BUCDC Center for Disease Control @ Boston University.

BUCDBUCDCC

Center for Disease Control @ Boston University

BUCDCBUCDC 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

BUCDCBUCDC 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

BUCDCBUCDC Lab Safety

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

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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”

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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 Goals1. Protect researcher from the sample2. 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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Trash

BUCDCBUCDC 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!

1

BUCDCBUCDC BUCDC Ice Breaker

Your Station

Towel 1

Towel 2

Towel 3

White Powder

Towel 4

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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

Round 1

Round 2

Write your ID#

Use your RIGHT hand - hold for 5 seconds

Round 3

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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

BUCDCBUCDC Round 1 – START!

Your #

Shakee’s #

1 242 233 224 215 206 197 188 179 1610 1511 1412 1313 1214 1115 10

• 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 #

Your #

Shakee’s #

16 917 818 719 620 521 422 323 224 1

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

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Round 1: Make a handprint and write down Shakee’s #

Round 2

Round 1

Write Shakee’s #

Round 3Your ID#

Self Test


• Shake hands with your new assigned “Shakee”

• Make hand print on next towel

• Write down Shakee’s #

Your #

Shakee’s #

1 122 113 104 95 86 77 68 59 410 311 212 113 2414 2315 22

Your #

Shakee’s #

16 2117 2018 1919 1820 1721 1622 1523 1424 13


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Write Shakee’s #

Round 3

Self Test

Round 1Your # Shakee’s #

Round 2


• Shake hands with your new assigned “Shakee”

• Make hand print on last towel

• Write down Shakee’s #

Your #

Shakee’s #

1 82 73 64 55 46 37 28 19 2410 2311 2212 2113 2014 1915 18

Your #

Shakee’s #

16 1717 1618 1519 1420 1321 1222 1123 1024 9


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Write Shakee’s #

Round 1 Round 2

Your # Shakee’s # Shakee’s #

Self Test

Round 3

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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 3Your

#Shakee’s #

Shakee’s #

Shakee’s #

Self Test

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WARNING!! WARNING!!!

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WARNINGAir Born Pathogen Detected Initiate Containment

ProtocolPressurizing LaboratoryLaboratory SealedLock Down Complete

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Possible Pathogenic AgentsBacteria/

Sample #Symptoms Prognosis

Mycobacterium tuberculosis (A)

Severe cough, fever, inflammation

If left untreated, patient prognosis is very good

Streptococcus pneumoniae (B)

Stiffness of the joints, fever, severe cough and migraines , short of breath


Escherichia coli (C)Fever, lose of equilibrium, twitching in the extremities


Staphylococcus aureus (D)

Inflammation of the joins, leading to numbness of the extremities, severe coughing, blue-black lesions

If left untreated, prognosis is poor with 98% fatality. Antibiotics should be administered within 6 hrs.

Unknown (U) ? ?

BUCDCBUCDC 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 expectedPatient Zero: First known case of disease

• Epidemiologists try to figure out:H

ow (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

BUCDCBUCDC 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 is infected


• Patient Zero is infected– Interacts with others,

passes on infection


• Patient Zero is infected– Interacts with others

• Patient Zero dies, neighbors infect


• Patient zero is infected– Interacts with others

• Patient Zero dies, neighbors infect – Neighbors interact with

others, infecting them


• Patient zero is infected– Interacts with others

• Patient zero dies, neighbors infected– Neighbors interact

with others, infecting them

• Disease reaches epidemic levels


• 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

BUCDCBUCDC 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

BUCDCBUCDC 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?


• 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?


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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)

Employee ID

Infected Individuals

Round 3 Round 2 Round 1 Initial1 1 1 1 1

2 2 2 2 2

3 3 3 3 3

4 4 4 4 4

5 5 5 5 5

6 6 6 6 6

7 7 7 7 7

8 8 8 8 8

9 9 9 9 9

10 10 10 10 10

11 11 11 11 11

12 12 12 12 12

13 13 13 13 13

14 14 14 14 14

15 15 15 15 15

16 16 16 16 16

17 17 17 17 17

18 18 18 18 18

19 19 19 19 19

20 20 20 20 20

21 21 21 21 21

22 22 22 22 22

23 23 23 23 23

24 24 24 24 24

25 25 25 25 25

26 26 26 26 26

Total # Infected

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Employee ID

Infected Individuals

Round 3 Round 2 Round 1 Initial

1 1 1 1 1

2 2 2 2 2

3 3 3 3 3

4 4 4 4 4

5 5 5 5 5

6 6 6 6 6

7 7 7 7 7

8 8 8 8 8

9 9 9 9 9

10 10 10 10 10

11 11 11 11 11

12 12 12 12 12

13 13 13 13 13

14 14 14 14 14

15 15 15 15 15

16 16 16 16 16

17 17 17 17 17

18 18 18 18 18

19 19 19 19 19

20 20 20 20 20

21 21 21 21 21

22 22 22 22 22

23 23 23 23 23

24 24 24 24 24

25 25 25 25 25

26 26 26 26 26

Total # Infected

BUCDCBUCDC Tracking the infection

• How quickly does the infection spread?

