BIOBUGS

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Center for Disease Control _at_ Boston
University
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Welcome

• B.U.C.D.C is the Boston University Center for
  Disease Control
• Founded 1968
• Mission The storage and study of pathogenic
  agents for the purposes of understanding disease
  transmission in order to control and eradicate
  potential outbreaks

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B.U.C.D.C. Staff

• Director of Pathogen Storage Jan Blom
• Director of Laboratory Safety Matthew Walker
• Director of Technology Support Xiaojuan Khoo
• Technical Assistants
• Derek Stefanik
• Ysabel Milton
• Kevin Yu
• Angela Seliga

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Lab Safety
http//www.mbio.ncsu.edu/MB451/lab/labSafety.jpeg
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Lab Safety Whats So Dangerous?
Pathogenic To cause disease

• Pathogenic Agents
• Bacteria Necrotizing Fasciitis, TB, Diptheria,
  Anthrax
• Viruses Swine Flu, Measles, Mumps, AIDS
• Parasites Tape worm, Guinea worm, Flea, Hook
  worm
• Prions Kuru, Creutzfeldt-Jakob Disease (CJD)

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Lab Safety Eliminate the Transmission of Disease

• Communicable Disease Can pass between
  individuals
• Cutaneous
• Direct skin to skin contact is required for
  transmission
• Anthrax, Athletes Foot
• Ingestion
• Transmission occurs when then the pathogenic
  agent is eaten
• Kuru, Tape worm
• Bodily fluid
• Direct contact with the following fluids is
  required saliva, blood, lymph, urine
• AIDS, Hepatitis
• Inhalation
• Agent is airborne enters via the lungs
• SARS, Influenza, The common cold

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Lab Safety Guidelines (Pg. 3-4)

• Primary Goals
• Protect researcher from the sample
• Protect the sample from the researcher
• Physical Barriers
• Lab coats
• Gloves
• Tie hair back

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Lab Safety Guidelines (Pg. 3-4)

• Other
• No eating/drink/headphones/cell phones
• Waste goes into appropriate bins
• Report all spills
• All backpacks/jackets should be placed on the
  counter away from the lab space
• Wash all working spaces with 70 ethanol
• ASK QUESTIONS IF YOU ARE NOT SURE!

Refer to the provided Lab Safety Guide and sign
the Lab Safety Contract (Pg. 4) before proceeding.
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Lab Safety Quiz What is Unsafe About These
Pictures?
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What is Unsafe About This Picture?
http//www.chem.unl.edu/chwang/orgsafety/jun17_12.
jpg
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What is Unsafe About This Picture?
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What is Unsafe About This Picture?
Trash
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BUCDC Ice Breaker!

• Put on lab coat, gloves
• Touch powder
• Blot hand
• Shake hands with assigned individual
• Blot hand on paper
• Repeat

FOLLOW THE DIRECTIONS AS THEY ARE GIVEN TO YOU
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BUCDC Ice Breaker Lab Safety

• Put on lab coat and gloves
• Note your ID number
• Dispose of gloves in generic waste
• DO NOT DISCARD THE PAPER TOWELS WITH HAND PRINTS!

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BUCDC Ice Breaker

• Your Station

Towel 1
Towel 2
Towel 3
Towel 4
White Powder
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Step 1 Place RIGHT HAND in powder
Powder

• Rub hands together, making sure you get lots of
  powder on your RIGHT hand.
• Make sure you stand away from table DO NOT
  CONTAMINATE your work bench and materials.

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Step 2 Make a handprint and write down your ID
Self Test
Use your RIGHT hand - hold for 5 seconds
Write your ID
Round 2
Round 3
Round 1
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Step 3 Series of Handshaking

• For each Round
• Shake hands for 5 seconds using RIGHT hands
• Make a handprint on towel
• Total of 3 Rounds of Handshaking
• We will tell you who to shake hands with

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Round 1 START!

