The overall goal of the following experiment is to examine the survival and dissemination of methicillin resistant staphylococcus aureus or MA as well as the chemokine and cytokine levels at the infection sites. This is achieved by removing the fur from the backs of mice to allow a clear area of observation. As a second step.
MRSA in a suspension will be administered to mice subcutaneously three days post-infection, infection pathologies, and bacterial counts can be assessed by determining the number of colony forming units from tissue homogenates on agar plates. Hello, how are you? My name is De Lordon.
I'm a post researcher. Hi, my name is Mariel Sanchez. I am a PhD student And I am Andrea Huda.
I am a research associated, we are part of George Lee's lab in the pediatric department at Cedar-Sinai Medical Center. This method can help answering key questions in the medical field, such as modeling of severe infection, including myositis, polymyositis, and necrotizing fascitis. Though this method can provide insight into severe emergency infections, it can also be applied to other system such as group A streptococcus and other organisms.
Additionally, we can study the function and pathogenesis of other organisms that cause myositis and polymyositis. Generally, individuals new to this procedure may struggle because one of the staff of caucus areas global regulator is highly mutable. If precautious is not taken, data collected may have significant variation which will result in date difficulty to interpret the data.
Let's get started. In a BSL two facility, prepare a sheep blood agar plate in TSA base and a sterile loop using a frozen block. Transfer the stock Mercer culture out of minus 80 degrees Celsius storage.
Then using the sterile loop, break a small chip of ice from the stock culture onto the plate. After acquiring a small chip of Mercer stock culture immediately transfer the stock culture back to the freezer to avoid thawing because repeated freezing and thawing will damage the stock culture. Use the loop to streak the agar facilitating the growth of individual Mercer colonies.
Incubate the plate at 37 degrees Celsius overnight without CO2 supplement. The next day. Prepare and label both a new sheet blood egg, TSA plate, and a 15 milliliter snap cap tube with three milliliters of THB.
Retrieve the overnight cultured M SSA plate and examine the hemolytic phenotype of the colonies. Using a sterile loop, choose a colony that has a phenotype consistent with previous experimental colonies. Inoculate the THB with the selected colony using the same loop streak.
The plate incubate the THB solution at 37 degrees Celsius overnight with shaking at 220 rotations per minute. These bacteria will be used to inoculate the culture for subcutaneous infection of the mice in later steps. Place the blood agar plate in the incubator as well.
The plate will be used the following morning to confirm that the hemolytic phenotype of the bacteria in the THB solution matches that of the originally selected colony. If the hemolysis phenotype is not consistent with the original clone, do not start the experiment. Transfer the mouse to a platform and shave the fur off its back.
If the animal is overly aggressive, sedation with isof fluorine may be necessary. Apply approximately five cubic millimeters of hair remover cream such as NA to the shaved area. NA may be purchased from the local drugstore.
Make sure to purchase the hair removal cream containing baby oil because the product without the baby oil will be too stimulatory for the mice. Allow the cream to incubate on the skin's surface for approximately one minute while the hair remover cream is incubating wet a few paper towels with double distilled water after a minute. Use the wet paper towels to wipe the shaved area clean.
Make sure the skin is free from the hair removal cream prewarm 10 milliliters of THB in a 50 milliliter screw cap tube at 37 degrees Celsius in a bacteria incubator for four to 10 hours. Transfer 20 microliters of the overnight bacteria culture to the prewarm THB. This will dilute the overnight merc.
A suspension at one to 500. Place the tube at 37 degrees Celsius in a shaking incubator at 220 rotations per minute for approximately two and a half to three hours after the incubation period. Use more THB to further dilute the bacterial suspension to one to 10 and measure the suspension with a spectrophotometer.
With absorbency at 514 nanometers. Keep re incubating the suspension at 37 degrees Celsius with shaking at 220 rotations per minute until the one to 10 diluted suspension reaches 0.25. To collect the M er centrifuge, the 50 milliliter tube at 3, 225 times gravity for 10 minutes at four degrees Celsius, discard the supinate Resus.
