Author Archives: boswells

Meet my microbes! Sofia

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Two of my original plates with my serial dilutions from my soil sample. I plates my dilutions on R2A, LB, AC, and 10% TSA. I had the widest variety of shape, size and color on R2A. With later tests I also have the best growth, and the most inhibition on R2A, so the majority of later tests were done using R2A plates.

My second patch plate of the 15 colonies picked from my original R2A plates. Patches 4, 6, and 11 exhibited signs of inhibition. These are the isolates I used for further testing.

LB plates: top two are the plates used for the first test against the ESKAPE pathogens; no inhibition. Bottom left is my serial dilution plate. Bottom right is Patch plate #2.

10% TSA plates: top two are the plates used for the first test against the ESKAPE pathogens; no inhibition. Bottom left is my serial dilution plate. Bottom right is Patch plate #2. Similar growth to the R2A plates, however nothing that was patches exhibited any signs of inhibition.

AC plates: top two are the plates used for the first test against the ESKAPE pathogens; no inhibition. Bottom left is my serial dilution plate. Bottom right is Patch plate #2.

Second set of LB plates used to test against a Gm +/- strain of a “safe” ESKAPE pathogen. Lots of slimy/ gooey yellow growth, but no signs of inhibition.

Second set of AC plates used to test against a Gm +/- strain of a “safe” ESKAPE pathogen. Also had lots of slimy/ gooey yellow and orange growth and the patch plates had very large and funky shaped patches; no signs of inhibition.

R2A plates with patches to test against Gm +/- strains of “safe”ESKAPE pathogens. There was not much inhibition against the Gm+ strain, but there was signs of inhibition against the Gm – strains. This is consistent with the results of the second ESAKPE pathogen test, in which we tested against all 8 strains. Signs of inhibition were more commonly seen against the Gm – strains.

Gram stain for the Gm + control (S. epidermis)

Gram stain for the Gm – control (E. coli)

 

Gram stain for isolate #4; Gm -; appears to be a small cocci but further examination concludes that the bacteria is a very, very small and thick rod, that appears somewhat round.

Gram stain for isolate #4; Gm  (purple in color); small cocci, some are diplococci and there are a few  short streptococci (chains).

Gram stain for isolate #11; Gm – (pink in color); appears to be a small cocci but further examination concludes that the bacteria is a very, very small and thick rod, that appears somewhat round.

One of my plates from the extract test; My three isolates produced five different extracts, however none of them exhibited signs of inhibition. This is indicates that none of my isolates are antibiotic producers. Signs of inhibition may possibly be visible if incubation time was longer. Additionally, it is possible that something went wrong with the extract, which is why there was no visible inhibition. A repeat of this test could provide more definitive conclusions.

Streak plate for isolate #6 with visible single, isolated colonies.

And the name of my microbe is… Sofia

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Sequence data was provided for all three of my isolates, however isolates 4 and 6 could not be identified. I was able to identify isolate number 11 though. I ran a sequence of 677 base pairs with BLAST. The closest match that came up was Klebsiella aerogenes (also known as Enterobacter aerogenes), with 98% identity and 10% query coverage (Accession number NR_102493.2).

Klebsiella aerogenes is a very small, Gram negative rod. It is such a small rod, that is may sometimes appear to be a cocci at first glance. When I first looked at the Gram stain of isolate #11, I thought I was seeing cocci, however I was able to enlarge the image and what appear to be cocci, are actually very, very small rods (difficult to see in this image). Klebsiella bacteria tend to be rounder and thicker than other members of the Enterobacteriacecae family. The Gram negative pink color is consistent between my Gram stain and that of K. aerogenes.

Gram stain for isolate #11

Gram stain of Klebsiella aerogenes

Image: http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab12/Entaerogenes.html

Members of the Enterobacteriaceae family, Klebseilla are facultative anaerobic, nonmotile bacteria. The Klebsiella genus consists of Gram-negative, oxidase-negative, rod-shaped bacteria with a prominent polysaccharide-based capsule (1). Klebsiella species can be found everywhere in nature, including in soil and water, on plants, and some strains are even considered to be a part of the normal flora of the gastrointestinal tracts of humans (1). One of the most well-known strains within this genus is K, pneumoniae, and ESKAPE pathogen, and a common pathogen of the human respiratory system that causes pneumonia (1). Some strains of Klebsiella have the ability to fix atmospheric nitrogen into a form that can be used by plants (2).

