 2014 Scientific American:

The Proton Radius Problem. By: Bernauer, Jan C., Pohl, Randolf, Scientific American, 00368733, Feb2014, Vol. 310, Issue 2