• Stand up
• Find assigned Shakee
• Stand by Shakee
• Shake hands for 5 seconds
• E.g., 1 shake 26
• Return to seat
• Make handprint on next towel
• Write Shakees

DO NOT TOUCH ANY OTHER PERSON OR SURFACE TO AVOID
CONTAMINATION!
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Round 1 Make a handprint and write down Shakees

Round 1
Write Shakees
Self Test
Round 2
Round 3
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Round 2 START!

• Shake hands with your new assigned Shakee
• Make hand print on next towel
• Write down Shakees

DO NOT TOUCH ANY OTHER PERSON OR SURFACE TO AVOID
CONTAMINATION!
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Round 2 Make a handprint and write down Shakees

Round 2
Write Shakees
Self Test
Round 1
Round 3
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Round 3 START!

• Shake hands with your new assigned Shakee
• Make hand print on last towel
• Write down Shakees

DO NOT TOUCH ANY OTHER PERSON OR SURFACE TO AVOID
CONTAMINATION!
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Round 3 Make a handprint and write down Shakees

Round 3
Write Shakees
Round 1
Round 2
Self Test
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After 3 rounds of handshaking

• Throw out gloves In generic trash
• Keep paper towels in front of you

Round 1
Round 2
Round 3
Self Test
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WARNING!! WARNING!!!
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WARNING

• Air Born Pathogen Detected
• Initiate Containment Protocol
• Pressurizing Laboratory
• Laboratory Sealed
• Lock Down Complete

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Possible Pathogenic Agents
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Action Plan

• Who was exposed to the pathogen?
• Who was Patient Zero?
• How is the disease passed from person to person?
• What bacteria have we been exposed to?
• What is the best treatment for that pathogen?

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Epidemiology - The Basics

• Epidemic When the number of newly reported cases
  in a population is larger then expected
• Patient Zero First known case of disease
• Epidemiologists try to figure out
• How (the disease is transmitted)
• When (was it transmitted)
• Where (did it start)
• What (what is the cause)
• Who (has the disease)

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Real World Example Swine Flu
Last updated on May 2, 2009
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Patient Zero to Epidemic

• Known Patient Zeros
• 1854 Baby at Louis House. Cholera in Soho
• 1915 Mary Mallon (A.K.A Typhoid Mary). Typhoid
  outbreak in N.Y.C.
• 1976 Mabalo Lokela Ebola viruses
• 2003 Liu Jianlun. SARS in Hong Kung
• 2009 swine flu

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Patient Zero to Epidemic

• Patient Zero is infected

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Patient Zero to Epidemic

• Patient Zero is infected
• Interacts with others, passes on infection

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Patient Zero to Epidemic

• Patient Zero is infected
• Interacts with others
• Patient Zero dies, neighbors infect

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Patient Zero to Epidemic

• Patient zero is infected
• Interacts with others
• Patient Zero dies, neighbors infect
• Neighbors interact with others, infecting them

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Patient Zero to Epidemic

• Patient zero is infected
• Interacts with others
• Patient zero dies, neighbors infected
• Neighbors interact with others, infecting them
• Disease reaches epidemic levels

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Patient Zero to Epidemic

• Patient Zero is infected
• Interacts with others
• Patient Zero dies, neighbors infected
• Neighbors interact with others, infecting them
• Disease reaches epidemic levels
• Response Isolate those infected
• Treat or let the disease run its course
• Quarantine

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Whos infected?

• How do doctors test for bacteria?
• Hint How do you test for Strep Throat?
• Take sample, and let it grow
• Problem Lack of time

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Whos infected?

• How do doctors test for bacteria?
• Hint How do you test for Strep Throat?
• Take sample, and let it grow
• Problem Lack of time
• Fortunately, the bacteria lights up under black
  light.
• Use light as a detection method
• Have you been infected?

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Action Plan

• Who was exposed to the pathogen?
• 8 people
• We have an outbreak situation
• Who was Patient Zero?
• How is the disease passed from person to person?
• Which bacteria have we have been exposed to?
• What is the best treatment for that pathogen?

