in vitro culture and isoenzyme analysis of giardia lamblia

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Assay for Susceptibility of Giardia to Ciprofloxacin A 100!-lg/ml Ciprofloxacin (Bayer) stock solution was prepared (from a 2 mg/ml intravenous fluid concentrate) in sterile TYI-S-33 medium. Various dilutions of the stock solution were prepared to give final concentrations of 5; 2,5; 1,25; 0,625 and 0,3125 !-Ig/ml in 7ml of TYI-S-33 in the culture tubes. The two reference strains (WB and H7) were retrieved from liquid nitrogen (as described in Chapter 6) and re-cultured in TYI-S-33 medium. Following 48 hours of incubation, the cultures were ice- chilled for 15min, centrifuged at 400xg for 10min at 4°C and the supernatant medium decanted. The pelleted cells were resuspended in 1ml of cold sterile medium and counted using a Nuebauer counting chamber. Aliquots of 0,1 ml containing 1000 cells/ml of each isolate were distributed to 6 Kimax glass tubes containing the TYI-S-33 culture medium. Each of the twelve cultures was incubated with each of the concentrations of Ciprofloxacin. Two tubes (one of each strain) incubated without the antibiotic served as control tubes. The tubes were incubated and maintained at 35,5°C at a 5° angle. Growth rate and attachment to glass were monitored in the twelve tubes for 20 and 48 hours by modifying the adhesion assay (Meyer, 1976; Farbey et al. 1995) as follows: After 20 and 48 hours incubation, all tubes were observed for adherent cells under a 40x microscope field in the entire inner surface of the culture tubes. A number · (growth rank) was assigned to each culture tube (including control tubes). This number represented the average number of adherent trophozoites present per 40x-microsope field in the lower inner surface of a culture tube using the guide outlined in Table 5.1. 109
Table 5.1 Outlines a relative semi-quantitative assessment of growth (ranking) of Giardia trophozoites in culture which could be used to monitor growth rate over time by counting adherent trophozoites on the lower inner surface of a culture tube under a 40x microscope field. (Adapted from Meyer, 1976). 1 1-5 2 5-50 3 50-250 4 250-500 The assay was repeated with higher concentrations of the drug ranging from 10- 50 J..Ig fml of Ciprofloxacin. Data obtained from the 8ayer electronic publication (1997) indicated a minimum inhibitory concentration (MIC) of 4pg fml for X. maltophilia. This concentration of Ciprofloxacin was subsequently incorporated into the contaminated cultures until no bacterial growth was detected on subcultures. 5.2.2 Propagation of Trophozoites In Vitro. 5.2.2.1 Culture of in vitro excysted trophozoites Cultures were initiated with 67 trophozoite batches derived from in vitro excystation (Chapter 3). These were aseptically transferred to sterile glass tubes containing TYI-S-33 medium with antibiotics. The tubes were incubated at 35,50C at a 5° angle to facilitate adherence of excysted trophozoites. After 48-72 hours, old medium was decanted (without dislodging the trophozoites from the tube walls) and replaced with fresh TYI-S-33. Culture tubes were maintained at 35,50C 110
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IN VITRO CULTURE AND ISOENZYME ANAL
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(j)edicated to my {ate parents :No
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the positive specimens by sucrose g
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Declaration This study represents o
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ACKNOWLEDGEMENTS The author wishes
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Table of Contents Page CHAPTER 1 -
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4.2.1. Animal Sourcing and Care ...
