Order 626741: Microbiology Staph Lab report

Introduction

               Staphylococci are gram-positive cocci (bacteria) typically about 1µm in size and grow in clumps, pairs, or occasionally short chains. The staphylococci genus is home to around 40 different strains of the bacteria, some being extremely common and harmless relative to humans while others are much less present and can cause serious infections when penetrating the barriers of the human body(Staphylococci,2017). Staphylococci epidermidis is one of these common types of staphylococci that is part of the normal human flora and is typically found on the skin and slightly less commonly in mucosal tissue due to it’s ability to withstand higher salt concentrations. It typically forms colonies 1-2mm in diameter and will test positive in the catalase test, but negative in a coagulase one. Another characteristic of this strain is that it is a facultative anaerobe, meaning that it can grow utilizing both aerobic respiration and fermentation. Being that S. epidermidis is typically found on humans, it does not normally pose a threat. However, out of all coagulase-negative staphylococci, s. epidermidis cause the most amount of infections, typically through the use of medical instruments, prosthetics, and catheters. This is due to the fact that there is a vast amount of s. epidermidis on the skin and can be moved into the organism through the instruments, posing threat for infection(Otto, 2009). . Another common coagulase negative staph is S saprophyticus. It is normally found in the flora of the female genital region and inside the gastrointestinal tract. S saprophyticus does not normally pose a threat but is contributed to causing around 10-20% of urinary tract infections in females.  S. Aureus is a strain less common but still found on the skin, in the nose, and the respiratory tract as it is also tolerant to higher salt concetrations. It is a coagulase positive strain, the only other coagulase positive staphylococci being s. intermedius. Although common and can work commensally in the body, S aureus is the main cause of staph infections and poses many virulent factors. A main threat of s. aureus is that it has gained the ability to become resistant to methicillin antibiotics and is the bacteria responsible for MRSA (methicillin-resistant S aureus). S aureus is responsible for superficial skin lesions, deep-seated infections, food poisoning due to the release of enterotoxins, and toxic shock syndrome by releasing of super antigens into the bloodstream. Other common staphylococci include S lugdunensis, S haemolyticus, S warneri, S schleiferi, S intermedius but they are rare pathogens (Kobayashi, 2015).

In order to identify and classify bacteria, physiological characteristics of the bacteria must be determined. Through the use of multi-test media, different physiological characteristics can be observed. MSA plates were used to determine that the strains taken from the nose and bottom of one show were not S aureus, as they did not ferment the mannitol and would not turn yellow.

16s rRNA is approximately 1500 base pairs and is used as the main method for phylogentic purposes when it comes to identifying bacteria. This method is used due to the fact that the 16s rRNA sequence is highly conserved between different species of bacteria. The 16s rRNA subunit is a main component in the 30s ribosomal complex in prokaryotes. The structural importance of this gene is what has slowed the evolution of it and led to it’s conservation. Through the method of PCR, the 16s rRNA sequence can be replicated and sequenced to determine the genus of staphylococci present. There are different PCR methods that can be used to extract and replicate the 16s rRNA sequence, the one used in this lab consisted of using an 8F primer and 1492R primer to result in a rRNA sequence of approximately 1484 base pairs, which can be sequenced and compared to identify the genus of staphylococci present (Janda,2007).

Our experiment attempted to determine the species of two unknown staphylococci that were not S aureus using the method of 16S rRNA sequencing and analyzing the sequence compared to other bacterial species called BLAST® by NCBI . One strain was taken from the nose and the other from the bottom of one shoe. After evaluation, we concluded that both the strain from the nose and the strain from the bottom of the shoe  was S epidermidis.

Materials and Methods

    Staph Lab Part I-Obtaining Specimens

The two Staphylococcus specimens were obtained from our nose and the bottom of our shoe. We obtained the samples by first sterilely pipeting 5ml of m-staphylococcus broth into 3 x 15 ml screw top cap tubes. The tubs were then inoculated separately by using sterile swabs to first swab the inside of one of our nose’s and inoculating the tube and then the bottom         of one our shoes. The swab was gently moved about the area of concern to pick up bacteria, then placed in the tube with the m-staphylococcus broth and moved around for a short period of time to transfer the microbes. They were then incubated at 37°C for one week. The m-staphylococcus broth consisted of using 20 grams of powder and mixing it with 80ml of water and vacuum filtrating it using .2µm filter to sterilize.

