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BUCDC. Center for Disease Control @ Boston University. Welcome. B.U.C.D.C is the Boston University Center for Disease Control Founded: 1968

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slide1

BUCDC

Center for Disease Control @ Boston University

welcome
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
b u c d c staff
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
lab safety
Lab Safety

http://www.mbio.ncsu.edu/MB451/lab/labSafety.jpeg

lab safety what s so dangerous
Lab Safety: What’s So Dangerous?
  • 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)

Pathogenic: “To cause disease”

lab safety eliminate the transmission of disease
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
lab safety guidelines pg 3 4
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
lab safety guidelines pg 3 41
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.

what is unsafe about this picture
What is Unsafe About This Picture?

http://www.chem.unl.edu/chwang/orgsafety/jun17_12.jpg

bucdc ice breaker
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

bucdc ice breaker lab safety
BUCDC Ice Breaker – Lab Safety

1

  • 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!
bucdc ice breaker1
BUCDC Ice Breaker

Towel 1

Towel 3

Towel 4

Towel 2

White Powder

Your Station

step 1 place right hand in powder
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.
step 2 make a handprint and write down your id
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

step 3 series of handshaking
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
round 1 start
Round 1 – START!

DO NOT TOUCH ANY OTHER PERSON OR SURFACE TO AVOID CONTAMINATION!

  • 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 Shakee’s #
round 1 make a handprint and write down shakee s
Round 1: Make a handprint and write down Shakee’s #

Round 1

Write Shakee’s #

Self Test

Round 2

Round 3

Your ID#

round 2 start
Round 2 – START!

DO NOT TOUCH ANY OTHER PERSON OR SURFACE TO AVOID CONTAMINATION!

Shake hands with your new assigned “Shakee”

Make hand print on next towel

Write down Shakee’s #

round 2 make a handprint and write down shakee s
Round 2: Make a handprint and write down Shakee’s #

Round 2

Write Shakee’s #

Self Test

Round 1

Round 3

Your #

Shakee’s #

round 3 start
Round 3 – START!

DO NOT TOUCH ANY OTHER PERSON OR SURFACE TO AVOID CONTAMINATION!

Shake hands with your new assigned “Shakee”

Make hand print on last towel

Write down Shakee’s #

round 3 make a handprint and write down shakee s
Round 3: Make a handprint and write down Shakee’s #

Round 3

Write Shakee’s #

Self Test

Round 1

Round 2

Your #

Shakee’s #

Shakee’s #

after 3 rounds of handshaking
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

Your #

Shakee’s #

Shakee’s #

Shakee’s #

warning
WARNING

Air Born Pathogen Detected

Initiate Containment Protocol

Pressurizing Laboratory

Laboratory Sealed

Lock Down Complete

action plan
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?
epidemiology the basics
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)

real world example swine flu
Real World Example: Swine Flu

* Last updated on May 2, 2009

patient zero to epidemic
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

patient zero to epidemic1
Patient Zero to Epidemic

Patient Zero is infected

patient zero to epidemic2
Patient Zero to Epidemic

Patient Zero is infected

Interacts with others, passes on infection

patient zero to epidemic3
Patient Zero to Epidemic

Patient Zero is infected

Interacts with others

Patient Zero dies, neighbors infect

patient zero to epidemic4
Patient Zero to Epidemic

Patient zero is infected

Interacts with others

Patient Zero dies, neighbors infect

Neighbors interact with others, infecting them

patient zero to epidemic5
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

patient zero to epidemic6
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

who s infected
Who’s 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

who s infected1
Who’s 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?

action plan1
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?
who is patient zero how fast is it spreading
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)
tracking the infection
Tracking the infection

How quickly does the infection spread?

26

24

22

20

18

16

14

12

10

8

6

Rd 3

Rd 2

Rd 1

Initial

Rd 4

4

2

Total # Infected

tracking the infection1
Tracking the infection

How quickly does the infection spread?

26

24

22

20

18

16

14

12

10

8

6

Rd 3

Rd 2

Rd 1

Initial

Rd 4

4

2

Total # Infected

emergency alert
Emergency Alert!

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

ice breaker discussion pg 8
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?
action plan2
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?
how to identify bacteria
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?
how to identify bacteria1
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?
bacterial morphology
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)

two cocci what s the difference
Bacteria 1

Morphology: Coccus

Pathology: mild cold

Bacteria 2

Morphology: Coccus

Pathology: death

Two Cocci, What’s the difference?
bacteria cell wall stained
Bacteria Cell Wall - Stained

Strong purple stain

Gram Stain

Strong pink stain

gram positive or negative bacteria morphology
Gram positive or negative?Bacteria Morphology?

