BIO 120.42 | Module 0: Lab Safety Guidelines & Operating Procedures

4 important aspects of Lab Safety Guidelines & Operating Procedures

rmam

• Risk groups and biosafety levels

• Microbiology lab SOP

• Aseptic technique

• Media prep calculations

Which risk group?

• Could be of local / exotic origin

• Involves respiratory transmission and can cause serious or lethal diseases where treatment and/or vaccines may or may not be available

• e.g., Bacillus anthracis, Mycobacterium tuberculosis

Risk group 3

Which risk group?

• Commonly encountered in the community

• Poses moderate environmental and/or health hazard

• Causes opportunistic/nosocomial infections

• Associated with treatable human diseases

• e.g., sspvlc

  • Salmonella, S. aureus, P. aeruginosa, Listeria, Campylobacter

Risk group 2

Which risk group?

• Has great potential for lethal infection

• Aerosol / droplet spread

• e.g., Ebola virus

Risk group 4

Which risk group?

• Nonpathogenic

• Poses minimal threat to environment / lab personnel

• e.g., B. subtilis, E. coli

Risk group 1

Set of minimum standards and practices established to ensure safe handling of microorganisms in lab environments

Biosafety levels

Biosafety levels are determined based on which 2 factors

pp

• Potential risks associated with the microorganisms being handled

• Protective measures needed to safeguard lab personnel, people outside the lab, environment lpe

Which BSL?

• srsb

• Special ventilation system

• Restricted access

• Specially trained personnel

• BSC

• Can be used to handle RG1,2,3

BSL-3

Which BSL?

• Standard microbial practices

  • Handle in the open

  • No special equipment

• Can be used to handle RG1

BSL-1

Which BSL?

• ispb

• Isolated lab (strictly controlled access, ventilation, waste management)

• Specially trained personnel

• Wearing positive pressure one-piece body suit

• BSC

• Can be used to handle RG1,2,3,4

BSL-4

Which BSL?

• BSC: only if splashes or aerosols are generated

• Signages posted

• Designated waste container for biohazards

• Autoclave (which may not necessarily be within the room but near it)

• Can be used to handle RG1, RG2

BSL-2

Which BSL?

BSL-2

• BSC

• Signages

• Biohazards waste container

• Autoclave (not necessarily within room but near it)

Which BSL?

BSL-4

• Isolated lab (strictly controlled access, ventilation, waste management)

• Specially trained personnel

• Positive pressure one-piece body suit

• BSC

Which BSL?

BSL-1

• Handle in the open

• No special equipment

Which BSL?

BSL-3

• Special ventilation system

• Restricted access

• Specially trained personnel

• BSC

5 safety equipment that must be present in lab (Microbio Lab SOP)

3 fire ee first

• Fire extinguisher

• Fire blanket

• Fire exit

• Emergency shower

• Eye-wash station

• First aid kit

Enumerate some Microbiology Lab SOP

1. Refrain from working in the lab when immunocompromised (e.g., undergoing chemotherapy treatment, organ transplant, have autoimmune conditions, pregnant)

2. No eating

3. No applying makeup

4. No smoking

5. No using of contact lenses

6. No running/horseplay

7. No sleeping

8. Cover wounds with a band-aid, especially if on the hands

9. Keep equipment and materials away from the edge of lab benches

10. Do not let electrical cords hang over the edge

11. Keep the floor free of slip hazards

Accident resulting from inadvertent contact with fixed object

Trips

Accident resulting from slip, trip, or fall from height

Falls

Accident resulting from loss of traction between shoe and walking surface

Slips

• Never leave any _ unattended

• If alcohol in a beaker ignites, put out the fire by _

• Do not try to _

• Treat all chemicals as _, unless your instructor tells you otherwise

• Heat source

• Smothering or covering with petri plate top cover

• Move the fire into the sink

• Poisonous, corrosive, flammable pcf

• Do not take cultures _

• When transporting cultures to incubator, use _

• Treat all cultures as _

• Out of the lab

• Plastic tub / test tube rack

• Potentially pathogenic

How to remove gloves properly

1. Spray gloved hands first with disinfectant, preferably 10% bleach

2. Pull out 1 glove by pinching from outside of glove and removing it out of your hand

3. Then insert ungloved hand into the inside of the other gloved hand, pull the hand free from the gloves, and make a bag out of the glove

4. Discard gloves in the regular trash

How to dispose chemical waste

• Discard chemical waste in designated waste containers

• This includes alcohol-stain waste but ensure there’s no culture media to prevent contamination or unintended chemical reactions

• Collected waste is then sent for hauling and treatment

How to dispose contaminated pipette tips & Pasteur pipette

• Autoclave or use 10% bleach (30 - 60 mins contact time)