    Staph Lab Part II-Streaking MSA Plates

               After the m-staphylococcus broths had been incubating at 37°C for a week, Mannitol Salt Agar plates were streaked using each specimen.  The bacteria growing in the tubes were transferred to its own MSA plate through the use of a sterilized swab. The plates were then incubated at 37°C. After a week of incubation, the bacteria growing on the plates were first analyzed to conclude that they were not S aureus by noting that the specimens did not ferment the mannitol and no color change to yellow agar was observed.  Then, one isolated colony from each plate was transferred from the MSA plate to its own MSA plate using an inoculating loop. These were then also incubated at 37°C.

    Staph Lab Part III-Gram Stain and 16s rRNA PCR

               The bacterium on the MSA plates were Gram –stained and observed under a microscope to securely indentify that they were Gram-positive Cocci. The procedure of the Gram-Stain and result were comparable to other research of such strains, in that the bacteria resulted in clustering, purple, cocci.( link 1,Link2).Bacterium from each plate were then used to inoculate a Microgen Staph ID test strip using an inoculating needle. These were then incubated at 37°C and examined the next week to gain an ID to match with the sequencing performed in the next step.

               16s rRNA sequencing was performed by first putting .5ml of sterile saline in two separate centrifuge tubes and labeling them.  An inoculating loop was then used to transfer a miniscule amount of each bacterium into each of the centrifuge tubes, using separate tubes for each of the different samples. A 50ml PCR reaction was then set up using the following concentrations of the additives:The components, once in the tubes, were mixed by pipeting up and down. The tubes were placed in a thermocycler that ran the denaturing, annealing, and extension phases using the following cycle:This cycle was repeated 40 times with a 5 minute 94°C step prior to the cycles for first denaturation and a 7 minute 72°C step post cycles to secure any other needed elongation.  This provided us with a theoretical yield of 240 copies of DNA.

    Staph Lab Part IV

               Cleaning Product

Since the PCR was run in part III, the mixture needs to be cleaned up in order to be used for DNA sequencing. The reaction contains unused primers and nucleotide, and a buffer for PCR. The mixture is run through a small column, BioRad Quantum Prep in this lab, which separates the molecules of he contents by size. Resin beads inside the columns will trap the smaller molecules such as the primers, nucleotides, and buffer while allowing the large PCR product molecules to flow through.  This was done following the 7 step procedure provided by BioRad; Step 1 was vortexing the column to resuspend the resin making sure none was settled, Step 2 was snapping off the tip and placing the whole column in a 2.0 ml wash tube, we then spun the column for 1 minute in a microcentrifuge at 735 x g. The column was then place in a clean 1.5 ml collection tube to collect the cleaned sample as we then added 50µl of the sample to the top of the column and spun it for 2 minutes at 735 x g. The purified sample had then passed through the resin beads and remained in the bottom of the 1.5ml collection which was sent out to Macrogen,Inc in Maryland for sequencing after it was tested for sound results through gel electropherosis.

Gel Electropherosis

After the product was cleaned and was in the correct state to be sequenced, we first ran the product on a gel electropherosis to see if the PCR had been run successfully.  It was expected that all PCR products would be 1484 base pairs long, as the reverse primer used was 1492R and the forward was 8F resulting in 1484 base pairs.  Each cleaned DNA segment was run in 1% agarose gel running in 1xTBE buffer and 10µL of the DNA was used. The 1x TBE buffer consisted of 89 mM Tris base, 89 mM Boric acid, and 2 mM EDTA. The stock was then diluted 10x to make 500ml total volume. This was used with a 50ml gel that was made using the following guidelines; .5g agarose was weighed and put in a 25ml flask, 50ml 1xTBE buffer was added and the agarose was then microwaved approximately 1 minute until it melted. The hot agarose was then poured into a 50ml blue cap tube and water was added to bring he volume back up to 50ml. We then added 5µl of 10mg/ml ethidium bromide and this was poured into the gel tray with an 8-well comb where it was left to harden.

