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1. Cultivation and Behavioral Assessment of Crithidia fasciculata and Leishmania tarentolae Alicia Werner Clemson University December 16, 2014 2. Introduction The purpose…
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  • 1. Cultivation and Behavioral Assessment of Crithidia fasciculata and Leishmania tarentolae Alicia Werner Clemson University December 16, 2014
  • 2. Introduction The purpose of this study was to develop a technique for culturing the parasites Leishmania tarentolae and Crithidia fasciculata. This involved mixing a batch of media, measuring parasite concentrations, calculating growth rates, and diluting the cultures when they became overcrowded. This technique would be used to create a continuous supply of the parasites for use in future experiments testing their chemotactic properties. C. fasciculata is a species of trypanosomatid parasite that infects various species of mosquitos.1 It is nonpathogenic to humans and exhibits a complex life cycle, alternating between two distinct morphological forms within the gut of the mosquito: the haptomonad form and the nectomonad form.1 The haptomonad form is round in shape and nonmotile, attaching to the surface of the host’s gut in clusters.1 It varies in length from 3 to 4 µm and lacks a flagellum.2 The nectomonad form is the infectious, elongated form of the parasite.1 It swims freely in the lumen of the host’s gut and can be expelled with the host’s feces.1 It ranges in length from 6 to 8 µm and possesses a long flagellum.2 L. tarentolae is a species of trypanosomatid parasite belonging to the order Kinetoplastida that infects lizards – primarily geckos.2 They are transmitted by phlebotomine sand flies and, like C. fasciculata, they are nonpathogenic to humans.2 They exhibit a complex life cycle consisting of two distinct morphological forms: a motile, extracellular promastigote form and a nonmotile, intracellular amastigote form.2 The slender, flagellated promastigote form typically ranges from 11-20 µm in length, and the oval-shaped, nonflagellated amastigote form generally measures approximately 4–5 µm in length.2 Inside their lizard hosts, they exist predominantly in the promastigote form and reside primarily in the bloodstream and lumen of the cloacae and intestine.2 They exist as promastigotes within their sand fly vectors, where they attach to the inner walls of the midgut.2 Although they are nonpathogenic to humans, they are capable of infecting mammalian phagocytic cells in vitro and differentiating into their amastigote forms once inside.2 Both L. tarentolae and C. fasciculata are capable of being cultured in standard bacteriological media such as Brain Heart Infusion broth, supplemented with Hemin. They should be grown at room temperature and their population densities restricted to 105 to 107 parasites per milliliter to ensure optimal growth. Materials and Methods All of the following steps were completed by personnel wearing lab coats, gloves, long pants, and closed-toe shoes. Preparation of BHI Nutrient Broth The nutrient broth was prepared by dissolving 7.4 g of Brain Heart Infusion (Sigma, Cat. #53286) in 200 mL of distilled water in a 250 mL Fisherbrand™ glass bottle. This was done three times, producing a total of 600 mL of BHI nutrient broth. Shortly after, the broth was autoclaved for 15 minutes at 121°C using a 50/50 mixture of
  • 3. tap water and distilled water. After it had cooled to room temperature, it was brought inside the Labconco Xpert Filtered Balance System biosafety cabinet, which had been running for at least five minutes prior. Inside the cabinet, the nutrient broth was transferred into plastic centrifuge tubes in 20 mL aliquots, and these were stored in a refrigerator until they were ready to be used. The biosafety cabinet was left running for another five minutes before being turned off, and all cabinet surfaces were wiped down with a solution of 10% Clorox bleach. Preparation of Hemin Solution The Hemin solution was prepared by combining 100 mL of distilled water with 200 mg of sodium hydroxide (Sigma, Cat. #S-5881) and 200 mg of Hemin from bovine (Sigma, Cat. #H-9039) in a Fisherbrand™ glass bottle. The Hemin solution was then autoclaved for 15 minutes at 121°C using a 50/50 mixture of tap water and distilled water. Once it had cooled to room temperature, the Hemin solution was brought inside the biosafety cabinet, which had been running for at least five minutes prior. Inside the biosafety cabinet, the Hemin solution was transferred to plastic centrifuge tubes in 20 mL aliquots. The centrifuge tubes were then wrapped in aluminum foil to protect them from light, and stored in a freezer until they were ready for use. The biosafety cabinet was left running for another five minutes before being turned off, and all cabinet surfaces were wiped down with a solution of 10% Clorox bleach. Sample Preparation Once the samples of Leishmania tarentolae and Crithidia fasciculata had been obtained, a centrifuge tube of Hemin solution and a bottle of Penicillin-Streptomycin (Sigma, Cat. #P-4333) were removed from the freezer and placed inside the biosafety cabinet along with the parasite samples. Prior to this, the biosafety cabinet had been left to run for at least five minutes. A centrifuge tube of BHI nutrient broth was then removed from the refrigerator and placed inside the biosafety cabinet as well, and the Hemin solution, PenStrep, and BHI broth were left