Biochemistry Lab Safety - PowerPoint Presentation

Slide1

Biochemistry Lab SafetySlide2

Personal

Protective Equipment: What must be worn when you work in the laboratory.

2

1

. PPEEye Protection

Lab CoatLong PantsClosed Toed Shoes – no exposed skin around feetLab gloves – when requiredSlide3

Eye Protection

Contact lenses are OK as long as glasses/goggles are worn

Prescription glasses – you must wear goggles over themSafety goggles are provided in organic labs in UV irradiating cabinetsEye wash stations are present in all labs

3Slide4

Clothing must cover all exposed skin including legs/ankles

Stockings or leggings do not provide good coverage

Sandals, flip

-

flops, Crocs, open-toe and open-top (i.e. ballet flat) shoes and canvas shoes (i.e. Toms) are not appropriate. These are not going to protect your feet if you drop a piece of glass with a liquid chemical reagent in it.





Clothing and Foot Protection

4

Slide5

Use of Gloves

Remove

gloves before handling objects such as doorknobs, telephones, pens, computer keyboards, pH meter or other electronic buttons, or phones while in lab. It might be convenient to have one gloved hand and one ungloved hand to do procedures where these kinds of things are used.

Throw away gloves anytime you take them off.You should expect to use several pairs of gloves in any given lab period.

Glove video5Slide6

Eyewash / Safety Shower

The

eyewash is on the left. Pull the handle and a fountain of water will appear that you can use to bathe your eyes.

The

safety shower

is on the right. Pull the handle and water will start spraying from the shower head on the ceiling. There’s no drain in the floor – we only do this in emergencies, because a flood of water will have to be cleaned up.

6Slide7

7

Eye WashSlide8

Safety Shower

8Slide9

Using the Fume Hoods properly

If this is not saying NORMAL, then the hood

is not protecting you. Keeping the sash

a

nd sliding panels in proper position keeps thisNORMAL, otherwise the alarm goes off. If the alarm goes off, you need to reposition t

hings to the correct positions, then press the “mute” button to reset the controller.The sash should never be raised abovethe green “operation” level when youa

re working in the hood.

This

window/bar

is called

the sash.

9Slide10

×

✓

✓

✓

Closed, not in use

In use, side-to-side panel

used as shield

In use, sash (window) raised

to less than 18 inches

Don’t open side shields

to make one big window.

10Slide11

When using a laboratory hood,

Check that the airflow is in the normal range on the digital displayTurn on the hood lightSet the equipment and chemicals back at

least 6 inches.Never lean in and/or put your head in the hood when you are working. This is worse than doing the experiment with no hood at all.It’s a good idea to put liquid

reagent containers in trays to catch all spills and drips

11Slide12

Fire Alarms –

know the location of one close to your lab

12Slide13

Fire Extinguishers – we have several in the labs and in the hallways.

13Slide14

14Slide15

Types of Fire Extinguishers

Most of our fire extinguishers are

ABC

.

It contains a dry powder to put out the kinds of fires we might encounter in the chemistry labs where we have class.

This is a special fire extinguisher for combustible metal fires. It is a type

D fire extinguisher. You won’t need to use this unless you work in a research lab with combustible metals.

15Slide16

Student Reaction in a Fire

Although we want you to be informed on the operation of a fire extinguisher, we do not expect you to use it. If a fire is ignited in your area, the proper STUDENT response is to:

Notify everyone in the roomIf possible shutdown any reaction in progress by removing heat/energy source

Proceed to the nearest exit and pull the nearest fire alarm

Evacuate the buildingAssemble in front of the library or in the YWCA parking lot for a positive headcount16Slide17

Keep your lab area clean.

×

×

×

×

Throw away used paper towels and used gloves,

immediately.

Don

’

t block the floor in front of the eyewash/shower station.

Don’t leave things in the floor because someone will trip over it.

Don’t leave cords dangling because someone will trip over them.

17Slide18

843-953-5611

Please take a moment now to program this number into your cell phone.

