laboratory standard operating...

LABORATORY STANDARD OPERATING PROCEDURES

1. Personal Protective Equipment including: gloves, eye protection, and a lab coat, must be worn when working with human cells or bacteria. Do not wear personal protective equipment outside of the lab. Open-toed shoes may not be worn in the lab. 2. No food or beverages in lab. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only. 3. Disposable labware is to be discarded in biohazard bags. Samples containing ≤ 1 mL volume can be discarded directly into biohazard bags. Petri dishes containing cultures are discarded directly into biohazard bags. 4. Sink area must be kept reasonably clean throughout the day. Eyewash station must be readily accessible. Sink must be free of all labware prior to leaving for the night. 5. Lab benches where work has been conducted must be decontaminated at the completion of work or at the end of the day and after any spill or splash of viable material with 70% EtOH, 10% Bleach, or Lysol. 6. If a spill occurs spray Lysol, or 70% EtOH, or a 10% Bleach solution on spill and place a paper towel on top of the spill to prevent further spreading. After ≥ 30 min remove paper towel and thoroughly decontaminate area using more of the disinfectant. If you are unsure about how to handle a spill contact Dr. Voyich or a science assistant. 7. Personnel must wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory. Bacteria can be easily passed on by human contact, and is commonly spread by the hands.

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P a g e | A2 Activity 1: Identify microbiota in your nose and throat Goal: Students will identify normal flora microorganisms from both nose and throat and look for Staphylococcus aureus and Streptococcus species. Day 1 Throat swab: 1. Get on your lab coat, goggles, and gloves. 2. Obtain an aliquot of sterile phosphate buffered saline (PBS), and sterile swabs. Obtain 2 plates each of Blood Agar (BA), Tryptic Soy Agar (TSA) and MacConkey Agar (MAC), and Mannitol Salt Agar (MSA). 3. Wet the swab with PBS. Say “Ahhhhhh.” Swab both of your tonsils. Place swab in sterile 15 mL tube containing a small amount of PBS. Discard swabs in biohazard. 4. Take the swab containing your partner’s normal microbiota and streak for isolation (page A3) on BA, put the swab back in the 15 mL tube with the PBS, repeat the procedure and streak for isolation on TSA, CNA, and MAC. Follow the directions for streaking for isolation on the board and ask for help if you need it. Discard all materials used for streaking for isolation in the biohazard. 5. Let the science team know when you are done and one of them will put your plates in the incubator. Nasal swab: 1. Get on your lab coat, goggles, and gloves but you don’t want a partner for this one! 2. Obtain an aliquot of sterile PBS. Swab one of your nostrils as demonstrated by the science team and streak for isolation on MSA, followed by TSA, CAN, and MAC. 3. Let the science team know when you are done and one of them will put your plates in the incubator.

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Streaking for Isolation

1. After dipping your nasal or throat swab into sterile PBS (in the 15 mL conical tube labeled PBS), move it rapidly over the full-width of the plate as indicated below in Circle 1. Place your swab in the 15 mL conical.

2. Turn the plate 90°, use an inoculating loop and start your streak at the end of the primary inculation zone. Make 3- 4 passes over the primary inoculation zone as shown in Circle 2. Dispose of your inoculating loop in the biohazard bag.

3. Get a new inoculating loop, turn the plate again 90° and repeat, this time make sure you have a portion of the streak that does not re-enter the previous streaks (see Circle 3).

4. Invert and incubate your plates overnight (science assistants will take your plates to the incubator).

P a g e | A4 Day 2

Use the charts below to record your observations from the nose and throat experiments.

For the recording growth use the plus system. This can be estimated by determining the zone where you ended up with single colonies (indicated on the Circle diagram page A3).

1 – 2+ = growth in the initial 1/3 to 1/2 of the plate.

3+ = growth in the middle 1/3 of the plate

4+ = growth in all zones

For the recording of the characteristics refer to the sheets in your notebook demonstrating growth characteristics of the different agars. Record color changes and types of hemolysis plus any other observations you note (different types of bacterial colonies shapes and sizes).

NASAL

TSA

Are the colonies all

the same size?

MSA

Did the colonies ferment

mannitol?

BA

What kind of hemolytic

reaction did you observe?

MAC

Did the colonies ferment lactose?

EMB

Did the colonies create a green sheen?

CNA

Are the colonies all the same size?

Characteristics of growth on

plate

Amount of Growth (plus

system)

Throat

TSA


the same size?

MSA


mannitol?

