Preston and Morrell modification(1962)

Transfer a loop full of Streptoccocus Pneumoniae (Sigma Aldrich) to a slide and mix with a drop of saline (Sigma Aldrich).

Dry on a heat plate (Sigma Aldrich) until it goes cloudy approx 2.5mins

Flood with crystal violet and wait for 30 sec.

Rinse with water thoroughly

Flood with Lugol Iodine (Sigma Aldrich) and wait for 30 sec.

Rinse with water thoroughly

Flood with Iodine/Acetone (Sigma Aldrich) and wait for 30 sec.

Rinse with water thoroughly

Flood with Safronin (Sigma Aldrich) and wait for 30 sec.

Rinse with water thoroughly and blot dry

Wait until completely dry

Observe under microscope (Olympus CX23) using 100X oil

Preparation of film(liquid/solid)

Use a sterile loop/pasteur pipette to transfer a Ioopful/drop of culture to the centre of the slide.

For liquid culture, place two more drops of bacteria on the same area on the slide.

Fix the preparation by placing on a hot plate at 37°C for 2½ minutes

Staining Procedure

After heat fixation, the slide was sequentially stained with Crystal Violet, Lugol’s Iodine, Iodine-Acetone, and Safranin, each applied for 30 seconds. After each staining step, the slide was rinsed with distilled water. The slide was blot-dried with absorbent paper. The previous chemicals were obtained from Sigma Aldrich.

For the microscopic analysis of the Streptococcus pneumonia sample, Olympus CX23 microscope was used. The prepared slide was placed on the stage and a drop of immersion oil was added to the centre of the film which was then placed under 100x objective lens to observe the bacteria.

Gram stain of Streptococcus pneumoniae

A single bacterial colony (S. pneumoniae) was picked up and emulsified with a drop of saline on a clean, wiped microscope slide. These were then dried on a hot plate, before gram staining. The procedure used was similar to that employed by Preston and Morrell (Preston and Morrell, 1962). This involved adding Crystal-Violet stain, Luqol’s Iodine, Iodine Acetone, and Safranin, covering the bacteria and leaving each staining solution on for 30 seconds before washing thoroughly with distilled water. The slides were then left to dry completely before being examined at x100 magnification with a drop of immersion oil on an Olympus CX23 microscope.  The reagents were purchased from Sigma Aldrich, UK at a stock concentration.

Statistical Analysis

Data were processed using Excel software (Microsoft), and analysed with Prism (GraphPad software). Each experiment was performed in triplicate, and data analysed using a one-way analysis of variance (ANOVA) as well as Dunnett’s multiple comparison (to compare test groups to the control group) and Tukey tests (when comparing all groups).

Figure Legend

Epithelial cell barrier integrity varies upon infection with different serotypes. Coverslips with human primary alveolar epithelial cells (HPAEpiCs) were washed, fixed, and immunostained, before viewing under a Nikon Eclipse 80i fluorescent microscrope. The data were processed using FIJI (ImageJ) before being analysed with Prism GraphPad, employing a one-way ANOVA test. Data are presented as mean +- standard error *, P<0.05, as determined by a one-way ANOVA test (n=3).

Results

To enable differences between the tight junction integrity of epithelial cells under infection by differnt serotypes, the tight junction protein ZO-1 was stained and visualised under fluorescent microscope.

S. pneumoniae serotype ST217 impacted barrier integrity the most, as was shown by the significant drop in corrected total cell fluorescence (CTCF) (Fig.1, p<0.05).

Materials List

Chemical Reagents:

1. Crystal Violet (dye for primary staining)

2. Lugol's Iodine (mordant)

3. Iodine-acetone solution (decolouriser)

4. Safranin (counterstain)

Additional Materials:

5. Microscope slides

6. Heat source (hot plate for heat-fixing bacterial culture)

7. Distilled water

8. Staining rack 

9. Wash bottle 

10. Blotting paper 

11. Immersion oil

Equipment:

12. Microscope (Olympus CX23)

13. Timer 

Gram staining methods

Following aseptic technique, a saline preparation of Streptococcus pneumoniae was taken from the original plate and spread on a labelled glass slide before being heat-fixed at 37 C for two and a half minutes. Utilising Sigma Aldrich© UK gram staining reagents, the slide was stained with crystal violet for 30 seconds, followed by Lugol’s iodine for 30 seconds and rinsed with distilled water at a 45-degree angle between each solution. The slide was decolourised with iodine-acetone until clear before being counter-stained with safranin, again for 30 seconds. Next, the slide was rinsed with distilled water, blotted with bibulous paper and allowed to air dry at room temperature. 

