Experiment 8A
1. The living cells adhered to the bottom of the plate and looked dark and small. I saw what looked like organelles as I squinted and looked very close at the grainy intracellular contents. Dead cells floated about, unadhered to the bottom of the plate and brighter. The cells of the new culture looked a little smaller and seemed to be vibrating (although this was likely just the microscope itself).
2. No time point and no dilution produced a confluent monolayer. This is because our starting culture was too dilute and our incubation time was too limited. Perhaps if we could “grow up” the culture in several consecutive rounds we could have produced a confluent monolayer.
3. There is no evidence of contamination. No fungi, bacteria nor hair.
4. Humidity prevents the media from evaporated, and CO2 is necessary for maintaining pH of the cell cultures.
Experiment 8B
1. The inducer could be an intracellular parasite that halts protein synthesis, thus prohibiting GFP expression. Alternatively, it could be too dilute or require certain conditions to become pathogenic.
2. The results in the handout are consistent — unfortunately, our positive control did not work.
3. No, our group did not detect any contamination.
4. E. coli is an extracellular pathogen and thus would elicit an extracellular response. For example, recruiting more macrophages. M. smeg is an intracellular pathogen and thus would elicit an intracellular response, such as apoptosis.
Experiment 8C
1. LPS and E. coli induced GFP.
2. LPS and E. coli were about equal, while M. smeg did not induce GFP.
3. The FACS results are not particularly novel for M. smeg — it is likely just an older culture that could not infect the macrophages. Alternatively, the strain of macrophages used might not be less susceptible than other macrophages to M. smeg infection. Overall, there was more likely something irregular with the M. smeg (most probable), conditions (also propable) or macrophages (somewhat probable) to explain the lack of GFP induction by M. smeg.
4. By concentration, a single microgram of LPS and fifty microliters of E. coli induced the strongest response. However, there was a signicant drop in induction with a hundred microliters of E. coli — this could be due to either macrophages dying from E. coli exposure, or possibly faulty lab technique. a deeper knowledge of interaction between E. coil and macrophages is necessary to confirm which concentration of E. coli gave the strongest response. After all, a hundred microliters of E. coli might induce the strongest and most widespread GFP expression initially, followed by a sharp drop-off as the cells die.
Part I
M1 peaks represent live cells without GFP expression (based on negative controls0 and M2 peaks represent live cells with GFP expression. E. coli shows a dose response at 100µl, but I would prefer to repeat the experiment to disprove experimental error since the M2 peaks for lower concentrations are about equal. For M. smegmatis is is impossible to tell whether dose response occurs, since there is only a single FACS diagram available to describe its induction of GFP expression. The lack of GFP induction by M. smeg is surprising because it is an intracellular pathogen. However, it is possible that the strain used lacked virulence factors or that it somehow halted host protein synthesis.
Part II
Sample A depicts T cells from the thymus. My reasoning is that there are lots of CD4+CD8+ cells and a few others scattered throughout; this majority of double-positive cells indicates is consistent with what I would expect from cells isolated in the thymus. Sample C depicts splenic T cells. My reasoning is that there are lots of CD4-CD8- cells (either non-thymocytes and/or dead T cells) and a few helper as well as cytotoxic T cells. A majority of double-negative cells is consistent with what I would expect when isolating T cells from the spleen.
Pseudomonas spp. are common soil-dwelling aerotactic Gram-negative proteobacteria with the unique ability to utilize exotic carbon sources for energy. While the majority of species can only perform respiratory metabolism, certain Pseudomonad species can anaerobically also use nitrate, nitrite or nitrous oxide as a terminal electron acceptor. Given a culture of Pseudomonad (Pseudomonas 80), I performed physiological, biochemical and molecular (Experiments 3C & 3D) analysis to determine its species.