BA



MAC


EMB


CNA



plate


system)

P a g e | A5 Activity 2: Methods to determine if a bacterial isolate has antibiotic resistance. Antibiotic resistance is a growing problem. When a bacterium has antibiotic resistance it limits the treatments we can use to successfully eradicate an infection. Therefore, with some microorganisms that have a high likelihood of harboring genes that confer antibiotic resistance (like Staphylococcus aureus) we do tests to look for specific genes. For today’s activity you will isolate DNA and look for a specific gene, mecA, that confer antibiotic resistance in Staphylococcus aureus. Goal: To understand basic techniques for determining if a pathogen has antibiotic resistance. Day 1 1. Get on your lab coat, goggles, and find a partner. 2. Obtain an aliquot of lysed bacteria. Also obtain all reagents for DNA isolation (science team will assist). Record the number that identifies your bacterial aliquots in your lab notebook. Both aliquots are from Staphylococcus aureus but one of the aliquots has antibiotic resistance while the other does not. Since the bacteria are lysed they are already dead and are therefore not infectious. Do all procedures for both samples. 3. Add 25μl Proteinase K, vortex. 4. Add 200μl Buffer AL from DNeasy kit and give samples to a science team member to incubate it for 30min @ 56°C. 5. Add 300μl 100% ethanol (EtOH), vortex. 6. Transfer sample to DNeasy mini spin column, spin @ max RPM for 1min, and discard flow through (in the bottom tube there will be liquid, pour it in the appropriate container labeled “DNA Discard”). 7. Place column in clean collection tube, add 500ul Buffer AW1, spin @ max RPM for 1min, discard flow through (in the bottom tube there will be liquid, pour it in the appropriate container labeled “DNA Discard”). 8. Place column in clean collection tube, add 500μl buffer AW2, spin @ max RPM for 3min. 9. Obtain a snap capped tube (1.5 mL microcentrifuge tube), and label the tube with the number of the sample and your initials. Transfer the column to your snap capped tube and add 40μl buffer AE (elution buffer). Incubate 1min @ RT and spin for 1min @ max RPM. THIS IS THE DNA. Discard the column.

P a g e | A6 Day 2 Determining the presence of mecA by polymerase chain reaction (PCR) 1. Get two PCR tubes each containing a bead. Label one tube with a “G” and one tube with a “M”. 2. Into the tube labeled “G”, pipette 10 µl of gyrB forward primer and 10 µl of gyrB reverse primer. 3. Into the tube labeled “M”, pipette 10 µl of mecA forward primer and 10 µl of mecA reverse primer. 4. Into each PCR tube, pipette 5 µl of your isolated DNA. 5. Place the lid on each tube and hand it to the science mentor. 6. The science mentor will run the PCR on your samples and run the samples through an agarose gel to separate the pieces of DNA in your sample (gel electrophoresis). You will receive a photo of your gel so you can determine if you did your procedure correctly. This will be indicated by amplification of gyrB a gene present in all Staphylococcus aureus strains. You will also determine if your sample had the mecA gene which confers presence of antibiotic resistance in Staphylococcus aureus. Tape your photo here and indicate your results.

P a g e | A7 Activity 3: Solve a microbiology case study Goal: To use your knowledge gained in the previous activities to identify the microorganism from a mixed culture and present your diagnosis to the other students. Day 1 1. Obtain a mystery sample and a case study from the science team. The mystery may contain Gram positive or Gram negative microorganisms (or both). 2. Using a swab streak your unknown on each agar and using an inoculating loop streak for isolation on BA, MSA, TSA, MAC, EMB, and CNA. Also streak on the agar that contains an antibiotic (science team will explain). 3. Give your plates to the science team to incubate overnight. Day 2 1. Prepare a Gram stain of your mystery sample. A. Draw 3 circles on a slide using a wax pen. B. In circle 1 take a single colony from the Staphylococcus plate this will be your control demonstrating

a Gram-positive result (ask a science team member for help).

C. In circle 3 take a single colony from the Escherichia coli plate this will be your control demonstrating a Gram-negative result (ask a science team member for help).

D. In circle 2 take a single colony from your unknown sample. E. Follow the steps on the Gram Stain Procedure Handout (following page). 2. Perform catalase test (page A8). 3. Write down your results and discuss your findings with your team. 4. Report the most likely culprit based on laboratory results and based on the clues given in the case study (i.e. how did the person get infected, what area of the body is experiencing the illness).

?

