in vitro culture and isoenzyme analysis of giardia lamblia

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5.2 MATERIALS AND METHODS 5.2.1 Culture Medium TYI-S-33 was utilised for initiation and maintenance of Giardia cultures. Essentially it was prepared as described by Diamond et al. (1978) and modified by Keister in 1983. The main ingredients are Biosate (a pancreatic digest of casein and yeast extract), bile, serum, salts, glucose and iron. Details of it's preparation appear in Appendix 7. 5.2.2 Optimisation of Culture System Since biological products vary in their ability to support growth of cells in vitro (Diamond et al., 1978), several trials using different brands of reagents were necessary to identify suitable lots of the different products. Two strains of Giardia, (WB, isolated from a patient infected in Afghanistan; and the H7 clone of the GS isolate obtained from a scientist from the National Institutes of Health (NIH) who had camped in Alaska) were obtained from Or Nash at the NIH, USA. These have been well established as axenic cultures and hence were used as control reference strains to assess good growth-supporting reagents. 5.2.1.1 Sera Several batches of bovine sera (lots: CN 1872; CN 1399 and CN2290) from Highveld Biological, Gauteng, (SA); fetal bovine serum (lots: 35-603-240 -1419; 35-603-240-1410 & 34-511-248-75) from Delta Bioproducts, Gauteng, (SA); fetal bovine serum from Sigma St. Louis USA, lots 25H4602, 46H4648 & 65H4664 107
were evaluated. 5,2,1,2 Biosate Biosate from BBL- Becton Dickinson and Company, USA, lots 1000L 7DESR and 1000E9DGNP were evaluated. 5,2,1,3 Antibiotics Because of expected widespread penicillin/streptomycin resistance among local bacterial strains, various antimicrobial agents (such as Gentamycin, Vancomycin, and low dose Amikacin) were used over and above the penicillin/streptomycin originally incorporated into the culture medium. While the optimisation experiments were undertaken, an aerophilic laboratory contaminant, Xanthomonas maltophilia (identified by biochemical reactions in the Api 20E system (BioMerieux) was detected on some culture tubes of the WB reference strain (the trophozoites continued to grow and multiply in the presence of these microbes). A disk-diffusion susceptibility assay revealed that the organism was resistant to the antibiotics incorporated in the medium, including Amikacin but sensitive to Ciprofloxacin. Subsequently, this antibiotic was required to eliminate the bacterial contaminants without inhibiting the growth of Giardia trophozoites. An assay to determine the Ciprofloxacin concentration tolerated by trophozoites was therefore performed, as literature citing use of this quionolone on Giardia cultures was not found. 108
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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
Page 77 and 78: 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: Majewska, 1985) United States (Nash
Page 131 and 132: Table 5.1 Outlines a relative semi-
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
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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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in vitro culture and isoenzyme analysis of giardia lamblia

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