in vitro culture and isoenzyme analysis of giardia lamblia

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Appendix 2 Preparation of 8eemer's stain Solution A Methylene blue (Polychem, Durban South Africa) 0.2g Sodium citrate Sodium chloride (Polychem, Durban, SA) (Polychem, Durban, SA) 1.1g 0.55g Saturated mercuric chloride (Merck, Darmstadt, Germany) Distilled water 0.1ml 100ml Solution B Eosin (BOH) 0.15 M Sodium Chloride (normal saline) 19 100ml The working solution is prepared by mixing equal volumes of solutions A and B. 173
Appendix 3 Excystation and culture of cysts from symptomatic and asymptomatic patients stools using the modified Rice and Schaefer method (Hamilton & Jackson, 1990). Total cyst counts and percent viabilities are indicated. Some cultures were contaminated while others died without evident contamination (indicated as sterile) 12 56 5 24hrs contaminated 34 30 Nil 10 70 10 24hrs contaminated 79 44 *8 24hrs contaminated 11 82 5 72hrs contaminated 8 100 nil 110 27 *2 6hrs contaminated 18 75 8 6hrs contaminated 10 88.2 10 24hrs Sterile 5 97 nil 8 87.5 2 12hrs contaminated 103 90.9 *36 72hrs contaminated 14 63 3 < 12hrs contaminated 11 71 12 < 12hrs contaminated 3 94 5 24hrs Sterile 13 87 nil 21 64 33 72hrs Sterile 2 41 nil 7 70 10 24hrs contaminated 11 55 2 < 12hrs contaminated 23 94 20 72hrs Sterile 9 43 nil 8 36 nil 17 33 2 174
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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
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Plate 2.1. An eosin-stained prepara
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2.4 DISCUSSION In terms of yield, i
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examination of wet preparations. Th
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ecognised (on unstained preparation
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cultivation in vitro (Kasprzak & Ma
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3.2 MATERIALS AND METHODS Three exc
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3.2.3 The modified Acid Pepsin meth
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• After 15 minutes incubation: Ma
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Plate 3.2 (b) A completely excysted
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Twenty-four batches of cysts (71 %
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3.3.3 The modified Acid Pepsin excy
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3.4 DISCUSSION Since our source of
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an unreliable indicator of viabilit
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propagating Giardia for subsequent
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4.2 MATERIALS AND METHODS 4.2.1. An
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A litter comprising of 7 C57BU6 mic
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for attached trophozoites and maint
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days pi. However, accurate quantifi
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Table 4.4. Summary of the inoculati
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not be infected in comparison with
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A proportionately higher percentage
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These findings strongly suggest tha
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CHAPTERS IN VITRO CULTURE OF GIARDI
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Diamond et al.'s (1978) medium. He
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Majewska, 1985) United States (Nash
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were evaluated. 5,2,1,2 Biosate Bio
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Table 5.1 Outlines a relative semi-
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dislodged by chilling the culture t
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Table 5.2. Sensitivity to Ciproflox
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Table 5.3. Summary results of in vi
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Once established in culture for a l
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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
Page 181 and 182: Heidelberg pp.397-398. Hiatt RA, Ma
Page 183 and 184: patients with X-linked agammaglobul
Page 185 and 186: Press, Calgary. pp 57-58. Nash TE,
Page 187 and 188: of Entamoeba histolytica in a group
Page 189 and 190: Wolfe M. S.1979. Giardiasis. Sympos
Page 191 and 192: APPENDICES APPENDIX 1 Information t
Page 193: Unayo imvume yokunqaba ukungenela l
Page 197 and 198: Appendix 4 Results of excystation a
Page 199 and 200: Appendix 5 Results of in vitro (aci
Page 201 and 202: Key to Appendix 5 *Replicated inocu
Page 203 and 204: Appendix 7 Preparation of culture m
Page 205 and 206: Appendix 8 List of enzyme systems u
Page 207 and 208: Appendix 9 Appendix (9a) Preparatio
Page 209: Appendix 10. Quantitative descripti
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