CHAPTER 3 MATERIALS AND METHODS
3.7 Transmission electron microscopy (TEM)
on numerous images, and the accuracy of the resultant phase definitions confirmed using the phase colour coding function which colour-coded the green phase blue, and the red phase pink. The original image was compared with the colour-coded image to ensure that all the correct areas were selected, and the undesired areas omitted. The ranges were then broadened or restricted until the colour thresholds were optimal. The area occupied by C. trachomatis and the area occupied by cells was calculated using the phase analysis function on each 100× image captured.
Chlamydial inclusion size was measured for the HaCaT cells at 33°C for days 2 to 5 post infection. Inclusions were too small to be visually detected 1 day after infection under these conditions. The detection parameters were setup to include particles with a minimum size of 60 pixels. Inclusions touching the border were excluded. Each image was separated into its red, green and blue components and the new image of the green component selected. The detect function was utilized to select chlamydial inclusions.
Each image was carefully compared with the original to ensure that only chlamydial inclusions were selected. Since the software could not discriminate between 2 or more inclusions in contact with each other, any inclusions that were touching each other were excluded from the analysis using the delete particle function. The particle results function was used to analyse each selected inclusion and measure various parameters, including the area (µm2), diameter (µm) and mean level of green that was detected per inclusion.
Results were output in a Microsoft Excel spreadsheet and the inclusion parameters of the chlamydial strains compared.
The ultrastructural development of the chlamydial inclusion in human keratinocytes and cervical epithelial cells, and the effect of the organism on the infected and exposed cells compared to an unexposed negative control over 48 hours, was investigated using transmission electron microscopy.
3.7.1 Monolayer preparation and infection
HaCaT and ME-180 cells were grown in tissue culture flasks. Once confluent cells were trypsinised and 1 x 105 cells seeded to 24 well plates (Greinier) to which 12mm round Thermanox coverslips (Nunc) had been inserted into each well. After 2 days, the cells were about 90% confluent. Trypsinization and enumeration using the Trypan Blue dye exclusion assay and a haemocytometer revealed a cell count of approximately 2.5 x 105 cells per well. The residual CCM was aspirated, the monolayer washed once with PBS, then chlamydial growth medium added. The chlamydial inoculum was thawed immediately prior to use, and diluted with SPG buffer. Cells were infected with 100 µl chlamydial suspension at an MOI of 0.25, which equated to 1 chlamydial EB per 4 cells to ensure the presence of both infected and uninfected exposed cells in each well. For the unexposed negative control, cells were treated with 100 µl sterile SPG buffer. Cells were immediately centrifuged at 1200 x g for 1 hour, then transferred to an incubator. This point was defined as T0. HaCaT cells were incubated at 37°C and 33°C to compare the effect of C. trachomatis on the host cell at core body temperature (37°C) versus the temperature of human skin (33°C), while the ME-180 cells were only incubated 37°C because these cells could not survive at 33°C. The residual inoculum was removed after 2 hours in the incubator and replaced with 1 ml fresh CGM-E.
3.7.2 Monolayer processing
At 1, 3, 9, 18, 24, 36 and 48 hours post-infection, 1 well for each strain, cell type and temperature combination was fixed and processed for transmission electron microscopy (TEM). After washing with EMEM, the cell monolayers were transferred to a fresh 24- well plate using sterile forceps and a dissecting needle. The original plate was returned to the incubator, while the harvested coverslips with their cell monolayers were processed within the 24-well plate until the final step. Cells were fixed with 2% gluteraldehyde in EMEM for 30minutes, then washed with EMEM twice for 5 minutes. The fixed monolayers were covered by a layer of sterile EMEM and refrigerated until they could be processed (no more than 20 hours). Cells were post-fixed, dehydrated and infiltrated directly in the 24-well plate (table 1). In order to embed the cells in Spurr resin, a beam capsule was filled with Spurr resin and the coverslip placed on top of the beam capsule with cell-side down touching the resin. The beam capsules were incubated at 60°C for 48 hours to allow the resin to solidify.
After 48 hours the coverslip was quickly removed while the resin was still warm allowing the cells to remain embedded in the Spurr resin.
Table 2. Processing schedule for TEM
Step Process Solution Temperature Time
1 Fixation 2% gluteraldehyde in EMEM 24°C 30 min
2 Wash EMEM 24°C 5 min
3 Wash EMEM 24°C 5 min
4 Wash Sodium cacodylate buffer 24°C 5 min
5 Post-fixation 1% osmium tetroxidea 24°C 45 min
6 Wash Sodium cacodylate buffer 24°C 5 min
7 Wash Sodium cacodylate buffer 24°C 5 min
8 Dehydration 50% ethanol 24°C 10 min
9 Dehydration 70% ethanol 24°C 10 min
10 Dehydration 90% ethanol 24°C 10 min
11 Dehydration 100% ethanol 24°C 10 min
12 Dehydration 100% ethanol 24°C 10 min
13 Dehydration 100% ethanol 24°C 10 min
14 Infiltration Ethanol : Spurr resin (1:1)b 24°C 30 min
15 Infiltration Spurr resinb 60°C 1 hour
16 Infiltration Spurr resinb 60°C 1 hour
17 Embedding Spurr resin 60°C 24-48 hours
a protected from light
b procedure carried out uncovered to allow polypropylene to evaporate
3.7.3 Ultramicrotomy
The section of the resin block containing cells was sawed off and trimmed to produce a
“mesa” with a trapezoidal shape. Ultrathin sections (50-60nm) were cut and collected onto uncoated copper 200 mesh grids, then double stained with uranyl acetate and Reynold’s lead citrate for 3 and 2 minutes respectively (Reynolds 1963). Uranyl acetate and Reynolds lead citrate was decanted and centrifuged to pellet any debris or solute crystals.
Grids were floated on a drop of uranyl acetate for 3 minutes, rinsed twice with triple distilled water and blotted dry on Whatman filter paper. They were then placed in a drop of Reynolds lead citrate, rinsed twice with triple distilled water and blotted dry.
3.7.4 Visualization and photography
Sections were viewed using a Jeol 1011 transmission electron microscope at an accelerating voltage of 100 kV. The TEM was interfaced with a Megaview III Software Imaging Systems camera unit. Images were captured digitally and measurements performed using iTEM analySIS (Germany) image analysing software.