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Who is Patient Zero?How Fast is it Spreading?

• In the worksheet, identify the individuals that
  are infected for each round of handshaking. Pg.
  7)

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(No Transcript)
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Tracking the infection
Total Infected

• How quickly does the infection spread?

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Tracking the infection
Total Infected

• How quickly does the infection spread?

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Emergency Alert!

• We have 2 HOURS to identify the pathogen and find
  a cure

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Ice Breaker Discussion (Pg. 8)

• Who is Patient Zero?
• What is the most likely route of transmission?
  Explain.
• How would the transmission of the disease change
  if it were airborne? Waterborne?

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Action Plan

• Who was exposed to the pathogen?
• 8 people
• The rest of the lab will be infected in 2 hours
• Who was Patient Zero?
• The first known infected patient is 5
• How is the disease passed from person to person?
• Most likely skin to skin contact
• Which bacteria have we have been exposed to?
• What is the best treatment for that pathogen?

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How To Identify Bacteria

• DNA sequencing
• Order of nucleotide (ACGT) varies between
  bacteria
• Culture
• Specific bacteria can only grow in certain
  environments
• Morphology
• Classify bacteria via shape or other physical
  properties
• Physiology (Enzymatic Processes)
• Certain bacteria can carry out different
  reactions
• Other?

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How To Identify Bacteria

• DNA sequencing
• Order of nucleotide (ACGT) varies between
  bacteria
• Culture
• Specific bacteria can only grow in certain
  environments
• Morphology
• Classify bacteria via shape or other physical
  properties
• Physiology (Enzymatic Processes)
• Certain bacteria can carry out different
  reactions
• Other?

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Bacterial Morphology

• Staphylococcus aureus, (MRSA)
• Neisseria gonorrhoea (Gonorrhea)
• Chlamydia trachomatis (Chlamydia)
• Bacillus anthracis (Anthrax)
• Bacillus cereus (Food poisoning)
• Bacillus subtilis
• Escherichia coli,
• Mycobacterium tuberculosis (Tuberculosis )
• Spirillum minus (Rat-bite fever)
• Treponema pallidium (Syphilis)
• Corynebacterium diphtheriae (Diphtheria)

Coccus (Round)
Bacillus (Rod)
Spirilla (Spiral)
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Two Cocci, Whats the difference?

• Bacteria 1
• Morphology Coccus
• Pathology mild cold

• Bacteria 2
• Morphology Coccus
• Pathology death

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Two Cocci, Whats the difference?
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Bacteria Cell Wall Structure
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Bacteria Cell Wall - Stained
Strong purple stain
Gram Stain
Strong pink stain
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Gram positive or negative?Bacteria Morphology?
Gram Positive ()
Gram Negative ()
Coccus
Rod
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Gram Stain The Basics
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Gram Positive or Negative?
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Gram Staining Lab Safety

• Put on lab coat, goggles and gloves
• Place triangle frame over sink/beaker
• All liquids can be emptied down the drain
• Place slides in glass disposal container
• Place gloves in generic waste

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Gram Stain - Protocol (Pg. 12-13)
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Light Microscope (Pg. 10)
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Power of Magnification
To calculate the power of magnification, multiply
the power of the ocular lens by the power of the
objective.
What are the powers of magnification for each of
the objectives we have on our microscopes? Fill
in the table in your worksheet. (Pg. 11)
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Power of Magnification
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Gram Stain - Protocol (Pg. 12-13)
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Looking at the Gram Stains (Pg. 13-14)

• Use the microscope to view Gram stained slides
  under various objectives
• 4x tiny specks/spots
• 10x faint smear of colored spots
• 40x start to see shape better
• STOP. Raise your hands at this point.
• A volunteer will help you set up oil immersion
  using the 100x objective.
• Draw and label all FIVE bacteria samples using
  the 10x and 100x objectives.
• The colors and shapes should be as accurate as
  possible
• Determine if the bacteria are Gram positive or
  Gram negative.