After the gel was made, the DNA had to be prepped to run on the gel. This was done by placing 10 µl of the DNA in a 1.5ml tube and had 2µl 6x gel loading buffer added to it. This then allowed us to place it in the gel box and fill the box with 1x TBE buffer until it was submerged , once the gel had hardened.  The gel was then loaded with our samples with a combined 5µl of DNA size markers. The size markers used were BioRad EZ Load 100 bp PCR Markers. These markers provide bands at every 100bp between 100-3000bp, with thick bands at 1000 bp and 3000 bp that contain triple the concentration. These markers allow for a template to gauge the size of the DNA fragments in which our products resulted in. The gel was hen run at 140V for approximately 30 minutes to allow the fragments to move through the gel. Once complete, the gel was viewed using an ultraviolet transilluminator to see that our DNA fragments had moved through the gel and using the markers we were able to determine that the size of the segments were approximately 1484 base pairs. This indicated that our PCR had been run successfully and could be sequenced. We prepared the sample for sequencing by placing 15µl of the cleaned PCR DNA in a tube labeled by specimen (shoe, nose) and sent to Macrogen,Inc. in Rockville, Maryland to be sequenced.

Materials and Methods

Staph Lab Part I-Obtaining Specimens

The two Staphylococcus specimens were obtained from our nose and the bottom of our shoe. We obtained the samples by first sterilely pipeting 5ml of m-staphylococcus broth into 3 x 15 ml screw top cap tubes. The tubs were then inoculated separately by using sterile swabs to first swab the inside of one of our nose’s and inoculating the tube and then the bottom of one our shoes. The swab was gently moved about the area of concern to pick up bacteria, then placed in the tube with the m-staphylococcus broth and moved around for a short period of time to transfer the microbes. They were then incubated at 37°C for one week. The m-staphylococcus broth consisted of using 20 grams of powder and mixing it with 80ml of water and vacuum filtrating it using .2µm filter to sterilize.

Staph Lab Part II-Streaking MSA Plates

After the m-staphylococcus broths had been incubating at 37°C for a week, Mannitol Salt Agar plates were streaked using each specimen. The bacteria growing in the tubes were transferred to its own MSA plate through the use of a sterilized swab. The plates were then incubated at 37°C. After a week of incubation, the bacteria growing on the plates were first analyzed to conclude that they were not S aureus by noting that the specimens did not ferment the mannitol and no color change to yellow agar was observed. Then, one isolated colony from each plate was transferred from the MSA plate to its own MSA plate using an inoculating loop. These were then also incubated at 37°C.

Staph Lab Part III-Gram Stain and 16s rRNA PCR

The bacterium on the MSA plates were Gram –stained and observed under a microscope to securely indentify that they were Gram-positive Cocci. The procedure of the Gram-Stain and result were comparable to other research of such strains, in that the bacteria resulted in clustering, purple, cocci.( (Namvar, 2014. Uconn,2017).Bacterium from each plate were then used to inoculate a Microgen Staph ID test strip using an inoculating needle. These were then incubated at 37°C and examined the next week to gain an ID to match with the sequencing performed in the next step.

16s rRNA sequencing was performed by first putting .5ml of sterile saline in two separate centrifuge tubes and labeling them. An inoculating loop was then used to transfer a miniscule amount of each bacterium into each of the centrifuge tubes, using separate tubes for each of the different samples. A 50ml PCR reaction was then set up using the following concentrations of the additives:

The components, once in the tubes, were mixed by pipeting up and down. The tubes were placed in a thermocycler that ran the denaturing, annealing, and extension phases using the following cycle:

This cycle was repeated 40 times with a 5 minute 94°C step prior to the cycles for first denaturation and a 7 minute 72°C step post cycles to secure any other needed elongation. This provided us with a theoretical yield of 240 copies of DNA.