In Experiment 3A, brightfield and phase-contrast microscopy were used to determine the size, motility, aerotactic behavior and Gram state of P. 80. Later in Experiment 3A, an oxidase test and oxidation/fermentation test gave me basic knowledge of P. 80’s metabolic pathway and ability to anaerobically ferment and deaminate (use amino acids for energy). Experiment 3B illuminated further physiological characteristics of P. 80, such as its optimal temperature, as well as tremendous biochemical information. Various growth media — both replica- and streak-plated — were invaluable instruments to confirm that I had a Pseudomonad and also narrow down the species. Lastly, Experiments 3C and 3D further described P. 80 via PCR, electrophoresis and sequence analysis.
An important part of identifying my P. 80 was confirming it was even a Pseudomonad. To do this, I used Gram-stains, brightfield microscopy, wet mounts, and phase-contrast microscopy to identify a Gram-negative and motile 3-5µm bacillus with aerotactic behavior. I used standard tap water, ethanol, iodine, purple and pink stain on a heat-fixed glass slide for Gram-staining, and 10x, 40x and 100x magnification for microscopy. With my findings so far consistent with known data on Pseudomonads, I performed an oxidase test by smearing P. 80, S. epidimidis (negative control) and P. aeruginosa (positive control) to verify that cytochrome c was present in the cellular respiratory pathway. With a positive oxidase result, I confirmed that cytochrome c was present. Lastly, I confirmed that my sample could not anaerobically ferment glucose (a unique inability of Pseudomonads). I used the oxidation/fermentation (o/f) test, whereby I stab inoculated a glucose-rich media, and then either poured oil over the media (anaerobic conditions) or did not (aerobic conditions) before incubation. Sugar catabolism alters the pH, yielding a yellow color from a pH indicator; deamination of the added peptone amino acids yields a blue color. My sample could not catabolize sugar anaerobically (fermentation) but was able to deaminate anaerobically, as expected.
After examination to confirm I had a Pseudomonad, I went about determining what species was P. 80. I probed deeper into the nature of my P. 80 by streak- and replica-plating onto a myriad of agars.
| Streak Plates | Streaks approximately one inch long were made on each plate for P. 80 and controls. |
| Starch Plate | After incubation on agar laden with starch, pouring iodine over the plate visualizes occurrence (lighter brown) or absence (darker brown) of starch hydrolysis. I found no evidence of starch hydrolysis for P. 80, although my Bacillus subtilis positive control was functional. |
| Gelatin Plate | After incubation on agar laden with gelatin, pouring trichloroacetic acid over the plate visualizes occurrence (clear) or absence (opaque) of gelatin hydrolysis. I found evidence of gelatin hydrolysis for P. 80 and my Bacillus subtilus positive control. |
| Egg Yolk Plate | After incubation on agar laden with egg yolk, I examined for evidence of phospholipase secretion. Phospholipases hydrolize lecithin (a major phospholipid in egg yolk) to form insoluble long chain fatty acids that look like white buildup around the colonies. My P. 80 and P. fluorescens positive control both had white precipitate around their colonies, indicated egg yolk hydrolysis. |
| Replica Plates I | I replica-plated in the following order. Beginning with MSA (containing no carbon source) and ending with YTA (containing yeast extract and tryptone, offering everything from amino acids to sugar) means that my positive control does not contaminate my negative control, and that oft-used nutrients such as glucose and fructose are not transferred to plates containing rarely-used nutrients. |
| MSA Plate | My negative control replica plate had no growth. |
| + acetamide | There was no growth on MSA+acetamide, a crystalline amide. |
| + maltose | There was no growth on MSA+maltose, a two-glucose disaccharide. |
| + lactose | There was no growth on MSA+lactose, a common sugar. |
| + fructose | There was moderate growth on MSA+fructose, another common sugar. |
| + glucose | There was moderate growth on MSA+glucose, yet anther common sugar. |
| YTA Plate | There was significant growth on the YTA plate, my positive control. |
| Replica Plates II | I performed replica-plating as described above, from MSA to YTA. |
| MSA Plate | My negative control replica plate had no growth. |
| + p-hydroxybenzoate | There was minimal growth on MSA+p-hydroxybenzoate, a carbon source. |
| + glycine | There was no growth on MSA+glycine, a crystalline organic carbon source. |
| + nicotinate | There was no growth on MSA+nicotinate, an organic vitamin and carbon source. |
| + geraniol | There was no growth on MSA+geraniol, a volatile carbon source that is absorbed from the air. |
| + tryptophane | There was no growth on MSA+tryptophane, an amino acid carbon source. |
| YTA Plate | There was significant growth on the YTA plate, my positive control. |
To determine P. 80’s optimal temperature, I inoculated three tubes of YTB (similar to YTA, described above) and incubated at 4°C (no growth), 30°C (significant growth) and 40°C (moderate growth). Also, I expanded on the oxidation/fermentation test by streaking (aerobic conditions) and stabbing (anerobic conditions) King’s medium B agar supplemented with 0.2% KNO3. Examining the incubated tubes under UV light revealed that P. unkown did not fluoresce (no glow), reduce nitrate to N2 gas (which would form bubbles) nor reduce nitrate to nitrite (which causes a red color upon addition of a nitrite reagent). Now clear that P. 80 did not reduce nitrate at all nor express fluorescent pigments, I confirmed that my positive and negative controls (P. aeruginosa and P. fluorescens) did and did not, respectively, reduce nitrate fully to N2 gas and secrete a fluorescent pigment.