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Gram Stain Discussion (Pg. 16)

• What characteristics can be determined using a
  Gram stain?
• What can happen to make Gram positive cells
  appear Gram negative?

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Two Cocci, Whats the difference?

• Bacteria 1
• Morphology Coccus
• Gram
• Pathology mild cold
• Differences in Physiology Protein A

• Bacteria 2
• Morphology Coccus
• Gram
• Pathology death
• Differences in Physiology Protein B

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Catalase Test The Basics

• Identify differences in the physiology of
  bacteria
• Catalase Enzyme
• Function To remove toxic byproducts
• Chemical reaction
• No bubbles negative for catalase
• Bubbles positive for catalase

2
2
Oxygen (gas)
Water (liquid)
Hydrogen Peroxide (liquid)
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Catalase Test
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Catalase Test Lab Safety

• Put on lab coat, goggles and gloves
• Hydrogen peroxide is corrosive
• Place wooden stick and gloves in generic waste
• Place slides in glass disposal container
• Return Petri dishes to instructors

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Catalase Test - Protocol (Pg. 18)

• Using a wooden stick, smear a small amount of
  bacteria from the Petri dish onto a clean
  microscope slide
• Label the slide using the Sharpie marker

Transfer bacteria
Bacteria dish
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Catalase Test - Protocol (Pg. 18)

• Using a wooden stick, smear a small amount of
  bacteria from the Petri dish onto a clean
  microscope slide
• Label the slide using the Sharpie marker
• Add 3 drops of H2O2 solution onto the smear.
• Record your observations in the worksheet (Pg.
  19).

Transfer bacteria
3 drops
Bacteria dish
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Catalase Test - Protocol (Pg. 18)

• Using a wooden stick, smear a small amount of
  bacteria from the Petri dish onto a clean
  microscope slide
• Label the slide using the Sharpie marker
• Add 3 drops of H2O2 solution onto the smear.
• Record your observations in the worksheet (Pg.
  19).
• Repeat steps 1-4 for all FIVE bacteria samples,
  using new materials each time.
• When done, dispose of materials in the correct
  bins

Transfer bacteria
3 drops
Bacteria dish
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Catalase Test Discussion (Pg. 19)

• Which bacteria tested positive for catalase?
  Which tested negative?
• What is the purpose of using new glass slides and
  sticks for each bacteria species?

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Cracking the Case

• Fill out the summary table in your worksheet (Pg.
  21)

Scientific names of given bacteria samples A
Mycobacterium tubercolosis C Escherichia
coli B Streptococcus pneumoniae D
Staphylococcus aureus
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Identifying the Unknown Pathogen Discussion
(Pg. 21)

• Write a 1 paragraph summary describing the
  characteristics of the unknown pathogen.
• By comparing these characteristics to known
  bacteria samples, A-D, we have identified the
  unknown pathogen that has infected our facility
  as _________________________.

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Action Plan

• Who was exposed to the pathogen?
• 8 people
• The rest of the lab will be infected in 2 hours
• Who was Patient Zero?
• The first known infected patient is 5
• How is the disease passed from person to person?
• Most likely skin to skin contact
• Which bacteria have we have been exposed to?
• Gram positive cocci
• Catalase positive
• Bacteria is Staphylococcus aureus
• What is the best treatment for that pathogen?

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Potential Treatments

• Suggestions?

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How do Antibiotics Kill?

• Antibiotic against life
• Derived from natural products
• Attack specific parts of the cell

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Example Neosporin

• Contains
• Bacitracin
• Attacks the cell wall (gram)
• Neomycin
• Binds to ribosome, prevents translation
• Polymyxin B
• Binds to and disrupts cell membrane (gram-)

Why so many different drugs in one?
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Antibiotic Testing
Why is there a clear zone immediately around the
antibiotic disk?
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Zone of Inhibition

• Compare diameters of the zones of inhibition to
  classify bacteria as
• R resistant
• Small/no zone of inhibition
• I intermediate
• Small to medium zone of inhibition
• S susceptible
• Large zone of inhibition

