TITLE: Staphilococcus Aureus Sampling
AUTHORS: Pol   Roca Cugat, Olga Sánchez

[DESCRIPTION]
This protocol is intended to study the affectation of Staphilococcus Aureus, including the MRSA variant. It outlines the basic protocol for a multi-subject study.

[GUIDELINES]
This protocol is intended to study the affectation of Staphilococcus Aureus, which is a Biosecurity Level 2 bacterial agent. As such, the laboratory should be adequate  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 betweeen the sampler and the subject, but not enough distance as for the sampling to be uncomfortable.
The environment should be comfortable, within this range of temperatures: 10 °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. Grab a permanent marker and divide each plate in three equal parts. You can help yourself with a guide.
SECTION: Sampling
2. Observe the subject. If their nails are longer than 3-6mm (white part that can be accessed). 
Bitten-down nails could lead to invalid results. Too long nails could lead to cross-contamination.
Ask the subject for his identificative information if this has not been done previously.
SECTION: Sampling
3. Place a Petri dish on the side of the Bunsen burner. Open the swab below the Bunsen burner.

 


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 (non--removable part) of the Petri dish.

SECTION: Sampling
4.1. Bring the subject's hands below the effect area of 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 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. 
 
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: 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
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. 
 unti l




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