Gram Positive (+)

Gram Negative (­–)

Coccus

Rod

gram stain the basics

Stains bacteria purple

Helps stain bind to peptidoglycan

Gram positivestained purple

Gram negative unstained

Gram positivestays stained as purple

Gram negativestained pink

Gram Stain – The Basics
gram staining lab safety
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
gram stain protocol pg 12 13

1. Crystal Violet 90 seconds

3. Iodine 60 seconds

2. Distilled Water (gentle) 5 sec

6. Safranin 60 seconds

  • 95% Ethanol 5 seconds (wash until colorless)

5. Distilled Water (gentle) 5 sec

Each person in the group stains one sample:

A, B, C, D, unknown

8. Blot dry without touching bacteria

7. Distilled Water (gentle) 5 sec

Gram Stain - Protocol (Pg. 12-13)
slide61

Ocular lens(Eyepieces)

Nosepiece

Arm

Objective lenses (4x – 100x)

Mechanical Stage

Coarse Focus (large)

Stage Clip

Fine Focus (small)

Condenser

Illuminator control (brightness)

Lamp (light source)

Base

Always carry a microscope with one hand holding the arm and one hand under the base.

Light Microscope (Pg. 10)

slide62

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)

slide63

We can see better details with higher the powers of magnification, but we cannot see as much of the image.

Which of these images would be viewed at a higher power of magnification?

Power of Magnification

gram stain protocol pg 12 131

1. Crystal Violet 90 seconds

3. Iodine 60 seconds

2. Distilled Water (gentle) 5 sec

6. Safranin 60 seconds

  • 95% Ethanol 5 seconds (wash until colorless)

5. Distilled Water (gentle) 5 sec

Each person in the group stains one sample:

A, B, C, D, unknown

8. Blot dry without touching bacteria

7. Distilled Water (gentle) 5 sec

Gram Stain - Protocol (Pg. 12-13)
looking at the gram stains pg 13 14
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.
gram stain discussion pg 16
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?
two cocci what s the difference2
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

Two Cocci, What’s the difference?
catalase test the basics
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)

Hydrogen Peroxide (liquid)

Water (liquid)

catalase test lab safety
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
catalase test protocol pg 18
Catalase Test - Protocol (Pg. 18)

B

Transfer bacteria

Bacteria dish

  • 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
catalase test protocol pg 181
Catalase Test - Protocol (Pg. 18)

A

A

Transfer bacteria

3 drops

Bacteria dish

  • 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).
catalase test protocol pg 182
Catalase Test - Protocol (Pg. 18)

A

A

Transfer bacteria

3 drops

Bacteria dish

  • 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
catalase test discussion pg 19
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?

cracking the case
Cracking the Case

Scientific names of given bacteria samples

A – Mycobacterium tubercolosis C – Escherichia coli

B – Streptococcus pneumoniae D – Staphylococcus aureus

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

identifying the unknown pathogen discussion pg 21
Identifying the Unknown Pathogen – Discussion (Pg. 21)

Sample D: Staphylococcus aureus

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: _________________________.

action plan3
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?
how do antibiotics kill

Cell Membrane

Cell Wall

DNA

DNA to RNA

(Transcription)

RNA to Protein

(Translation)

How do Antibiotics Kill?
  • Antibiotic: “against life”
  • Derived from natural products
  • Attack specific parts of the cell
example neosporin
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?

antibiotic testing
Antibiotic Testing

Layer of Bacteria

Antibiotic disk (contains drug)

Zone of inhibition

Why is there a clear zone immediately around the antibiotic disk?

zone of inhibition
Zone of Inhibition

Antibiotic 1

Antibiotic 5

Antibiotic 2

Antibiotic 3

Antibiotic 4

What conclusions can you draw from the above picture?

  • 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
antibiotic testing lab safety
Antibiotic Testing – Lab Safety
  • Put on lab coat and gloves
  • Place gloves in generic waste
  • Return plates to instructor
antibiotic testing protocol pg 24
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

antibiotic testing discussion pg 25
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?

course of action completed
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)
alert
ALERT

Air Sampling – No Pathogen Detected

Treatment Protocols Successful

- Antibiotics administered

Quarantine No Longer Necessary

Lab Lockdown Terminated

Outbreak Contained and Eliminated

outbreak contained
Outbreak...Contained
  • Summarize what we did today
  • Conclusions
  • Why is this important?
we hope you enjoyed biobugs
We hope you enjoyed BIOBUGS!

Fill out evaluation survey.

Lunch is served at 12 pm in Room 352.

Panel discussion will start at 12:25 pm.

for reference handshake partners will change depending on student numbers
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 won’t shake with him/herself.

slide91
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?)
  • In which organelle is catalase mostly likely found in human cells?
  • Catalase like many enzymes is temperature dependent. How would you design an experiment to test the activity of this enzyme? Assuming you are using catalase isolated from human livers, at which temperature do you think the enzyme would be most efficient? Draw a graph of enzyme activity versus temperature
  • Catalase is an enzyme; therefore what is it made of?
  • Catalase is present in ALL animals and in all tissues. Furthermore, it has nearly identical structures. Comment on the evolution history of this enzyme, when and where did it List 2 targets of an antibiotic
  • Neusporin contains 3 different antibiotics, what is the benefit
  • Why don’t antibiotics work for a viral infection?
  • Draw a plate with 3 antibiotic disks. Describe what is happen to the bacteria in the region of each disk
    • Disk 1 resistant,
    • Disk 2 susceptible
    • Disk 3 intermediate
  • When a doctor prescribes antibiotics, it is strongly recommended that the patient take the entire dosage, even if they begin feeling better. Why is this important
  • When dealing with a patient that has a bacterial infection, doctors usually prescribe older antibiotics first. Newer drugs are reserved for the most serious patients, why is this necessary?
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