Sep 19, 2022
Version 10
Staphylococcus Aureus Sampling V.10
- Mar Roca Cugat1,
- Olga Sánchez2
- 1Institut Jaume Vicens Vives;
- 2Universitat Autònoma de Barcelona
- Olga Sánchez: Peer review;
Protocol Citation: Mar Roca Cugat, Olga Sánchez 2022. Staphylococcus Aureus Sampling. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb6pk1lpk/v10Version created by Mar Roca Cugat
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 19, 2022
Last Modified: September 19, 2022
Protocol Integer ID: 70244
Keywords: Microbiology, sampling, swab, staphilococcus aureus
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
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.
Materials
PPE
- Face shield or protective goggles
- FFP2/KN95 or higher-rated mask
- Latex, non-powdered gloves
- Lab coat
Sampling material
- Clean and sterile cotton swabs (n+n/10 being n the number of tests required)
- MSA Agar Petri dishes (n/(2 to 4) being n the number of dishes required)
- Sterile Ringer solution
- Permanent marker
Support material
- Bunsen burner
- Incubator
- Ethanol: >80%
- Bleach solution at 50%v in water.
- Reagents to make LB
- Inactivation buffer
- Photospectrometer
Safety warnings
This protocol requires interaction with people, possibly infected with a pathogen (especially in 2022, when this protocol was written and put into practice, as COVID-19 was still going strong). As such, there is a risk of infection which can be reduced with proper PPE use.
Proper ventilation is recommended at all times, even when the pandemic situation is over. However, the sterile field must be preserved at all costs, so try to direct the airflow in order for it not to affect the results.
Preparation
Preparation
1h
1h
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.
10m
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.
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.
Sampling
Sampling
1d 6h
1d 6h
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.
Place a Petri dish on the side of the Bunsen burner. The burner will be the center of our sterile field.
Safety information
Watch out so as not to break the sterile field
Nail sampling7 steps
For cases where the nails are 3-6mm long
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.
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.
"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.
Repeat n times
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 24:00:00 .
1d
Study
Study
1d 2h
1d 2h
Expected result
It is expected to one of these results. A bright red colour means the sample was uninoculated. A pinkish colour with translucent streaks means there is Staphylococcus epidermis present. A faint yellow colour or a bright yellow colour means there is Staphylococcus aureus present
The samples should be taken out of the incubator, the results introduced into the database and comunicated to the subject.
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).
Safety information
Watch out for impurities/contaminations
Prepare an LB dillution (20 mL LB ) with as many pure colonies as possible. Incubate at37 °C for24:00:00 .
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.
Protocol
NAME
Calibration Protocol - Conversion of OD600 to Colony Forming Units (CFUs)CREATED BY
Paul Rutten
1d