Suspend the bacterial palette in 10 milliliters of DPBS and wash the bacteria again by repeating the centrifugation step for this severe infection model. Re suspend the bacterial pellet in approximately one milliliter of DPBS resulting in approximately one times 10 to the 10 colony forming units per milliliter. Assemble sheep blood A-R-T-S-A plates serially dilute the bacteria 10 to the one to 10 to the eightfold in DPBS plate.
10 microliter drops of the serially diluted bacterial suspension onto a sheet blood agar plate in triplicate. Incubate the plates at 37 degrees Celsius overnight. After incubating the plates for 16 to 18 hours, observe and record the hemolysis phenotype of the inocular.
Count the colony forming units on the plates. The infection inocular will be calculated based on the colony forming unit. Number on the plates.
Prepare a syringe with 100 microliters of the bacterial suspension in DPBS prepared earlier. Prior to injection, mice should be sedated so that their breathing rate is about once per second. For demonstration purposes, a euthanized mouse is shown in this video.
Inject the M ser into one flank after injection. Transfer the mouse back to its cage. Observe the animals for three to five hours after injection to make sure the mouse is alive before transfer back to the animal room.
Monitor the lesions on the backs of the animals daily after the animals have been sacrificed. Measure and record the size and condition of the lesions on the skin. Both skin and muscle lesion areas are quantified by multiplying the length and width of the lesion in millimeters.
Irregularly shaped lesions must be broken down into smaller symmetrical pieces and each piece measured by the same method. Prepare 1.5 milliliter micro centrifuge tubes with 100 microliters of DPBS for tissue collection. Nick the skin with a sterile razor blade and excise the lesion with a pair of sterile scissors.
When excising the lesion, make sure that approximately two to five millimeters of healthy skin surrounding the area is also excised. Place the lesion into the tissue collection tubes for analyzing the muscle lesions. Peel the skin off carefully to measure the size and to record the condition of the lesion on the muscle tissue.
Collect the spleen and kidneys. Place these tissues in their own individual collection tubes. Homogenize the tissues by agitating with a one milliliter syringe for approximately 30 seconds.
Add 900 microliters of DPBS to each tube. Assemble THA plates vortex the homogenized tissue samples for five minutes. Serially dilute the sample.
Suspensions tend to the one to 10 to the six fold. In 96, well plates dispense 10 microliters of each serially diluted suspension onto the plates. Incubate the plates in a 37 degrees Celsius bacteria incubator overnight.
The next morning, count the colony forming units from the plates and calculate the number of colony forming units based on the dilution factors. If the infection is done correctly, a bleb will be observed on the skin surface immediately after the subcutaneous injection. If no bleb is observed, the injection may be too deep, which may affect the outcome of the infection.
Lesion size and bacterial survival may be assessed at various points post-infection with MSA. The following skin lesions were examined on day three post-infection and are examples of a lesion on a CD one mouse infected on one flank with one times 10 to the seven colony forming units. Muscle lesions are typically found in the tissue below the skin lesions.
The following are examples of the coinciding muscle surface lesions for the previously shown skin lesion photos. These are muscle lesions observed at day three post infection in a mouse infected with one times 10 to the seven colony forming units. The lesion area is determined as the length times width of the lesion as measured in millimeters and may be measured to assess the tissue damage of skin.
The colony forming unit number of the bacteria localized at the infection site can be quantified on excise lesions. The colony forming unit count can indicate whether a virulence factor provides the MSA with a survival advantage. While attempting this procedure, it is important to remember to use the standard safety precautions due to use of needles and highly pathogenic microbes Following this procedure.
Last as the abscess model we uses 1, 000 fold lower inocular compared to our model can be used to answer additional questions such as what are the S factors that promote persistent RSA infection? Don't forget that working with a syringes filled with a Mars A can be extremely dangerous and precautions such as discarding the syringe directly in the sharp, it is important to avoid accidental stabbing by the needle. Also, for this experiment, it is important to follow a standard BS O2 protocols to assure proper disinfection of the work area.
So that's it. Thanks for watching. Hi.Hi, bye.