P a g e | A8

Catalase Test Some bacteria such as Staphylococcus aureus and Staphylococcus epidermidis, contain the enzyme catalase which reacts with hydrogen peroxide and forms water and bubbles of oxygen. Other types of bacteria, such as Streptococci species do not contain catalase. Thus, a drop of hydrogen peroxide on a sample of bacteria can help to differentiate what type of bacteria is present.

1. Obtain a single culture and pick up the culture with an inoculating loop. Spread the colony on a glass slide.

2. Ask for a science mentor to come and assist you with the catalase test. You will pipette a small amount of hydrogen peroxide onto your sample. Record the results.

P a g e | A9 Use the chart below to record your observations from your case study.

For the recording growth use the plus system. This can be estimated by determining the zone where you ended up with single colonies (indicated on the Circle diagram page A3).

For the recording of the characteristics refer to the info sheets in your notebook demonstrating growth characteristics of the different agars. Record color changes and types of hemolysis plus any other observations you note (different types of bacterial colonies shapes and sizes).

Unknown #

TSA


the same size?

MSA


mannitol?

BA



MAC


EMB


CNA



plate


system)

Additional observations:

Gram stain results:

Catalase Results:

Clues from the case study:

Diagnosis:

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P a g e | B2

Mannitol Salt Agar (MSA)

A selective and differential agar which contains 7.5% salt to select for certain Gram‐positive

bacteria such as Staphylococci. The high salt concentration selects for halophilic organisms

(organisms that like salt). It also contains mannitol sugar and phenol red (a pH indicator) which

will select for organisms that ferment mannitol. If an organism can use mannitol as its energy

source, the agar will turn yellow due to a drop in the pH which causes a color change. For

example, both Staphylococcus aureus (section A) and Staphylococcus epidermidis (section B) will

grow on MSA but only Staphylococcus aureus can ferment mannitol. Section C demonstrates

no growth.

Blood Agar (BA)

A nutrient rich medium that contains 5% sheep blood. This agar will grow most organisms and

will help you identify what bacteria is growing based on the ability of the bacteria to lyse blood

cells. Different bacteria have different types of hemolysins: α, β, and δ hemolysin. The blood

agar will look different depending on what type of hemolysins the bacteria have.

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MacConkey Agar (MAC)

A selective and differential medium used to select Gram‐negative bacteria. It can differentiate

bacteria based on their ability to ferment lactose. If bacteria can use lactose, they will turn a

pink color. If bacteria cannot use lactose, they will remain colorless or yellow. E. coli will

ferment lactose but Salmonella sp. cannot.

Eosin Methylene Blue Agar (EMB)

A selective and differential medium that allows growth of Gram‐negative bacteria. The agar

contains lactose and two dyes: eosin and methylene blue. When a bacteria is able to ferment

lactose, it produces acid and turns the bacterial colony a dark purple to black. Colonies that do

not use lactose will be colorless.

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Colistin‐Nalidixic Acid Agar (CNA)

A selective medium containing the antibiotics colistin and nalidixic acid which selects for Gram‐

positive bacteria. S. aureus, S. epidermidis, and Group A Streptococcus will grow on this

medium.

Tryptic Soy Agar (TSA)

A supportive medium which contains glucose and amino acids for the growth of most

microorganisms, it will not select for Gram‐positive or Gram‐negative (both groups will grow on

this agar).

Bacterialogical Media , http://faculty.mc3.edu/jearl/ML/ml‐8.htm

Gram‐negative rodFerments lactoseCatalase positive

Clinical PresentationoWatery or bloody diarrheaoUrinary Tract Infection (UTI)oSeptic Shock

E. coli on MacConkeyagar

Clinical Case ExamplePatients in a small town visit the hospital

complaining about bloody diarrhea, fatigue, and confusion. After interviewing the patients, the doctors discover that each patient frequents the

same fast‐food burger joint. The doctors identify the causative agent using serological

testing and stool cultures.

PathobiologyIn normal GI floraAnimal sources of infectionTransmits via fecal‐oral

C2

Gram negative rod Motile by flagellaProduces hydrogen‐sulfideDoes not ferment lactoseCatalase positive

Clinical PresentationoGastroenteritis

PathobiologyCarried in animals and humansTransmits fecal‐oral

Clinical Case ExampleA veterinary student complains to the doctor of diarrhea and abdominal

tenderness. He also had nausea and vomited the day before. He admits he

recently played excessively with his turtle.

