Insti-­‐Spore Si Protocol www.Insti-­‐Fluor.com Introduction: Weaponized endospores are coated with silica. This makes them resistant to fluorescent staining, and thus the Bacterial Endospore Technique (MAA or Insti-­‐
Spore test slides) fails. Endospores may be treated with hydrofluoric acid (HF) however, which strips the silica coating and allows fluorescent probe to enter the cell. There is a problem with this technique though, because the HF treatment permeabilizes the endospore wall and permits the fluorescent dye to penetrate the spore coat, and thus stain the entire cell. When this occurs the diagnostic “Ghost Football” of non-­‐coated spores is no longer present. The problem, therefore, is developing a technique for identifying whether endospores 1) are present, and 2) are silica-­‐coated. This problem may be solved using the microscope techniques of Phase Contrast followed by Fluorescence probing: 1) Phase Contrast imaging 2) HF Treatment followed by fluorescent probing 3) No HF Control fluorescent control using either the MAA or Insti-­‐Spore test. Together, these three rapid tests can identify putative Bacillus endospores and distinguish silica-­‐treated from non-­‐treated. Note: HF (hydrofluoric acid) is used in this test. Take extra precaution and avoid all contact with HF. Wear appropriate PPEs, especially eye protection. SOP: IDENTIFICATION OF SILICA-COATED BACILLUS ENDOSPORES
Phase Contrast Microscopy is a transmitted light microscope technique that exploits the large index of refraction exhibited by endospores. Using this technique endospores appear as bright “dots” against a gray background. Thus, the first step in ID of Weaponized endospores is to examine an untreated sample using Phase Contrast optics. If the sample contains bright bacteria-­‐size dots then you can be certain that the sample contains endospores. Following this, a similarly untreated sample should be examined using the Fluorescent Endospore test. If “ghost footballs” are observed, then the sample contains non-­‐weaponized endospores. Finally, a HF-­‐
treated sample is visualized using the Fluorescent Endospore test. If solid fluorescent endospores are visualized, then you can be certain that the sample contains silica-­‐coated endospores. Procedure: 1. Mix unknown with water and make two slides. 2. Place 10µl sample in the blank Insti-­‐Fluor slide. Label it “PC Control” 3. Place 10µl sample in the well of an Insti-­‐Spore slide. 4. Observe PC Control using Phase Contrast microscopy. Look for bright white (refractive) bacteria-­‐size dots. 5. Observe Insti-­‐Spore sample using Blue excitation. Look for either a) Ghost Footballs OR b) No fluorescent endospores. Evaluate: i.
ii.
iii.
iv.
Are endospores present? (using Phase Contrast) Are “Ghost Footballs” present (using fluorescence microscopy). If YES to both, STOP. You have non-­‐weaponized endospores. If Yes to (i) and No to (ii), proceed to step 6. 6. Add 0.5ml DI water to 25µl 1M HF (already in the vials) to make a 50mM HF working solution. WEAR GLOVES AND GOGGLES. 7. Add the HF working solution to the vial of sample (this is a toothpick-­‐end amount of silica-­‐coated spores). 8. Cap and incubate at RT for 15min. 9. Vortex for a second to resuspend spores. 10. Add 10µl HF-­‐treated sample to an Insti-­‐Spore slide. Apply coverslip. 11. Observe using Blue excitation. You will be looking for endospores that are fluorescent throughout the cell (not Ghost Footballs). Evaluate: i. Are fluorescent endospores present? ii. If YES then you have Weaponized Endospores Figure 1: Phase Contrast imaging of silica-­‐coated Bacillus endospores. Approximately 600x (40x oil immersion objective). Figure 2: A: Non-­‐silica coated Bacillus endospores showing the diagnostic “doughnut” fluorescence. B: Silica-­‐coated Bacillus endospores after HF treatment. Note that the entire endospore is fluorescent and that some silica powder remains.