• Discard in sharps container

How to dispose needles and syringes

• Discard in sharps container

• Do not recap needles by hand

• Do not remove needles from syringes by hand

• Do not bend or break needles by hand

• Do not manipulate needles by hand

How to dispose broken glassware

Discard in broken glassware container

How to dispose liquid waste

• Autoclave or use 10% bleach (30 - 60 mins contact time)

• Discard in sink as regular waste

How to dispose solid waste

• Autoclave

• Discard as regular waste

How to dispose contaminated tissue

• Preferably 10% bleach (30 - 60 mins contact time) to ensure all tissue surfaces are disinfected since bleach will be able to penetrate all throughout (instead of autoclaving)

• Discard as regular waste

#1 rule in decontamination of lab waste

Do it ASAP (no later than 8 days after generation when stored above freezing temperature)

Refers to the killing or removal of all viable organisms, including endospores and viruses

Decontamination

2 methods of decontamination

• Using moist heat (autoclaving)

• Using chemicals (10% bleach)

Decontamination method that denatures most macromolecules of microorganisms

Autoclave

Autoclave setting for decontamination

121 C, 15 psi, 30 mins

Autoclave setting for sterilization

121 C, 15 psi, 15 mins

What should you not autoclave?

fracer

• Flammable material

• Radioactive materials

• Acids

• Chlorine-based products / chlorine

• Explosive material

• Reactive, corrosive, toxic material

How to sterilize BSC / Laminar Flow Hood

1. Turn on BSC (warm up)

2. Turn on UV lamp (default time = 10 - 15 mins)

3. 10% fresh bleach solution

   1. Cover all surfaces

   2. 10-min contact time

   3. Evaporate or wipe it dry before using ethanol because when bleach + ethanol mixes, chloroform forms, which is corrosive

   4. Do not stick your head in the unit

4. 70% ethanol

   1. Cover all surfaces

   2. 10-min contact time

   3. Evaporate or wipe dry

   4. Do not stick your head in the unit

How to decontaminate BSC / Laminar Flow Hood

1. 10% fresh bleach solution

   1. Cover all surfaces

   2. 10-min contact time

   3. Evaporate or wipe dry

   4. Do not stick your head in the unit

2. 70% ethanol

   1. Cover all surfaces

   2. 10-min contact time

   3. Evaporate or wipe dry

   4. Do not stick your head in the unit

3. Turn on UV lamp (default = 10 - 15 mins)

4. Turn off BSC

How to handle biological spill

1. Cover any spilled cultures with paper towel

2. Spray disinfectant generously (starting from edges to avoid further spreading spillage across surface)

   1. Allow 30 - 60 mins contact time

3. Work concentrically to clean up absorbent material (from the outer edge of the spill towards the center)

4. Report all spills and accidents to instructor (incident report form)

Set of procedures followed in handling microbial cultures to prevent contamination of scsoe source of culture, culture media, self, other lab workers, and the environment

Aseptic technique

Aseptic technique refers to the set of procedures followed in handling microbial cultures to prevent contamination of _

scsoe

• Source of culture

• Culture media

• Self

• Other lab workers

• Environment

Give examples of aseptic techniques

1. Washing your hands thoroughly before and after every experiment

2. Disinfecting your work area using tissue and ethanol before and after every experiment

3. Turning off electric fans

4. Ensuring proper ventilation system within the lab

5. Removing bags, notebooks, gadgets, etc. from work benches

6. Following a “clean-to-dirty” workflow to ensure contamination from dirty areas does not spread to clean areas

   1. Position nondominant hand closest to the clean station; dominant hand nearest to the dirty station

   2. e.g., If you’re right-handed, then the clean station should be to your left and you start working there, while dirty station should be to your right.

   3. Once an item is used in dirty station, avoid bringing it back to clean station.

7. Properly labeling cultures

8. Working near an alcohol lamp when doing isolation techniques outside of BSC (convection current)

9. Holding loop/needle at an angle of 60 degrees (to heat large SA of wire), passing the entire length of loop/needle through flame until red-hot, then cooling this for 10 secs before use

Culture media prep

• Volume of agar plate

• Volume used for calculations

• 15 - 20 mL/plate

• 20 mL (this gives it a room for error)

When do you have to add 10% extra to culture media volume?

When culture media is to be dispensed into different containers (room for error)

Culture media prep

• Volume of test tubes

• Volume used for calculations

• 5 mL/tube

• 5 mL/tube + 10% (total volume)

When do you not add 10% extra to volume of media?

When media will not be dispensed into different containers

T/F: You heat media only if agar is dispensed after sterilization

FALSE

Heat media only if agar is to be dispensed before sterilization (e.g., when making NA tubes)

Culture media prep

• Maximum volume for flask/bottle

50 - 70% of max capacity

When do you have to heat media?

