Nasal Swab collection process:
Nasopharyngeal swabs are purposefully made in the form of long, flexible shafts that are prepared from metal or plastic, and contain rayon, flocked nylon, or polyester tips. Moreover, you would also require personal protective equipment (PPE), which includes gloves, protective masks, gown, and face shield. Prior to the procedure, label the sample tube and fill the required requisition forms. Follow the contact and respiratory precautions that are recommended by your institution and the Centers for Disease Control and Prevention (CDC). PPE must be worn carefully. First, put on the protective gown. Then, use either an alcohol-containing solution or soap and water to wash your hands. Put on the gloves after washing the hands. Next, as per the recommendations of the CDC, put on the N95 or higher-grade mask.
At last, to protect eyes and face, put on the face shield. First, request the individual to blow nose to get rid of excessive secretions. Take one nasal swab. Slightly tilt back the head of the patient to gain more access to the nasal passages and close the patient’s eyes to reduce the mild discomfort during the process. Next, insert nasal swab just above the nasal passage floor, along the nasal septum, into the nasopharynx till you feel a resistance. Insert the swab parallel to palate. However, in case of resistance, try to reinsert the swab closer to the nasal canal floor at a different angle. The depth up to which the swab is inserted must be the same as the distance between the outer ear opening and the nostrils. According to the recommendations of the CDC, retain this position of the swab for few seconds to allow it to absorb the secretions. Then, please take out the swab, while simultaneously rotating it, and put it into a collection tube. At the groove, break the swab and discard the remains. Next, close the tube, clean its surface using a wipe soaked with a surface disinfectant, and put it into a biohazard bag.
Then, remove PPE in proper order. First, remove the gown, and then, the gloves. Wash the hands as described previously. Next, wear a fresh pair of gloves, take off the face shield, either discard or clean and store it as per the institutional guidelines. Again, remove the gloves, rewash the hands, wear a new pair of gloves, and take off the face mask. Follow the guidelines of the institute to reuse or dispose of it. In the end, take off the gloves and wash your hands. The above process has previously been described by Marty, Chen, and Verrill, 2020, in a video .
Oropharyngeal Swab collection process:
After putting on PPE and labeling the sample as described previously, hold the tongue of the subject out of the way using a tongue depressor. Then, swab the tonsilar pillars and the posterior pharyngeal wall in a sweeping position. Ask the individual to say “aah” to allow uvular elevation. Avoid touching the soft palate and the tongue by the swab. We then put the swab into the pre-labeled viral transport media. This is the process recommended by the WHO .
Gram staining process:
The following equipment is used for this process: Bunsen burner, clean glass slides, inoculating loop, lens paper and lens cleaner, microscope, bibulous paper, distilled water, immersion oil, and microbial culture (18-24 h old).
The following reagents are used: crystal violet, Gram’s iodine, ethyl alcohol, and safranin.
1. Glass slide preparation: To prepare microbial smears, it is necessary to remove the oil or grease that comes out of our fingers during handling. For this, wash slides in soap and water and wipe them using a wipe dipped in an alcohol-based solution.
2. Slide labeling: It is often useful in marking the smear area by creating a circular area on the underside using a glassware-marking pen. Furthermore, you may also write the initials of source organisms using the same pen on edge. However, one must ensure no contact between the staining reagents and the label.
3. Smear preparation: Roll the swab on the clean slide surface.
4. Fixing: Heat fixing is useful for eliminating the bacteria from the smear, adhering smear to the slide, and allowing natural uptake of staining reagent. To heat fix the slide, first air-dry the smear. Next, hold the slide from one end and expose the rest of the slide to a Bunsen burner flame with smeared side up.
5. Gram staining: Put the slide in a staining tray. Fill the tray with crystal violet for 1 min. Then, rinse the tilted slide using tap or distilled water. Then, fill it with Gram’s iodine for 1 min. Again, rinse the tilted slide using distilled or tap water. The smear would be stained in purple color. Next, tilt the slide and pour acetone or 95% ethyl alcohol over the smear for 5-10 seconds drop by drop to decolorize the stain, although you must take care not to decolorize the stain excessively. Rinse with water immediately—counterstain using safranin for 45 seconds. Rinse the tilted slide using tap or distilled water. Blot-dry the slide, and view the smear under a light microscope.
6. The inoculation method for growth and culture assessment is described in general in figure 2 below .
Once we get growth, we will take samples from that part, for sensitivity analysis using the machine Vitek-2 compact ® which is as follows :
1. Choose the isolate.
2. Prepare microbial suspension and check the composition of the McFarland Standard.
3. Use ID suspension to make AST suspension.
4. Inoculate the cards within the instrument. Transfer them from filling door to loading door manually for processing.
5. Scan cassette worksheet at the workstation.
6. Obtain the results within 5 to 8 h.
We will use Gram Stain for routine assessment of the prevailing microbiome and then we will do a culture assessment. After getting culture and Gram stain report, we will match with the existing data of the prevailing microbiome in a healthy individuals as published in various papers.