Comparative study among lactophenol blue, lactophenol solution and proteinase-K lytic solution for rostellar hooks morphometry of Echinococcus granulosus protoscolices
Article Category: Research Note
Data publikacji: 25 sty 2020
Zakres stron: 63 - 70
Otrzymano: 15 lis 2019
Przyjęty: 13 gru 2019
DOI: https://doi.org/10.2478/helm-2020-0010
Słowa kluczowe
© 2020 S. J. Hammad, S. Cavallero, F. S. Al-Nasiri, S. D᾿Amelio, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.
For morphometric study of rostellar hooks from
Out of 34 fertile hydatid cysts collected from 18 infected sheep in Kirkuk slaughterhouse, Iraq during June of 2015, five fertile hydatid cyst samples were selected from liver to perform the present study. According to Perez-Serrano
The study was designed for the first time for comparing among three lytic solutions (Lactophenol blue, lactophenol and proteinase-K lytic) for preparation of microscopic slides. Lactophenol blue solution of Central Drug House company (India) was used. Lactophenol solution was prepared by mixing 50 ml of phenol (CDH, India), 10 ml of lactic acid (Solvo Chem, UK), 20 ml of glycerol (Solvo Chem, UK) with 900 ml of distilled water (Humason, 1979). Proteinase-K lytic solution was prepared according to Wu et al. (2007) with modification, by mixing 300 μl of TE9 buffer with pH 7.5 and 200 μl of 10 mg/ml of proteinase-K stock solution. TE9 buffer was prepared according to Wu et al. (2007) and Blasco-Costa et al. (2012) with modification on the volume of components. TE9 buffer contained 10 mM of tris-HCl (Scharlau, Spain), 125 mM of sodium chloride (SCRC, China) and 10 mM of EDTA (Panreac, Spain) with pH 8. Proteinase-K stock solution with 10 mg/ml (w/v)% was prepared according to Harvey (2000) by dissolving 50 mg of proteinase-K (Bio Basic, Canada) in 5 ml of distilled water (Alkem laboratories, India). Lactophenol blue, lactophenol and proteinase-K lytic solutions were used on all samples (for comparison) as in the following steps: i) one drop of protoscolices with ethanol 70 % was taken by 3 ml Pasteur pipette and laid on the middle of microscope slide for each solution and was examined under microscope before adding the lytic solutions to act time zero; ii) each slide was putted in separated Petri dish, and then a drop from each lytic solution was added on the specimens separately. All slides were incubated for 4 hours with repetitive microscopic examinations under 100x and 400x magnification every 30 minutes (time 30, time 60, time 90, time 120, time 150 and time 240 minutes) with continued repetitive additions of the all lytic solutions to prevent specimen drying. Slide with proteinase-K lytic solution was incubated at 56 °C, while, lactophenol blue and lactophenol solution slides were incubated at a room temperature; iii) at time 240, the incubation was stopped, and a drop of aqueous gelatin-glycerol was added to all specimens to preserve it for a long time. Aqueous gelatin-glycerol consisting of 5 g of gelatin (Panreac, Spain), 50 ml of glycerol (Solvo Chem, UK), 5 ml of phenol (CDH, India) and 50 ml of distilled water (Humason, 1979). In conclusion, a microscopic examination was carried out under 1000x with oil immersion and fine pressure on the coverslip was done to identifying the rostellar hooks.
Clustering behavior of protoscolices and visibility of components at different times after treated with lactophenol-based solutions and Proteinase-K lytic solution.