inside the biosafety cabinet until they had reached room temperature. Once the Hemin solution, PenStrep, and BHI nutrient broth had reached room temperature, 50 µl of Hemin solution, 100 µl of PenStrep, and 20 mL of BHI nutrient broth were combined in a 50 mL Erlenmeyer flask and gently swirled. In order to generate a 1:1000 dilution of the parasite sample, 20 µl of culture containing the parasite was added to 20 mL of new media and the mixture was gently swirled. The flask was then labeled with the time, date, parasite type, and concentration, and aluminum foil was fixed over the mouth of the flask. Determining Parasite Concentration The hemocytometer was prepared by administering a tiny amount of water onto the ridges on either side of the counting chamber. The coverslip was then affixed to these ridges using the surface tension of the water, and 10 µl of culture was injected into the inlet on the edge of the slide. The hemocytometer was then placed on the stage of a
  • 4. Nikon Eclipse LV100 microscope and viewed under 50, 100, 200, and  500× magnification using the program, NIS Elements Basic Research. The numbers of parasites present in each of the four corner squares of the grid were counted, and the average was taken of these numbers. This average represented the number of parasites present in one cubic millimeter, or one microliter of culture. Once the sample concentration had been calculated, the coverslip and slide were gently washed with soap and water and blown dry using a high-pressure air hose. Results Day 1 (November 14 at 5:20 pm) Day 4 (November 17 at 4:50 pm) Day 6 (November 19 at 9:00 pm) Day 8 (November 21 at 5:30 pm) Concentration (parasites/mL) Existing culture - 4,500,000 3,250,000 2,750,000 New culture 4,000 4,500 3,250 2,750 Table 1: Parasite concentrations obtained through direct counts using a hemocytometer. Figure 1: Graph of C. fasciculata concentration as a function of time using cultures of varying starting densities and periods of growth. 0   500000   1000000   1500000   2000000   2500000   3000000   3500000   4000000   4500000   5000000   0   44.5   52   71.5   Concentration  (parasites/mL)   Time  (hours)   Concentration  of  C.  fasciculata  as  a  Function  of  Time   Days  1-­‐4   Days  4-­‐6   Days  6-­‐8  
  • 5. Figure 2: Graph of the predicted exponential growth pattern of C. fasciculata between days 1 and 4 using the equation,  𝑌 = 4.0×10! (2! ) to predict the growth curve, where x is the number of times the population doubled and 4.0×10! is the starting concentration of the sample. 𝑌 = 4.5×10! represents the final concentration of the sample and was used to find the value for x at which 𝑌 = 4.0×10! 2! = 4.5×10! . Point of Intersection: (10.1, 4.5×10! ) Doubling Time (Td) = !".!  !"#$% !".!  !"#$%  !"#$%&! = 7.08  hours/population  double 0   1000000   2000000   3000000   4000000   5000000   6000000   7000000   8000000   9000000   0   1   2   3   4   5   6   7   8   9   10   11   Concentration  (parasites/mL)   Population  Doubles   Trial  1  (Days  1-­‐4)   Y=4000(2^X)     Y=4500000  
  • 6. Figure 3: Graph of the predicted exponential growth pattern of C. fasciculata between days 4 and 6 using the equation,  𝑌 = 4.5×10! (2! ) to predict the growth curve, where x is the number of times the population doubled and 4.5×10! is the starting concentration of the sample. 𝑌 = 3.25×10! represents the final concentration of the sample and was used to find the value for x at which 𝑌 = 4.5×10! 2! = 3.25×10! . Point of Intersection: (9.50, 3.25×10! ) Doubling Time (Td) = !".!  !"#$% !.!"  !"#$%  !"#$%&! = 5.47  hours/population  double 0   500000   1000000   1500000   2000000   2500000   3000000   3500000   4000000   4500000   5000000   0   1   2   3   4   5   6   7   8   9   10   Concentration  (parasites/mL)   Population  Doubles   Trial  2  (Days  4-­‐6)   Y=4500(2^X)   Y=3250000  
  • 7. Figure 4: Graph of the predicted exponential growth pattern of C. fasciculata between days 6 and 8 using the equation,  𝑌 = 3.25×10! (2! ) to predict the growth curve, where x is the number of times the population doubled and 3.25×10! is the starting concentration of the sample. 𝑌 = 2.75×10! represents the final concentration of the sample and was used to find the value for x at which 𝑌 = 3.25×10! 2! = 2.75×10! . Point of Intersection: (9.72, 2.75×10! ) Doubling Time (Td) = !!.!  !"#$% !.!"  !"#$%  !"#$%&! = 4.58  hours/population  double 0   500000   1000000   1500000   2000000   2500000   3000000   3500000   0   1   2   3   4   5   6   7   8   9   10   Concentration  (parasites/mL)   Population  Doubles   Trial  3  (Days  6-­‐8)   Y=3250(2^X)   Y=2750000  
  • 8. Figure 5: Graph of doubling times calculated for C. fasciculata as a function of time using data from trials 1, 2, and 3. y  =  10.958x  -­‐  6.5693   40   45   50   55   60   65   70   75   4   4.5   5   5.5   6   6.5   7   7.5   Culturing  Time  (hours)   Doubling  Time  (hours)   Calculated  Doubling  times  for  C.  fasiculata  as  a   Function  of  Culturing  Time  
  • 9. Figure 6: Photomicrograph of nectomonad form of Crithidia fasciculata viewed under 200× magnification on day 4 (November 17), prior to sample dilution. The concentration in this photograph was estimated to be 4.5×10! parasites per milliliter. Figure 6: Photomicrograph of nectomonad form of Crithidia fasciculata viewed under 100× magnification on day 4 (November 17), after sample dilution. The concentration was estimated to be 4.5×10! parasites per milliliter.
