TITLE: Staphylococcus Aureus Sampling
AUTHORS: Mar Roca Cugat, Olga Sánchez

[DESCRIPTION]
This protocol is intended to study the affectation of Staphylococcus Aureus, including the MRSA, VISA and VRSA variants, even if it makes the test more difficult to perform. It outlines the basic protocol for a multi-subject study, while using basic and minimal resources found in almost every biology lab.

[GUIDELINES]
This protocol is intended to study the affectation of Staphylococcus Aureus, which is a Biosecurity Level 2 bacterial agent. As such, the laboratory should be adequated to those standards or take measures in order to prevent infection, cross-contamination, or leaks.

[STEPS]
SECTION: Preparation
1. Wash your hands with soap. Put on your lab coat, your mask, and your goggles or face shield. Make sure your mask is airtight and air cannot escape through the sides.

SECTION: Preparation
1.1. Prepare the area where you are going to work. Disinfect the surfaces with the bleach solution.
The subjects should not be able to walk behind you, only to the side or to the front. Make sure to leave enough distance between the sampler and the subject, but not enough distance as for the sampling to be uncomfortable.
The environment should be comfortable, within the following range of temperatures: 20 °C--35 °C 
You should have a plastic, sealable box to your side or on the table to store the sampled Petri dishes. 
The Bunsen burner should be to the front of you, within a hand of distance. 
The fresh swabs and Petri dishes should never be accessible by the subjects.

SECTION: Preparation
1.2. Using a permanent marker, divide each plate into two to four equal parts. You should help yourself by using a guide, such as a ruler.
SECTION: Sampling
2. Observe the subject's hands. If their nails are longer than 1-4 mm (the white part of the nail that can overgrow). 
Bitten-down nails could lead to invalid results. Too long nails could lead to cross-contamination.
Ask the subject for their identificative and contact information if this has not been done previously.
SECTION: Sampling
3. Place a Petri dish on the side of the Bunsen burner. The burner will be the center of our sterile field.

 


SECTION: Sampling
4. Ask the subject to wash their hands thoroughly and below the nails with soap and lukewarm water. Note their subject ID on the bottom (agar side) of the Petri dish.

SECTION: Sampling
4.1. Bring the subject's hands below the sterile area generated by the Bunsen burner. Open the Ringer solution and soak the swab. Proceed by swabbing below every nail in both hands. Once done, the subject can be dismissed. 

SECTION: Sampling
4.2. "Paint" one half of the Petri dish with the swab, softly so as not to break the agar but firmly as to get the sample to transfer to the plate.
SECTION: Sampling
5.   
Once the plate has 3 samples, place it in the full plates box.
Once there are 20 plates in the box, group them together with tape, write an identificative group number and place it in the incubator.

The incubator should be set to 37 °C and left to incubate for 1440 min. 
SECTION: Study
6. 
 
The samples should be taken out of the incubator, the results introduced into the database and comunicated to the subject.
SECTION: Study
7. Using one of the extra MSA Agar plates, culture and purify a sample of Staphilococcus Aureus  This can then be treated with GRAM tinture in order to observe it under an optical microscope (ideally at x1000-x1200). 

 







SECTION: Study
8. Prepare an LB dillution (20 mL ) with as many pure colonies as possible. Incubate at37 °C for1440 min. 
Perform an OD600nm. The sample should be diluted to get around 1 OD in order for it to be accurate. When the result comes in, the solution should be diluted with LB to an OD of  8 OD to 12 OD.  With the OD600, we can check the result and contrast it to a known source.
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SECTION: Sampling
4. Ask the subject to blow their nose thoroughly. Note their subject ID on the bottom (non--removable part) of the Petri dish.

SECTION: Sampling
4.1. Open the Ringer solution and soak the swab. Proceed by swabbing circularly their nose. The swab should have its head fully inside the subject's nose, however if the bone can be felt, the swab should not go further in.

SECTION: Sampling
4.2. "Paint" one third of the Petri dish with the swab, softly as not to break the agar but firmly as to get the sample to transfer to the plate.
SECTION: Sampling
5.   
Once the plate has 3 samples, place it in the full plates box.
Once there are 20 plates in the box, group them together with tape, write an identificative group number and place it in the incubator.

The incubator should be set to 37 °C and left to incubate for 1440 min. 
SECTION: Study
6. 
 
Using one of the extra MSA Agar plates, culture a sample of Staphilococcus Aureus (incubate at37 °C for1440 min). This can then be treated with GRAM tinture in order to observe it under an optical microscope (ideally at x1000-x1200). 
SECTION: Study
7. Prepare an LB dillution (20 mL Calibration Protocol - Conversion of OD600 to Colony Forming Units (CFUs) ) with as many pure colonies as possible. Incubate at37 °C for1440 min. 
Perform an OD600nm. The sample should be diluted to get around 1 OD in order for it to be accurate. When the result comes in, the solution should be diluted with LB to an OD of  8 OD to 12 OD. 
 unti l




SECTION: Study
8. Send a sample to MicrobesNG using the given inactivation buffer.