The slide was then microscopically reviewed at 100x, oil immersion magnification with the Olympus CX23 for morphology and gram staining patterns. Gram staining patterns were consistent with known patterns for S. pneumoniae, ie, gram-positive cocci in pairs and chains.

Statistical Analysis

Data was processed using Excel software (Microsoft) and analyses were performed using Prism (Graphpad Software CA, USA). Assume all tests were performed at least in triplicate, unless otherwise stated. Considering the presence of more than two groups and assuming a normal distribution, the data was analysed using One-way analysis of variance (ANOVA) to compare the means of a single variable across multiple groups.

Results

To enable the damage done by the serotypes to be visualised fluorescent immunostaining was applied. Labelling the  ZO-1 protein to directly compare the decrease in fluorescence with the damage to the tight junctions. To conclude our results, while both D39 and ST217 strains showed a notable decrease in immunofluorescence when compared to our control, only ST217 showed a statistically significant (Fig. 1, P<0.05) impact on cell tight-junction integrity. Thus, ST217 may show increased pathogenicity driven by markedly decreasing the density of ZO-1 protein known to be localised at tight junction sites. 

Deep, Arpit, Arijit, Shantanu, Kshitija, Sejal

Materials and Methods

To perform Gram staining on Streptococcus pneumoniae, prepare a smear from a colony on a culture plate and heat-fix it on a glass slide and then apply a series of Sigma Aldrich reagents: crystal violet for 30 Seconds and Lugol's iodine for 30 seconds and Iodine acetone decolorizer for 30 seconds and Safranin counterstain for 30 seconds. After each step, you rinse with water. After air-drying, we examine the slide under an Olympus CX23 microscope at 100x magnification and we use oil immersion. This method differentiates bacteria based on cell wall composition, with S. pneumoniae, being Gram-positive, retaining the purple crystal violet-iodine complex. The entire process allows for visual identification of the bacteria's Gram staining and morphological characteristics.

Gram Staining

The slides were labeled using the sample ID number in pencil. A drop of saline was added through a sterile loop, and a single bacterial colony was emulsified onto the slides. The bacterial film was fixed by placing the slides onto a hot plate at 37 degrees Celsius. The fixation was done to kill the bacteria and ensure the sample's adherence. Then, the gram staining procedure was applied by using kits form Sigma Aldrich, UK. First, crystal violet was added and left for 30 seconds. Then, it was washed with tap water before being added by Lugol Iodin for 30 seconds. And finally using iodine acetone following the Preston and Morrel modification (1962).

wenjin henan meina xiaojie jiayi kun

Gram Stain of Streptococcus pneumoniae

A single colony of S. pneumoniae was selected and emulsified in a drop of saline on a clean microscope slide. The slide was dried on a hot plate before proceeding with the Gram staining. The method followed was adapted from Preston and Morrell (1962). The staining process involved sequentially applying Crystal Violet stain, Lugol’s Iodine, Iodine Acetone, and Safranin. Each reagent was left on the sample for 30 seconds before thorough rinsing with distilled water. After drying completely, the slides were examined under an Olympus CX23 microscope at 100× magnification using immersion oil. All reagents were obtained from Sigma Aldrich, UK, in stock concentrations.

CTCF data were processed using Microsoft Excel prior to performing analyses using Prism (GraphPad Software, CA, USA). For all experiments, at least three replicates were performed and averages were used for the analyses, unless otherwise stated. The data were then analysed using one-way analysis of variance (ANOVA) comparing sample values to the control value

Data were processed utilizing Excel software (Microsoft), and analyses were performed using Prism (GraphPad Software). All experiments were performed in triplicate and data were analyzed using one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison test (when comparing with the control group).