Staph Lab Part IV

Cleaning Product

Since the PCR was run in part III, the mixture needs to be cleaned up in order to be used for DNA sequencing. The reaction contains unused primers and nucleotide, and a buffer for PCR. The mixture is run through a small column, BioRad Quantum Prep in this lab, which separates the molecules of he contents by size. Resin beads inside the columns will trap the smaller molecules such as the primers, nucleotides, and buffer while allowing the large PCR product molecules to flow through. This was done following the 7 step procedure provided by BioRad; Step 1 was vortexing the column to resuspend the resin making sure none was settled, Step 2 was snapping off the tip and placing the whole column in a 2.0 ml wash tube, we then spun the column for 1 minute in a microcentrifuge at 735 x g. The column was then place in a clean 1.5 ml collection tube to collect the cleaned sample as we then added 50µl of the sample to the top of the column and spun it for 2 minutes at 735 x g. The purified sample had then passed through the resin beads and remained in the bottom of the 1.5ml collection which was sent out to Macrogen,Inc in Maryland for sequencing after it was tested for sound results through gel electropherosis.

Gel Electropherosis

After the product was cleaned and was in the correct state to be sequenced, we first ran the product on a gel electropherosis to see if the PCR had been run successfully. It was expected that all PCR products would be 1484 base pairs long, as the reverse primer used was 1492R and the forward was 8F resulting in 1484 base pairs. Each cleaned DNA segment was run in 1% agarose gel running in 1xTBE buffer and 10µL of the DNA was used. The 1x TBE buffer consisted of 89 mM Tris base, 89 mM Boric acid, and 2 mM EDTA. The stock was then diluted 10x to make 500ml total volume. This was used with a 50ml gel that was made using the following guidelines; .5g agarose was weighed and put in a 25ml flask, 50ml 1xTBE buffer was added and the agarose was then microwaved approximately 1 minute until it melted. The hot agarose was then poured into a 50ml blue cap tube and water was added to bring he volume back up to 50ml. We then added 5µl of 10mg/ml ethidium bromide and this was poured into the gel tray with an 8-well comb where it was left to harden.

After the gel was made, the DNA had to be prepped to run on the gel. This was done by placing 10 µl of the DNA in a 1.5ml tube and had 2µl 6x gel loading buffer added to it. This then allowed us to place it in the gel box and fill the box with 1x TBE buffer until it was submerged , once the gel had hardened. The gel was then loaded with our samples with a combined 5µl of DNA size markers. The size markers used were BioRad EZ Load 100 bp PCR Markers. These markers provide bands at every 100bp between 100-3000bp, with thick bands at 1000 bp and 3000 bp that contain triple the concentration. These markers allow for a template to gauge the size of the DNA fragments in which our products resulted in. The gel was hen run at 140V for approximately 30 minutes to allow the fragments to move through the gel. Once complete, the gel was viewed using an ultraviolet transilluminator to see that our DNA fragments had moved through the gel and using the markers we were able to determine that the size of the segments were approximately 1484 base pairs. This indicated that our PCR had been run successfully and could be sequenced. We prepared the sample for sequencing by placing 15µl of the cleaned PCR DNA in a tube labeled by specimen (shoe, nose) and sent to Macrogen,Inc. in Rockville, Maryland to be sequenced.

Kobayashi, S. D., Malachowa, N., & DeLeo, F. R. (2015). Pathogenesis of Staphylococcus aureus Abscesses. The American Journal of Pathology, 185(6), 1518–1527. http://doi.org/10.1016/j.ajpath.2014.11.030

Janda, J. M., & Abbott, S. L. (2007). 16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls . Journal of Clinical Microbiology, 45(9), 2761–2764. http://doi.org/10.1128/JCM.01228-07

Namvar, A. E., Bastarahang, S., Abbasi, N., Ghehi, G. S., Farhadbakhtiarian, S., Arezi, P., … Chermahin, S. G. (2014). Clinical characteristics of Staphylococcus epidermidis: a systematic review. GMS Hygiene and Infection Control, 9(3), Doc23. http://doi.org/10.3205/dgkh000243

(n.d.). Retrieved November 11, 2017, from http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.html

Otto, M. (2009). Staphylococcus epidermidis – the “accidental” pathogen. Nature Reviews. Microbiology, 7(8), 555–567. http://doi.org/10.1038/nrmicro2182

Staphylococcal Infections: MedlinePlus. (2017). Retrieved November 9, 2017, from https://medlineplus.gov/staphylococcalinfections.html