Media is heated if agar is to be dispensed prior to sterilization (e.g., when making NA tubes)

T/F: You add 10% extra to culture media volume if media is to be dispensed in different containers

TRUE

Why must media be heated if agar is to be dispensed before sterilization?

This is done to dissolve components and ensure that they are mixed thoroughly

What must be the total volume of dH2O added to your media if you have to make 10 NA plates?

• 1 plate = 20 mL / plate

• 20 mL/plate x 10 plates = 200 mL dH2O

What should be the amount of ff. components to prepare 200 mL of cultured media?

• dH2O = 1 L

• Beef extract = 3 g

• Peptone = 0.5%

• Agar = 17 g

• Beef extract

  • 3g / 1000 mL x 200 mL = 0.6 g

• Peptone

  • 0.5 g / 100 mL x 200 mL = 1.0 g

• Agar

  • 17 g / 1000 mL x 200 mL = 3.4 g

Formula for dilution vs. dilution factor

• Dilution (D) = vol transferred / total volume

  • raise to negative 10^-n

• Dilution factor (DF) = 1 / D

Compute for the dilution (D) and dilution factor (DF) of tubes A and B

• Tube A

  • D_A = vol transferred / total vol

    • 1 mL / 10 mL = 0.1 = 10^-1

  • DF_A = 1 / D

    • 1 / 0.1 = 10

• Tube B

  • D_B = Prev D (vol transferred / total vol)

    • 10^-1 (0.1 / 10 mL) = 0.001 = 10^-3

  • DF_B = 1 / D

    • 1 / 10^-3 = 1000 = 10³

General formula for concentration computations

C₁V₁ = C₂V₂

Formula for anhydrous / hydrous form substitution computations

• Weight anhydrous / MW anhydrous = Weight hydrous / MW hydrous

• MW = No. of atoms of each element x Atomic weight of element

  • C = 12

  • H = 1

  • O = 16

  • N = 14

Prepare 500 mL of 0.1 g / 100 mL reagent from a 2 g / 10 mL reagent

• C1V1 = C2V2

• 2g/10 mL (V1) = 0.1g/100mL (500 mL)

• V1 = 0.1g/100mL (500 mL) / 2g/10mL

• V1 = 0.5 g / (2g/10mL)

• V1 = 2.5 mL

• Total vol = 2.5 mL of 2g/10mL reagent + 497.5 mL dH₂O

You need the unhydrated form (12 g MgSO4), but only the hydrated form (MgSO4 × 5H2O) is available.

• MW of MgSO4 = 120.366 g/mol

• MW of H2O = 18.015 g/mol

• Weight anhydrous / MW anhydrous = Weight hydrous / MW hydrous

• 12 g MgSO4 / 120.366 g/mol = Weight hydrous / (120.366 g/mol + 5 × 18.015 g/mol)

• Weight hydrous = (12 g MgSO4 / 120.366 g/mol) (120.366 g/mol + 5 × 18.015 g/mol)

• Weight hydrous = 20.980 g = 21 g of hydrated form (MgSO4 × 5H2O)

Formula for computing for moles

Weight solute / MW solute

Formula for computing for molarity

• moles solute / L solution

• (Weight solute / MW solute) / vol of soln (L)

Molarity problem

• Given

  • 200 mL dH2O

  • 2 M solution

  • MW = 56.11 g/mol

• Compute for the amount of solute (g)

• Molarity (M) = moles / L

• M = (Weight solute / MW solute) / vol (L)

• 2 M = (Weight solute / 56.11 g/mol) / 0.200 L

• 2 mol/L (0.200 L) (56.11 g/mol) = Weight solute

• Weight solute = 22.44 g

Conversion units 10^-1 → 10^-18

dcmunpfa

• deci = 10^-1

• centi = 10^-2

• milli = 10^-3

• u (micro) = 10^-6

• nano = 10^-9

• pico = 10^-12

• femto = 10^-15

• atto = 10^-18

Conversion units 10¹ → 10¹⁸

dhk mg tph

• deca = 10¹

• hecto = 10²

• kilo = 10³

• mega = 10⁶

• giga = 10⁹

• tera = 10¹²

• peta = 10¹⁵

• hexa = 10¹⁸

Conversion

• ug → _ → _

• uL → _ → _

• ug → mg → g

• uL → mL → L

Convert 900 ug → kg

900 ug (1 mg / 10³ ug) (1 g / 10³ mg) (1 kg / 10³ g) = 9×10^-7 kg

Fill in the conversion table for % w/w, % w/v, % v/v

	w/w	w/v	v/v
pph or %
parts per thousand
ppm
ppb
ppt

	w/w	w/v	v/v
pph or %	g / 100 g	g / 100 mL	mL / 100 mL
parts per thousand	g / kg	g / L	mL / L
ppm	mg / kg	mg / L	uL / L
ppb	ug / kg	ug / L	nL / L
ppt	ng / kg	ng / L	pL / L