S. enterica on MacConkeyAgar

C3

a.k.a. Group A Streptococcus

Gram positive cocci in chainsTypically infects throat or skinΒ‐hemolytic on blood agarCatalase negative

Clinical PresentationoPharyngitis (Strept throat)oImpetigooCellulitis

PathobiologyTransmits human to human via respiratory droplets, salivaTrauma introduces bacteria into skin

Clinical Case ExampleA young child presents with a fever

and skin rash localized around the lips and on his arms. The rash appears to have pustules with yellow crusts. Cultures from the skin show Gram pos. cocci and are β‐hemolytic. The doctor administers penicillin G.

S. pyogenes on blood agar

C4

Gram‐positive grows in clustersCatalase positiveCoagulase negative

Clinical PresentationoInfection on indwelling medical devices such as catheters or prosthetic joints

PathobiologyNormal flora on skinForms biofilms and adheres to medical device

Clinical Case ExampleTen days after undergoing chemotherapy for cancer a, a middle‐aged man develops a fever. On exam, he has erythema and tenderness at the insertion site of the IV catheter. Blood cultures are positive for Gram‐positive bacteria. The original catheter is removed and the patient is

started on antibiotics.

S. epidermidis on MannitolSalt Agar (MSA)

C5

Gram positive clustersCatalase positiveCoagulase positiveAntibiotic resistance is a problem (MRSA = methicillin‐resistant S. aureus)

Clinical Presentationo Skin/subcutaneous: impetigo,

cellulitis, boilso Sepsis, Endocarditis

PathobiologyColonize skin or pharynx and immune system responds and causes inflammation and abscess developmentEntry into blood via ruptures in skin

Clinical Case ExampleA college basketball player presents to

the clinic with several red painful purulent boils on his upper arms. Gram stain of the purulent material reveals Gram‐positive clusters. Culture is

resistant to penicillin and methicillin.

S. aureus on Mannitol Salt Agar (MSA)

C6

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Tips for Your Project

Read this over before the ID virtual lab meeting. During the virtual meetings we will answer questions about testing a hypothesis and on your experimental design.

1. You can work in groups or work by yourself.

2. Develop a hypothesis to be tested. Think of a topic you are interested in learning more about. For example, have you ever wondered if hand sanitizers actually work? Or how fast they actually work? Do they work better than plain old soap and water? Figure out what is known about the topic – perform an internet search, read an article about it, interview an expert. Develop an educated guess i.e. a hypothesis, based on your knowledge.

I hypothesize that 60 seconds of rubbing my hands with waterless hand sanitizer will reduce bacterial numbers and diversity on my hands compared to washing my hands with soap and water for 60 seconds.

3. Write down your experimental procedure. How will you test your hypothesis? What will the variables be? How many plates do you need? Since we are shipping supplies in October you can tell us if you need more plates and or swabs to test your particular hypothesis. Of course, there is a limit on how large you can get these experiments but we can accommodate a few extra materials.

Experimental Procedure:

A. Identify your control. How do you obtain a baseline of bacteria from your hands? Should you wash your hands first before beginning the experiment? Should the test be done on the same day or on a different day? What is the more controlled experiment? The control will be compared to the experimental groups to assess how the treatment altered the outcome.

B. Design a procedure where the hands will be as equally dirty. Make sure you are consistent with how you dirty your hands! Some possible methods include washing your hands with soap and water (to keep the baseline bacteria on your hands similar between tests) and then:

1. touching raw chicken for 60 seconds.

2. brushing your horse without gloves for 5 minutes.

3. typing on a public computer for 5 minutes (at a library or computer lab at school).

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C. Decide how to sample the bacteria on your hands. Will you swab each finger? Will you swab 3 fingers on each hand – on one hand only? Will you combine the swab material? If so how – on the petri dish? Make sure you are consistent with how you collect samples!!

D. Standardize how you “wash” your hands. Your hypothesis says 60 seconds but doesn’t specify how vigorous you’ll be washing. Probably want to make sure the motion is very similar between washing with the waterless hand sanitizer and with soap and water. Now again you need to sample the bacteria on your hands – do this exactly as you did after touching the contaminated material.

4. Record exactly how you are doing your experiment so you can determine if you have introduced more variables that are complicating your results.

5. Record results, take pictures, and fill out table for growth on media: how much growth (use the plus system from the module, 1-2 +, 3+, 4+), and which plates supported growth (diversity of the microorganisms)?

6. If possible, repeat!

7. Was your hypothesis correct? What are the implications of your findings? How does this support or add to the information that is already available on soap and water versus waterless hand sanitizers? If you were to perform your experiment again what would you change?