| Time of exposure | Lactophenol blue or lactophenol solution | Proteinase-K lytic solution |
|---|---|---|
| Protoscolices aggregated/ Components and rostellar hooks not visible (Fig. 2a-b) | Protoscolices separated/ clearer vision of components (Fig. 2c-d) | |
| Protoscolices aggregated/ Components and rostellar hooks not visible (Fig. 3a-b) | Protoscolices separated/ clearer vision of components (Fig. 3c-d) | |
| Protoscolices aggregated/ clearer vision of components (Fig. 4a-b) | Protoscolices separated/ vision of components same as time 60 (Fig. 4c-d) | |
| Protoscolices partially separated/ components visible but with no lysis of membrane (Fig. 5a-b) | Protoscolices well separated/ starting of membrane lysis (Fig. 5c-d) | |
| Protoscolices partially separated/ components visible but with no lysis of membrane (Fig. 6a-b) | Protoscolices well separated/ increasing of membrane lysis (Fig. 6c-d) | |
| Protoscolices partially separated/ components visible (better with lactophenol) but with no lysis of membrane (Fig. 7a-b) | Protoscolices well separated/ membrane lysis semi-completed (Fig. 7c-d) Rostellar hooks free and visible (Fig. 8) |
Fig.1
Microscopic photography of protoscolices at different magnification (a, b) before use of lytic solutions (zero time), showing aggregation of protoscolices within the preservation solution (ethanol 70%).
Fig. 2
Microscopic photography of protoscolices after 30 min from zero time of addition of lactophenol blue (a), lactophenol solution (b), proteinase-K lytic solution (c, d).
Fig. 3
Microscopic photography of protoscolices after 60 min from zero time of addition of lactophenol blue (a), lactophenol solution (b), proteinase-K lytic solution (c, d).
Fig. 4
Microscopic photography of protoscolices after 90 min from zero time of addition of lactophenol blue (a), lactophenol solution (b), proteinase-K lytic solution (c, d).
Fig. 5
Microscopic photography of protoscolices after 120 min from zero time of addition of lactophenol blue (a), lactophenol solution (b), proteinase-K lytic solution (c, d); arrows indicated the sites of lysis in cellular membrane.
Fig. 6
Microscopic photography of protoscolices after 150 min from zero time of addition of lactophenol blue (a), lactophenol solution (b), proteinase-K lytic solution (c, d).
Fig. 7
Microscopic photography of protoscolices after 240 min from zero time of addition of lactophenol blue (a), lactophenol solution (b), proteinase-K lytic solution (c, d), arrows indicated the sites of lysis in cellular membrane.
Fig. 8
Microscopic photography of hooks after lysis the membrane of protoscolices after 240 min from zero time of addition of proteinase-K lytic solution to the protoscolices.
This article does not contain any studies with human participants or animals by any of the authors therefor the present study formal consent is not required.
During the first inspection, before adding the lytic solutions, the protoscolices resulted aggregated after examination using a microscope with 400x, thus not allowing a clear definition of the internal structures and rostellar hooks (Fig. 1a, b) Whereas, at the last step, after 240 min from the addition of lytic solutions, lactophenol blue provided still little clear vision and no lysis of protoscolices membranes even after putting a cover-slide on sample; lactophenol solution gave a more clear vision, the cellular components were noticed but also un-differentially and no lysis of cellular membrane even after putting a cover-slide on sample was observed; proteinase-K lytic solution determined the clearest vision with semi-complete lysis of cellular membrane and with complete lysis after putting a cover-slide on sample. After using proteinase-K lytic solution, the hooks appeared distributed in two separated rings alternatively between large and small hooks, and each one had three regions (blade, guard and handle). Also, after using proteinase-K lytic solution, it became easier to count the number of hooks (mean= 34, SD= 4) and to distinguish between large hooks (larger, less robust, had more pointed blade) and small hooks.
Clustering behavior of protoscolices and visibility of internal components at each time after time zero are summarized in Table (1).
In the present study, treatments of rostellar hooks of
According to Central Drug House (CDH), the manufacture company, lactophenol either in lactophenol blue or in lactophenol solution is harmful if swallowed or inhaled by humans, provoking severe skin burns and eye damage, and is suspected of causing genetic defects due to germ cell mutagenicity and may cause damages to organs following prolonged or repeated exposure.
Proteinase-K lytic solution is also frequently used in molecular studies for DNA extraction on various living cells and tissues. Also, proteinase-K lytic solution was used as enzymatic digestion technique to obtain and study the sclerotized structures of monogenean parasites as
The present study is the first study that evaluates lactophenol blue and proteinase-K lytic solution in digestion of
Based on the obtained results, the present study recommends the use of proteinase-K lytic solution in morphometric of rostellar hooks, as it provides a clearer vision of hooks if compared with lactophenol-based solutions and may guarantee safer conditions for operators, although a protective equipment must be used in any case.