  • 10. Figure 6: Photomicrograph of Crithidia fasciculata viewed under 200× magnification on day 22 (December 5), after sample dilution. The particles in this sample can be attributed to bacterial growth due to a contaminated batch of culture. Discussion In this study, we were successful in obtaining satisfactory growth of Crithidia fasciculata using the technique stated in the Materials and Methods section. We were not, however, successful in maintaining a viable culture of Leishmania tarentolae under the same conditions. A number of factors may have contributed to our inability to culture L. tarentolae, including contamination of media or glassware, culture conditions prior to our obtaining the sample, temperature fluctuations in the lab, or improper handling of the sample or lab equipment. Another problem encountered during this study was contamination of one of the portions of media used to culture C. fasciculata. As shown in Figure 6, the dilution produced on day 22 of the experiment was filled with microscopic particles, making it virtually impossible to identify individual parasites in the sample. These particles were attributed to bacterial growth in the BHI nutrient broth due to contamination during transfer of the broth into the centrifuge tubes. As shown in Figures 2, 3, and 4, the doubling times calculated for C. fasciculata, and thus the growth rates, showed considerable variation between the three trials, ranging
  • 11. from 4.58 to 7.08 hours per population double. This data suggests that the organism does not exhibit a simple exponential growth pattern, but rather a logistic pattern of growth. Logistic growth is a density-dependent growth model represented by a sigmoidal curve, in which the population density increases gradually at first, then increases rapidly, and finally levels off when the organism reaches the carrying capacity for its environment.3 Evidence for this pattern of growth is presented in Figure 5, which shows that the doubling times calculated for C. fasciculata vary almost directly with the amount of time each sample remained in a single culture. This suggests that the growth rate slowed once the population had reached a certain carrying capacity, and that this carrying capacity was reached sometime between 0 and 71.5 hours after the sample had been introduced to the new culture media. This decline in growth rate may have been due to overcrowding, limited resources, or a number of other factors. The logistic growth model is consistent with a typical microbial growth curve, which consists of four phases: a lag phase, an exponential phase, a stationary phase, and a death phase.4 The lag phase occurs when the sample is first introduced to the medium.4 During this phase, the cell number remains constant because the cells are busy synthesizing new components necessary for replication.4 The exponential phase occurs immediately after the lag phase, when the cells begin dividing.4 During this phase, the growth rate is at its maximum for the specific organisms and conditions being tested.4 The next phase, the stationary phase, occurs when the microbial population reaches its maximum carrying capacity, typically around 109 cells per milliliter, and remains constant.4 Finally, the death phase occurs when the total number of viable cells decreases, often at an exponential rate.4 In order to improve this experiment, we might measure cell concentration using spectrophotometry rather than a hemocytometer.4 Spectrophotometry is used to determine cell concentration by measuring the amount of light absorption that occurs when a beam of light is run through the sample. It would likely provide a more accurate estimate of parasite concentration than a hemocytometer because it eliminates human error. Furthermore, hemocytometers rely on the cells being evenly dispersed in the sample, which poses a problem because C. fasciculata tends to settle at the bottom of the culture. References 1. Wallace, F. G. Flagellate Parasites of Mosquitoes with Special Reference to Crithidia fasciculata, 1943, 29, 196 2. Raymond, F.; Boisvert, S.; Roy, G.; Ritt, J. F.; Legare, D.; Isnard, A.; Stanke, M.; Olivier, M.; Tremblay, M. J.; Papadopoulou, B.; Ouellette, M.; Corbeil, J. Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species, 2012, 40, 1131 3. Cain, M. L.; Bowman, W. D.; Hacker, S. D. Ecology: Second Edition, Sinauer Associates: Sunderland, MA, 2011, 214.
  • 12. 4. Willey, J. M.; L. M. Sherwood; Woolverton, C. J. Prescott’s Microbiology, 8th ed. McGraw-Hill: New York, NY. 2011.
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