The next step was to analyze P. 80 at a molecular level. To do so, a polymerase chain reaction (PCR) was performed using universal- and Pseudomonad-specific primers. Universal primers amplify regions supposedly present in both my experimental organism (P. 80) and negative control (Bacillus subtilis); Pseudomad-specific primers amplify regions unique to Pseudomonads. 
To visualize the PCR results, I performed gel electrophoresis (shown below) with each column corresponding to: ladder; P. 80 + Pseudomonad-specific primers; Bacillus subtilis + Pseudomonad-specific primers; P. 80 + universal primers; Bacillus subtilis + universal primers. I expected to see bands in all four experimental columns except Bacillus subtilis + Pseudomonad-specific primers, but instead only saw a band on the column corresponding to Bacillus subtilis + universal primers. Since P. 80 did not resolve any bands, either the PCR itself was aberrant or the strain is mutated or contaminated.
At this point, I was ready to analyze P. 80 directly at the nucleotide level. I submitted amplified universal regions of the P. 80 genome to South Korea for sequencing. A few weeks later, I received the sequence (shown below) and used NCBI BLAST to see if the sequence is homologous to other species. After a few false starts with vague vaginal epithelial cultures, I found that it corresponded to Comamonas spp (Score = 878/878), Comomonas testosteroni (Score = 878/878) and P. testosteroni (Score = 878/878).
Due to morphological, physiological, biochemical, metabolic and nucleotide consistencies, I identified P. 80 as P. testosteroni. Experiment 3A helped me confirm it was a Pseudomonad, and Experiment 3B explored which energy sources it could use as well as pigment production, nitrate reduction and hydryolase activities. Positive or negative results on the procedures of Experiment 3B were highly informative, narrowing down the strongest possibilities to P. putida, P. aeuruginosa and P. testosteroni. Each possibility conflicted on at least one criterion: gelatin hydrolysis and 42°C growth for P. putida; fluorescent pigment production for P. aeruginosa; and fructose catabolism and gelatin hydrolysis for P. testosteroni.
Sequence analysis Experiment 3D strongly indicated that I had P. testosteroni, which was surprising considering that until that point P. aeruginosa was the strongest candidate. If my culture had been P. aeruginosa, then the only inconsistency (no fluorescent pigment production) was possibly due to environmental conditions; after all, my P. aeruginosa control was not able to secrete fluorescent pigment either. A loss-of-function mutation is much more common than a novel or gain-of-functon mutation, as would be the case if my culture was P. testosteroni (as it was able to catabolize fructose and hydrolyze gelatin, two characteristics foreign to P. testosteroni. Alternatively, it is possible that there was an error in the PCR amplification, since I could not even resolve any bands upon electrophoresis of both universal- and Pseudomonad-specifc-primed P. 80 gene segments. Despite these inconsistencies between Experiment 3B and 3D, I chose to follow Experiment 3D’s results since it turned up multiple Pseudomonads, P. testosteroni-like species and even P. testosteroni itself.