Image: https://en.wikipedia.org/wiki/Klebsiella_pneumoniae

Image: http://microbe-canvas.com/Bacteria.php?p=422

Image: https://microbewiki.kenyon.edu/index.php/Klebsiella_pneumonia

There is little research that discusses the Klebsiella genus as antibiotic producers. In fact, the majority of the literature about this genus discusses how many species within the genus have become resistant to antibiotics, and produce beta-lactamases that have the ability to break down commonly used antibiotics (4). These are often referred to as extended-spectrum beta-lactamase (ESBL) producing bacteria (4). The most common ESBLs are E. coli and K. pneumoniae. In addition to ESBL production, there are certain Klebsiella strains that are cytotoxin producers, such as K. oxytoca (5). These strains of K. oxytoca have been implicated in antibiotic-associated hemorrhagic colitis. The clinical manifestations are usually preceded by antibiotic treatment with a Beta-lactam agent (5). Cytotoxins are chemical weapons that T-cells use to destroy infected cells, protecting healthy cells. Some cytotoxins works my making holes in the cell membrane, while others turn on a program that causes the cell to self destruct (5).

Sources:

  1. Parwanu, N., K. Rogers, and A. Tikkanen. 2010. Klebsiella Bacteria Genus. Encylopedia Britanica.
  2. Buckle, J. 2014. Learn more about Klebsiella. Science Direct.
  3. 2018. Klebsiella. Wikipedia Encylopedia.
  4. Provinvial Infection Control (PIC-NL). 2011. EXTENDED-SPECTRUM BETA-LACTAMASE (ESBL) PRODUCING BACTERIA. Health.gov.nl.ca
  5. Barson, W.J., and M.J. Marcon. 2012. Etiologic Agents of Infectious Disease. Principles and Practice of Pediatric Infectious Diseases. 4th ed.

 

Extract News! Sofia

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From my 3 (4, 6, and 11) different isolates, I obtained 5 different extracts. Isolates 6 and 11 produced two different extracts because the initial extract produced three different layers, two of which were of interest for extraction. To test my extracts for antibiotic production, I chose 2 different Gram positive and 2 different Gram negative strains to use. The Gram positive strains I chose were: S. epidermis (1) and E. raffinosis (4) and the Gram negative strains that I chose were: E. coli (2) and A. baylyi (5). I chose strains 2 and 4 because when we previously tested our isolates against the ESKAPE pathogens, I had observed inhibition of all three of my isolates against these two strains. I chose 1 and 5 because there had been slight signs of inhibition when previously tested, which may have become more prominent with a prolonged growth time.

Unfortunately when my extracts were tested against each of these strains, none of my extracts displayed antibiotic-producing activity against any of the tester strains. To further conclude that these bacterial isolates are not antibiotic producers, I could test the extracts against different strains, or test them on a different type of media. Additionally, the plates could be left to grow for a longer period of time. If the observations continued to be the same, it would be reasonable to conclude that none of my isolates are antibiotic producers.

Fun with Soil- Sofia Boswell

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Where did you obtain your soil sample? Why did you choose this location?
I obtained my soil from a flower bed located outside of my house. I chose this location partially out of convince, but also because it seemed like it would be a fairly rich soil, seeing as it was supporting a substantial amount of plant growth. To obtain my sample, I dug down about 1.5-2 inches, to collect some of the darker and wetter soil, and avoid some of the mulch on top.

Do you expect a lot of isolates? Why or why not?

Seeing as this is fairly rich soil, I was expecting a good amount of isolates to grow. The soil I chose was much more clay like, and was fairly wet since it had just rained. Since we had learned that clay-like soils tends to support the growth of more anaerobic microbes.
Have you initial observations supported this?

So far, I have observed a lot of growth on all of my plates. Each one of my plates produced growth, and there is a lot of diversity of color, shape, and size among the colonies on each plate!
What media did you choose? How did you sample differ on the different media? What dilutions? 

Initially, I chose to use LB, AC, and R2A, my reasoning being that the richer and more nutrient dense medias would support the rich and presumed microbe dense soil. I chose to plate my 10^-2, 10^-3, and 10^-4 dilutions on each type of plate. After a few days of incubation, observations indicated that R2A grew the most diversity of microbes, however there was significant growth on the LB and AC plates as well. The 10^-2 plates of all three media were fairly overgrown, but still had some very interesting colonies. The 10^-3 plates were all very diverse and gave me the most useable colonies for my patch plates. The 10^-4 plates all had low amounts of growth, however this allowed my to obtain a definitive colony count, from which the cfu could be calculated.


Will you need to redo any? 

I chose to redo all three of my 10^-3 plates to see if I got anymore interesting growth. I also decided to make two more 10^-3 plates, using the PDA and 10% TSA medias, since these are both minimal medias and my R2A plate had yielded the best results. I wanted to see if these media types would result in similar growth.