What conclusions can you draw from the above
picture?
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Antibiotic Testing Lab Safety

• Put on lab coat and gloves
• Place gloves in generic waste
• Return plates to instructor

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Antibiotic Testing Protocol (Pg. 24)

• Use a ruler to measure the zone of inhibition for
  the three antibiotics that we are testing.
• Take two measurements per antibiotic disk
• Fill in the table in the worksheet (Pg. 25)

Drug X
Drug Y
Drug Z
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Antibiotic Testing Discussion (Pg. 25)

• Determine if the bacteria is resistant,
  intermediate or susceptible to each of the three
  antibiotics.
• Which antibiotic has the most potential for
  treating the infectious disease?

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Course of Action - Completed

• Who was exposed to the pathogen?
• 8 people
• The rest of the lab will be infected in 2 hours
• Who was patient zero?
• The first known infected patient is 5
• How is the disease passed from person to person?
• Most likely skin to skin contact
• Which bacteria have we have been exposed to?
• Gram positive cocci
• Catalase positive?
• Bacteria is Staphylococcus aureus
• What is the best treatment for that pathogen?
• Recommended antibiotic is Drug Y (Ampicillin)

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ALERT

• Air Sampling No Pathogen Detected
• Treatment Protocols Successful
• - Antibiotics administered
• Quarantine No Longer Necessary
• Lab Lockdown Terminated
• Outbreak Contained and Eliminated

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Outbreak...Contained

• Summarize what we did today
• Conclusions
• Why is this important?

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We hope you enjoyed BIOBUGS!
Fill out evaluation survey. Lunch is served at 12
pm in Room 352. Panel discussion will start at
1225 pm.
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For reference Handshake partners will change
depending on student numbers
Round 1
Round 2
Round 3
Start with odd number if you have odd number of
students and even number if you have even number
of students. That way, student wont shake with
him/herself.
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• What are the two primary goals of lab safety?
• A student is working with a rob shaped, gram
  bacteria. (S)he puts a small amount of liquid
  containing that bacteria on a plate and places it
  in an incubator to grow. The following day, the
  plate yields both gram and cells. What
  happened to the sample?
• Even if a sample is not harmful to the scientists
  why should they always wears gloves?
• In the outbreak scenario, when the pathogen was
  detected, the lab was pressurized to BELOW
  atmospheric pressure. Why is this so? (HINT
  which direction will the air flow?)
• Assuming the bench top is clean, how could you
  test to see if any bacteria/contamination is
  present after it was cleaned?
• How does the advent of modern travel
  (cars/airplanes/trains/boats) influence the
  outbreak of a disease? (HINT review the case of
  the TB patient traveling to Europe)
• Name three methods that a disease can be
  transmitted. How are they similar? How are they
  different?
• During the Spanish Flu pandemic of 1918, those
  with the disease were isolated from the rest of
  the population. What is this called?
• How would geographical boundaries such as rivers
  and mountains protect against the spread of a
  disease?
• How would the time that a infection takes to kill
  a carrier influence the spread of that pathogenic
  agent. (HINT compare and contrast what would
  happen if the patient dies within 2 hours of
  infection, versus one that may not die for 2
  years) What are the two primary components of the
  peptidoglycan layer?
• Draw and label a gram-positive and gram-positive
  cell to scale.
• Which cell (gram-positive or gram-negative) shows
  the color of the COUNTER stain? What is the
  purpose of this counter stain?
• Why is staining required to tell if a cell is
  gram or gram-
• Label and draw the 3 types of bacterial
  morphology.
• How do is magnification of a microscope
  calculated
• When using a high powered objective, only the
  fine focus knob should be used, why?
• What is the clinical value of the gram staining
  protocol
• Draw the reaction (starting with hydrogen
  peroxide) that catalase is involved it. Label the
  states of each reactant and product.
• What is the purpose of the catalase enzyme in
  cells? (HINT Where does the hydrogen peroxide
  come from?)