8. Put together a presentation. You can either have a poster or oral presentation for our meeting at Montana State University in January 2013.

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Use this Outline to Design your Experiment

Hypothesis:

Background Facts:

Methods:

Variables:

Results:

P a g e |D 5

Which media supported growth? How much growth did you find? Review your materials from the hands-on module at Montana State University.

TSA MSA BA MAC EMB CNA

Additional observations:

Implications of your findings:

What would you do differently if you had the chance to re-do the experiment?

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Abscess: a collection of pus on the body that causes pain, swelling, and inflammation around it; typically due to an infection

Aliquot: a portion of a solution

Antibiotic Resistance: a microorganism that is able to survive exposure to an antibiotic

Antibiotics: a substance or compound that slows down or prevents the growth of bacteria

Bacteria (Eubacteria): a domain of microorganisms, only micrometers in length, with a primitive nucleus

Buffer AE: a solution that binds to DNA in order to remove purified DNA from a column

Buffer AL: a solution that disrupts protein structures

Buffer AW1: a solution that denatures (destroys) proteins in order to purify DNA

Buffer AW2: a solution that contains ethanol, which removes cellular salts from a sample in order to purify DNA

Carrier: an individual who is colonized (infected) with a pathogen, but free of disease, who is capable of acting as a source of infection for others

Catalase: an enzyme that degrades hydrogen peroxide into hydrogen and water

Catheter: a tube that can be inserted into a body cavity, duct, or vessel that allows for drainage, administration of fluids or gases, or access by surgical instruments; an example is a urinary catheter inserted in the bladder to drain urine

Cellulitis: a common skin infection caused by bacteria, symptoms commonly include: redness, inflammation, and soreness of the skin; also can cause fever-like symptoms

Centrifuge: a piece of equipment that puts an object in circular motion, causing denser substances to settle to the bottom of the tube- “the pellet”- while the less dense constituents remain on top- “the supernatants”

Coagulase: a protein produced by some microorganisms that converts fibrinogen to fibrin, resulting in clumping of blood; used to distinguish between different types of Staphylococcus species

Colonization: establishment of a microbial population in the animal/human host

Colony: a visible group or cluster of bacteria derived from one bacterium, you count colonies on an agar plate to quantify bacterial growth

Crystal Violet: a dye used in Gram staining; it remains inside bacterial walls containing higher amounts of peptidoglycan

Deoxyribonucleic acid (DNA): found in all cells and contains the genetic instructions used for development and function; consist of two sugar backbones (containing deoxyribose sugar) with linked nucleotides in between; the nucleotides can be one of four bases and the sequence of these bases determines genetic make-up of the organism

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Disease: a condition that is accompanied by an impaired body function

Echocardiogram: a type of ultrasound that images the heart; can provide an assessment of the state of heart tissue

Endocarditis: an inflammation of the inside lining of the heart chambers and heart valves

Endotoxin: a type of toxin found on Gram-negative bacteria that is made up of the lipopsaccharide chain found on the outer membrane of the bacterium

Erythema: redness of the skin occurring with skin infection, injury, or inflammation

Ethanol: an alcohol used for many biological purposes; can be used to disrupt protein structure and remove salts to help purify DNA; can be also used as a disinfectant due to its ability to penetrate bacterial cell walls and is used to decolorize bacterial cells in the Gram staining procedure

Exotoxin: a potent toxin secreted by some forms of bacteria

Flora: a population of microbes that inhabit the internal and external body surfaces of healthy humans and animals (see: microbiota)

Furuncles: a boil; results in a painful swollen area on the skin filled with pus and dead tissue

Gastroenteritis: a medical condition characterized by inflammation of the GI tract, including the stomach and small intestine; results in a combination of diarrhea, vomiting, abdominal pain, and cramping

Gel Electrophoresis: the separation of DNA, Ribonucleic Acid (RNA), or protein, based on size and charge; electric currents are applied to move molecules through the matrix

Gram Stain: a method of differentiating bacterial species into Gram-positive and Gram-negative bacteria based on the amount of peptidoglycan in their cell walls

Gram-negative: bacteria that do not retain the crystal violet stain due to a low amount of peptidoglycan in the cell wall; these bacteria typically stain red during Gram staining (they retain the color of safranin, the counter-stain)

Gram-positive: bacteria that retain the crystal violet stain due to a high amount of peptidoglycan in the cell wall; these bacteria typically stain purple during Gram staining

gyrB: a gene present in all Staphylococcus aureus; used as a control during PCR to confirm DNA was isolated from Staphylococcus aureus