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APPENDIX 8 List of enzyme systems u
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List of Tables Page Table 1.1 Commo
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List of Plates Page Plate 2.1 An eo
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ABBREVIATIONS AND UNITS OF MEASUREM
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agi/is in 1882. In 1914, Alexeieff
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unique about Giardia in humans, whi
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To-date, use of the different speci
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Differentiation into the cyst stage
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1.3 Transmission 1.3.1 Human hosts
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Owen, 1984) 1.3. 2 The Zoonosis of
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1.4 Distribution The morbidity and
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children by Schutte et al. in 1981
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of (a) the enormous absorptive capa
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Ig M (100% of patients), IgG (70%),
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chymotrypsin) between different iso
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impact on the physical growth of 29
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Figure 1.3. Transmission Electron m
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among children between the ages of
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where immunocompromised mice have p
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up to 4 distinct genotypes within e
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in initiation of infection (excysta
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In order to address these and many
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multiple stool examinations in 4 of
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2.2 MATERIALS AND METHODS 2.2.1 Spe
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Table 2.1. Semi-quantitative assess
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2.2.3.2 The modified Zinc sulphate
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Preliminary work with five samples
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% Viability = unstained cysts/total
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Table 2.3. Prevalence of Giardia in
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Table 2. 4. Monthly summary of stoo
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spp (59%). Other parasites less fre
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Observations • A large proportion
Page 79 and 80: Plate 2.1. An eosin-stained prepara
Page 81 and 82: 2.4 DISCUSSION In terms of yield, i
Page 83 and 84: examination of wet preparations. Th
Page 85 and 86: ecognised (on unstained preparation
Page 87 and 88: cultivation in vitro (Kasprzak & Ma
Page 89 and 90: 3.2 MATERIALS AND METHODS Three exc
Page 91 and 92: 3.2.3 The modified Acid Pepsin meth
Page 93 and 94: • After 15 minutes incubation: Ma
Page 95 and 96: Plate 3.2 (b) A completely excysted
Page 97 and 98: Twenty-four batches of cysts (71 %
Page 99 and 100: 3.3.3 The modified Acid Pepsin excy
Page 101 and 102: 3.4 DISCUSSION Since our source of
Page 103 and 104: an unreliable indicator of viabilit
Page 105 and 106: propagating Giardia for subsequent
Page 107 and 108: 4.2 MATERIALS AND METHODS 4.2.1. An
Page 109 and 110: A litter comprising of 7 C57BU6 mic
Page 111 and 112: for attached trophozoites and maint
Page 113 and 114: days pi. However, accurate quantifi
Page 115 and 116: Table 4.4. Summary of the inoculati
Page 117 and 118: not be infected in comparison with
Page 119 and 120: A proportionately higher percentage
Page 121 and 122: These findings strongly suggest tha
Page 123 and 124: CHAPTERS IN VITRO CULTURE OF GIARDI
Page 125 and 126: Diamond et al.'s (1978) medium. He
Page 127 and 128: Majewska, 1985) United States (Nash
Page 129: were evaluated. 5,2,1,2 Biosate Bio
Page 133 and 134: dislodged by chilling the culture t
Page 135 and 136: Table 5.2. Sensitivity to Ciproflox
Page 137 and 138: Table 5.3. Summary results of in vi
Page 139 and 140: Once established in culture for a l
Page 141 and 142: Plates 5.3 and 5.4 Confluent growth
Page 143 and 144: difficult to produce viable culture
Page 145 and 146: dominance of certain genotypes unde
Page 147 and 148: Cryopreservation and successful ret
Page 149 and 150: adjusted to 5°C per minute from am
Page 151 and 152: 6.3 RESULTS Trophozoites have been
Page 153 and 154: Table 6.1. A longevity record of sa
Page 155 and 156: Diamond (1995) stated that an impor
Page 157 and 158: into two categories. One group cont
Page 159 and 160: technique to the first local (South
Page 161 and 162: for Giardia Iysates. These included
Page 163 and 164: 4. To exclude the presence of bacte
Page 165 and 166: Plate 7.1 Banding pattern of 8 seri
Page 167 and 168: PGM activity was displayed by Giard
Page 169 and 170: Plate 7.6. Hexokinase Lane 1. Unino
Page 171 and 172: Plate 7.8. Phosphogluconate dehydro
Page 173 and 174: stably expressed between different
Page 175 and 176: techniques, which would allow diffe
Page 177 and 178: Belosevic M, Faubert GM, MacLean JD
Page 179 and 180: Farthing MJG, Mata L, Urrutia JJ, K
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Heidelberg pp.397-398. Hiatt RA, Ma
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patients with X-linked agammaglobul
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Press, Calgary. pp 57-58. Nash TE,
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of Entamoeba histolytica in a group
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Wolfe M. S.1979. Giardiasis. Sympos
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APPENDICES APPENDIX 1 Information t
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Unayo imvume yokunqaba ukungenela l
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Appendix 3 Excystation and culture
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Appendix 4 Results of excystation a
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Appendix 5 Results of in vitro (aci
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Key to Appendix 5 *Replicated inocu
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Appendix 7 Preparation of culture m
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Appendix 8 List of enzyme systems u
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Appendix 9 Appendix (9a) Preparatio
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Appendix 10. Quantitative descripti
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