in vitro culture and isoenzyme analysis of giardia lamblia

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APPENDIX 6 Preparation Of Excystation Media (Hamilton & Jackson, 1990) Appendix 6a Induction Medium Twenty-five ml of Hanks salt solution was made up to 100 ml with distilled water, autoclaved for 10 min at 103 kPa, cooled to 37°C and 0.40g L-cysteine hydrochloride (Polychem, Durban, South Africa) and 0.42 g sodium bicarbonate (Associated Chemicals Enterprise cc, Gauteng, South Africa) added. The pH was adjusted to 2,0 with 10M HCI (Polychem, Durban, and SA). Appendix 6b Excystment medium Forty millilitres of Hanks solution was made up to 100 ml with distilled water. One gram of Trypticase (Merck, Darmstadt, Germany) and 0,5g glucose (Polychem, Durban, SA) were added. The solution was autoclaved at 103 kPa for 10 min. Following cooling to 37°C, the following were added: 0,4 g L-cysteine hydrochloride (Polychem, Durban, SA.); 0,25 g Trypsin (Merck, Darmstadt, Germany) and 15 ml decomplemented horse serum *(Highveld Biological, JHB, SA.) The pH was adjusted to 7,5 with 1M NaOH (Polychem. Durban, South Africa) and the medium distributed in 3ml aliquots in glass tubes, overlaid with 0.5mlliquid paraffin and incubated at 37°C. Excess medium was used for washing cysts following the induction step. ~ *Later, fetal calf serum (Sigma, St Louis, USA) was used in place 0/ the Highveld brand 0/ horse serum. 181
Appendix 7 Preparation of culture medium - Trypticase-Yeast-Iron-Serum -33 (TYI-S-33) modified from Keister (1983). Bile and an increased amount of L-cysteine were the essential modifications added by Keister to the original preparation. Biosate Peptone (BBL, Cockeysville, USA) 30g/L Sodium chloride (Polychem, Durban, SA) 2g/L Di-potassium hydrogen phosphate (Polychem, Durban, SA) 19/L Potassium dihydrogen phosphate (BDH Chemicals) 0.6g/L Ascorbic acid (Polychem, SA) 0.2g/L Ferric ammonium citrate, brown granules (Merck, Germany) 0.023g/L D-Glucose (Polychem, SA) 10g/L L-cysteine monohydrochloride (Polychem, SA) 2g/L Decomplemented fetal bovine serum (Sigma, St.Louis, USA) 100ml Bovine bile (Sigma, St Louis, USA) 0.5g/L The pH is adjusted to 7.0-7.2 with 10M NaOH (Polychem, Durban, SA). The following antibiotics were added to the medium: Penicillin (Biowhittaker,Walkersville, MD) 100000 u/ml Streptomycin (Biowhittakker, Walkersville, MD) 1000 ug/ml Amphotericin B (Biowhittaker, Walkersville, MD) 5 ug/ml Amikacin 10ug/ml (Bristol Myers, Squibb, New Jersey, USA) Occassionally,Gehtamycin (Bayer) 40 ug/ml was also used. The medium is sterilised by filtration through a 0,22u filter (Millipore, Bedford, MA) and stored at 4°C. Modification (Diamond, 1995; Nash TE and Conrad JT, personal communication) 182
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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
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difficult to produce viable culture
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dominance of certain genotypes unde
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Cryopreservation and successful ret
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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 and 194: Unayo imvume yokunqaba ukungenela l
Page 195 and 196: Appendix 3 Excystation and culture
Page 197 and 198: Appendix 4 Results of excystation a
Page 199 and 200: Appendix 5 Results of in vitro (aci
Page 201: Key to Appendix 5 *Replicated inocu
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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