Hemolysis: from the Greek word meaning “release of blood”, is the rupturing of red blood cells followed by the release of hemoglobin into surrounding medium, there are three types of hemolysis: alpha (α) reduces the iron in the blood (bacteria colonies form “a green halo” on blood agar), beta (β) completely ruptures the blood cells (bacteria colonies form a “clear halo” on blood agar), and gamma (γ) is a lack of hemolysis (colonies often appear grey with no halo on blood agar)

Hypothesis: a proposed explanation for a phenomenon/occurrence, often based on previous observations that can be tested with a set of experiments

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Impetigo: a common skin infection characterized by a single, or many, blisters filled with pus and, when broken, leaves a reddish, raw-looking base

Infection: results from a microbe that penetrates body surfaces, gaining access to tissues and multiplying which then induces a host response

Iodine: a trapping agent that binds to crystal violet making it a larger molecule to trap the dye in the peptidoglycan

Lactose: a sugar found in milk formed by galactose and glucose

Lipopsaccharide chain: found on the outside of the outer envelope of a Gram-negative bacteria, often called endotoxin

Lyse: to burst or cause dissolution or destruction of cells

Lysostaphin: an antibacterial enzyme that can cleave components of Staphylococcus aureus cell wall; this is used in DNA extraction to release the genetic makeup (DNA) out of bacteria

Mannitol: a sugar alcohol, typically of a lower (acidic) pH; component of Mannitol Salt Agar (MSA); colonies of bacteria that can ferment mannitol appear yellow on the plate while those that cannot appear pink

mecA: a gene found in bacteria that confers resistance to a large class of antibiotics including penicillin and methicillin; Staphylococcus aureus strains that have this gene are called methicillin-resistant Staphylococcus aureus (or MRSA)

Media: a liquid or gel designed to support the growth of microorganisms or cells

Meningitis: a bacterial infection of the membranes covering the brain and spinal cord

Microbiota: a population of microbes that inhabit the internal and external body surfaces of healthy humans and animals (see: flora)

Microorganism: a microscopic organism that can consist of a single cell, cell cluster, or a multicellular complex organism; includes bacteria, viruses, algae, fungi, etc.

Morphology: the form and structure of organisms and their specific features

MRSA: methicillin-resistant Staphylococcus aureus a form of Staphylococcus that has developed resistance to a wide spectrum of antibiotics (see mecA)

Opportunistic Pathogen: microorganism that is free living or a part of the host’s normal microbiota but may become pathogenic under certain circumstances, such as when the immune system is compromised

Pathogen: a microorganism capable of producing disease in a healthy animal or human host

Pathogenicity: ability to cause disease

Peptidoglycan: a major component of the bacterial cell wall that contributes to the shape of the bacteria; amount of peptidoglycan is used to differentiate bacteria in the Gram stain (see Gram-positive and Gram-negative)

Pharyngitis: inflammation of the throat; also known as a sore throat

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Pharynx: the throat

Phosphate Buffered Saline (PBS): a buffer solution used in biological research; a water-based salt solution that is non-toxic to cells

PI Buffer: a buffer used in the isolation of DNA that prevents cellular clumping and degrades the RNA that is present

Polymerase Chain Reaction (PCR): a technique used to amplify DNA in order to generate thousands to millions of copies of a specific sequence; may be used for DNA cloning, identifying evolutionary ancestors, diagnosing genetic diseases, and identifying genetic fingerprints used in forensic science labs

Primer: a start point for DNA synthesis and replication; primers are typically short strands of nucleic acid (the building blocks of DNA) that are used to bind a specific sequence of DNA in order to amplify it during a PCR reaction

Proteinase K: destroys proteins that degrade DNA and RNA

Reagents: a substance or compound added to a system in order to cause a reaction

Ribonucleic acid (RNA): like DNA, found in all cells; consists of one sugar backbone (containing ribose sugar) with nucleotides bound to the backbone; RNA is created based on a DNA template and is then used to make proteins necessary for cellular function

Safranin: a biological stain used as a counterstain in Gram staining

Sepsis: a medical condition characterized by a whole body inflammatory response to an infection; also known as blood poisoning

Serological Testing: a test used to determine the presence of a microorganism in the blood

Supernatant: the liquid above a solid residue (pellet) after centrifugation

Virulence: attributes of a microbe that enhance its pathogenicity

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