Total # Infected

Initial

Rd 1

Rd 2

Rd 3

2

4

6

8

10

12

14

16

18

20

22

24

26

Rd 4

BUCDCBUCDC Tracking the infection

• How quickly does the infection spread?

Total # Infected

Initial

Rd 1

Rd 2

Rd 3

2

4

6

8

10

12

14

16

18

20

22

24

26

Rd 4

BUCDCBUCDC 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?


• 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?


BUCDCBUCDC 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?

BUCDCBUCDC 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?

BUCDCBUCDC 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, What’s the difference?

Bacteria 1

• Morphology: Coccus

• Pathology: mild cold

Bacteria 2

• Morphology: Coccus

• Pathology: death

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Bacteria Cell Wall Structure

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Bacteria Cell Wall - Stained

Gram Stain

Strong purple stain

Strong pink stain

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Gram positive or negative?Bacteria Morphology?

Gram Positive (+) Gram Negative ( –)

Coccus Rod

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Stains bacteri

a purple

Helps stain bind to

peptidoglycan

Gram positive stained purple

Gram negative unstained

Gram positive

stays stained as purple

Gram negative

stained pink

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)

1. Crystal Violet 90 seconds

2. Distilled Water (gentle) 5 sec

3. Iodine 60 seconds

4. 95% Ethanol 5 seconds (wash

until colorless)

6. Safranin 60 seconds

8. Blot dry without touching bacteria

Each person in the group stains one sample:

A, B, C, D, unknown



BUCDCBUCDC Light Microscope (Pg. 10)

NosepieceObjective

lenses (4x – 100x)Stage Clip

Lamp (light

source)

Ocular lens(Eyepieces)Arm

Mechanical StageCoarse Focus

(large)Fine Focus (small)

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

base.

Base

Condenser Illuminator

control (brightness)

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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)

Power of Magnification

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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

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Gram Stain - Protocol (Pg. 12-13)

1. Crystal Violet 90 seconds


3. Iodine 60 seconds

4. 95% Ethanol 5 seconds (wash

until colorless)

6. Safranin 60 seconds

8. Blot dry without touching bacteria

Each person in the group stains one sample:

A, B, C, D, unknown



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Looking at the Gram Stains (Pg. 13-14)

1. 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

2. STOP. Raise your hands at this point.

3. A volunteer will help you set up oil immersion using the 100x objective.

4. Draw and label all FIVE bacteria samples using the 10x and 100x objectives.

1. The colors and shapes should be as accurate as possible

2. 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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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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• 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

Oxygen (gas)Water (liquid)

+

Catalase Test – The Basics

Hydrogen Peroxide (liquid)

2 2

BUCDCBUCDC 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)

1. Using a wooden stick, smear a small amount of bacteria from the Petri dish onto a clean microscope slide

2. Label the slide using the Sharpie marker

BBacteria dish

Transfer bacteria

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2. Label the slide using the Sharpie marker3. Add 3 drops of H2O2 solution onto the smear.

4. Record your observations in the worksheet (Pg. 19).

ABacteria dish A

3 drops

Transfer bacteria

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2. Label the slide using the Sharpie marker3. Add 3 drops of H2O2 solution onto the smear.

4. Record your observations in the worksheet (Pg. 19).5. Repeat steps 1-4 for all FIVE bacteria samples, using new

materials each time.6. When done, dispose of materials in the correct bins

ABacteria dish A

3 drops

Transfer bacteria

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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?

BUCDCBUCDC Cracking the Case• Fill out the summary table in your worksheet (Pg. 21):

Bacteria

SampleScientific Name

Morphology

Gram Staining

(+/-)

Catalase test (+/-)

A

B

C

D

Unknown

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: _________________________.

Sample D: Staphylococcus aureus



• Who was Patient Zero?– The first known infected patient is #5


• Which bacteria have we have been exposed to?– Gram positive cocci– Catalase positive– Bacteria is Staphylococcus aureus


BUCDCBUCDC Potential Treatments

• Suggestions?

BUCDCBUCDC How do Antibiotics Kill?

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

Cell Membrane Cell Wall

DNA DNA to RNA

(Transcription)

RNA to Protein

(Translation)

BUCDCBUCDC 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?

BUCDCBUCDC Antibiotic Testing

Layer of Bacteria

Antibiotic disk

(contains drug)Zone of inhibition

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

BUCDCBUCDC 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

Antibiotic 1

Antibiotic 3

Antibiotic 2

Antibiotic 4

Antibiotic 5

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 patient zero?– The first known infected patient is #5


• 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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ALERTAir Sampling – No Pathogen

Detected Treatment Protocols Successful

- Antibiotics administered

Quarantine No Longer Necessary

Lab Lockdown Terminated

Outbreak Contained and Eliminated

BUCDCBUCDC 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 12:25 pm.

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For reference: Handshake partners will change depending on student numbers

Your #

Shakee’s #

1 152 143 134 125 116 107 98 89 710 611 512 413 314 215 1

Your #

Shakee’s #

1 72 63 54 45 36 27 18 159 1410 1311 1212 1113 1014 915 8

Round 1 Round 2

Your #

Shakee’s #

1 32 23 14 155 146 137 128 119 10

10 911 812 713 614 515 4

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.

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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?)• 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?

BUCDC Center for Disease Control @ Boston University.

Documents

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