Statistical analysis

The fluorescence intensity data for Streptococcus pneumoniae strains (Control, D39, ST306, and ST217) were collated and processed using Microsoft Excel for initial organization and cleanup. Data analysis was subsequently performed in GraphPad Prism (GraphPad Software, CA, USA). All experiments were conducted in triplicate. Data analysis was conducted using one-way ANOVA in Prism (GraphPad), an appropriate statistical method for comparing mean values across three or more independent groups. By using ANOVA, it is possible to identify significant variations in CTCF means across the different treatment groups Results were reported as mean ± standard deviation (SD), and p-values < 0.05 were considered statistically significant.

Data were processed utilizing Excel software (Microsoft), and analyses were performed using Prism (GraphPad Software). All experiments were performed in triplicate and data were analyzed using one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison test (when comparing with the control group).

Nadya , Omar , Elizabith and Fai

The data were processed using Excel software, facilitating precise organization and computation. For the subsequent analyses, we employed Prism software from GraphPad (CA, USA), which provides advanced statistical capabilities. Each experiment was conducted with a minimum of three replicates to ensure the reliability of the results, unless otherwise noted.

For data analysis, we utilized Student's t-test to compare differences between two independent groups. In cases involving three or more groups, a general linear model (GLM) was applied, followed by one-way analysis of variance (ANOVA) and Tukey’s test for post-hoc comparisons. This structured methodology allowed for a thorough examination of the experimental outcomes

Figure legends

The bar graph compares CTCF values across Control, D39, ST306, and ST217 groups. ST306 has the highest CTCF, significantly exceeding both D39 () and ST217 (**). The Control group shows moderate CTCF, higher than D39 () and ST217 (**), but below ST306. D39 is reduced compared to Control and ST306, while ST217 has the lowest fluorescence, significantly below Control and ST306.

Fig 1.0: There was a huge significant difference observed between the CTCF values of serotypes ST306, D39 and ST217. This shows that the serotypes ST217 and D39 impact the integrity of the cell tight junctions more than ST306. The immunostained cells were examined under a fluorescent microscope and CTCF values calculated. A One-way ANOVA was used to analyze the data and has been presented as mean +/- standard deviation. * is p < 0.05. CTCF =Corrected Total Cell Fluorescence.

Results

To demonstrate the effect of different serotypes of S. pneumoniae on barrier integrity, alveolar ZO1 were tagged with fluorescent anti-ZO1 (Rb polyclonal anti-ZO1 Abcam ab96586). These were observed under fluorescent microscope.

It was observed that ST217 had a significant reduction in Corrected Total Cell Fluorescence (CTCF) (Figure X p<0.05) compared to the control. So this suggests that the ST217 serotype was the most virulent and cause the most lysis of the alveolar cells.

Deep, Arpit, Shantanu, Arijit, Sejal, Kshitija

Result

Fluorescence intensities of biofilms treated with Control, D39, ST306, and ST217 revealed significant differences (ANOVA, P=0.0042). ST306 had the highest output, while D39 and ST217 showed lower values. ZO-1 fluorescence analysis indicated serotype-dependent epithelial barrier disruption: ST306 reduced intensity significantly (p<0.05), and ST217 caused the most severe loss (p<0.01), suggesting variations in structural stability and cellular impact among Streptococcus pneumoniae serotypes.

To reflect the potential impacts of the bacterial serotypes on epithelial barrier integrity, ZO-1 protein was used to mark tight junctions in epithelial cells, which allows the assessment of the integrity of cell junctions across different groups. ST217 showed the lowest ZO-1 expression, suggesting a potential downregulation of tight junctions (Figure 1, p < 0.05). ST306 exhibited significantly higher ZO-1 expression (p < 0.05), indicating enhanced tight junction upregulation compared to the D39, and ST217 groups. The differential ZO-1 expression highlights the varying impact of Streptococcus pneumoniae serotypes on epithelial junctions, with ST217 potentially impairing tight junction integrity. This finding suggests serotype-specific effects on barrier function that could have implications for infection and pathogenesis.