Once again, the number to call in an emergency is

:

18Slide19

Centrifuge SafetySlide20

Rotor Safety

Do not run rotors above their rated speed

Inspect rotor for imperfections and signs of wear that can eventually lead to catastrophic failureDo not drop rotorRinse the rotor after every useAvoid using abrasive brushes for cleaning

If you suspect rotor has been damaged, do not use itDo not use a rotor that is not compatible with your model centrifugeUse tubes and adapters that are rated for use in the rotor being used

Swinging

Bucketrotor

Disposable tubesNeed adaptors

Fixed angle rotorSlide21

Accident involving improper rotor usage

Centrifuges that malfunction can create projectiles out of the

rotor shards. If the centrifuge starts to make horrible noises,

cut the power and leave the roomSlide22

Loading the Centrifuge

Be certain that tubes are balanced with a partner

Don’t forget to include caps when weighing the tubes for balanceSecure the rotor on the spindle by tightening all knobs on the lidTug gently on the rotor to make sure it is secured to the spindle

Do not overfill bottles (3/4 full max)

Both knobs are tightened in some

models to secure rotor to the spindle

Counterbalance yourl

abeled sampleSlide23

Unloading Centrifuge

Take precautions if biohazards or other hazardous material is used as aerosols can form during vacuum cycles

Clean the chamber from condensation and any spillsNever try to open the centrifuge door before the rotor is done spinningNever reach a hand or anything else into the chamber when rotor is spinningNote: it is sometimes difficult to look at a spinning rotor and determine if it is spinningSlide24

Safety Overview

http://www.youtube.com/watch?v=q_0phA034n0

Note: A modern centrifuge will have low tolerance for mismatched tubes and will shut

itself off if tubes are not balancedAlso, most modern centrifuges will not allow the door to

unlock while the rotor is still in motion.Slide25

Autoclave Safety

25Slide26

What is an Autoclave?

An autoclave is a specialized piece of equipment designed to deliver heat under pressure to a chamber, with the goal of decontaminating or sterilizing the contents of the chamber. Slide27

Personal Protective Equipment

Autoclaves utilize steam, heat and pressure and therefore the risk of personal injury through scalding, burns and exploding glassware is great.

Personal protective equipment is absolutely required. 1) Safety Glasses 2) Lab Coat 3) Long pants

4) Closed Shoes 5) Long thermal gloves 6) Face shield recommended Slide28

What can be autoclaved?

Cultures and stocks of infectious

materialCulture dishesTips, pipettes, gloves, paper towels, aluminum foilCentrifuge bottlesGlassware -- all caps must be loosened

Media and other aqueous solutionsContaminated solid itemsSlide29

What CANNOT be autoclaved?

Solvents or volatilesChlorinated compounds (HCL, bleach

)CorrosivesRadioactive material Some plastics Slide30

Cycle Differences

Fluids must be autoclaved under a “liquid” settingItems such as pipette tips, test tubes, and centrifuge bottles are run under “dry” or “gravity” setting

The difference in settings is how the cycle is ventedLiquids must depressurize slowly and dry cycles conclude with a vacuum step to draw off condensationSlide31

Loading and Unloading the Autoclave

All screw caps must be loosened to prevent pressure changes in the glassware that can cause the container to burst

All items should be placed in an autoclave tray to prevent scald burns in the event of a spillReturn autoclave trays promptly so that other users do not skip using a tray because they can’t find one

Don’t skip using a trayDo not remove liquid that is still boilingIf possible, allow glassware to cool before removing

Loosen cap by several threadsSlide32

Door Safety

Never try to open a door that is under pressureNever try to speed up the venting process by tampering with the door, by turning on and off the machine, etc. Venting takes time.

Know where the pressure gauges are for the instrument you are usingIf possible, vent door slowlySlide33

Autoclaving Waste

Contaminated pipette tips and solid waste should be sterilized prior to disposal

Collect waste in a special autoclave-safe biohazards bagPlace bag in secondary containerVent the bag by openingDo not overfill bag

After removal place entire bag in a new trash bag so that “biohazard” signs are no longer showingSterilized waste can go into the normal trashAutoclave tape can be used to verify heat delivery but it does not guarantee proper sterilization

This bag is too fullSlide34

Container Choice

Pyrex glass, metal, polypropylene (PP) plastic and polycarbonate (PC) plastic are best choices

Polyethylene (PE), polystyrene (PS), and high density polyethylene (HDPE) will often melt and make a messSlide35

Autoclaving Tips

Add a 2 cm depth of water to trays with glassware; the water helps eliminate air pockets between the tray and the glass and helps prevent glass from breaking

Do not fill liquid past 75% volumeSeparate items to increase steam penetrationIncrease cycle time for large volumes of liquidTemperature must be maintained at 121°C for at least 30 minutesSlide36

Maintenance

Report any irregularities to your supervisorDo not operate if there is a steam outage

Failed runs should be reported and loggedSlide37

Overview

http://www.youtube.com/watch?v=T901F2W7wksPlease note: newer autoclaves such as the one in the New Science Center do not have pressure gauges and a chart recorder, but these parameters are displayed on the computer screen as the cycle is started.Slide38

Toxic Chemical SafetySlide39

Health Hazardous Chemicals

Categories:

IrritantsSensitizersCorrosivesCarcinogensTarget Organ Effects

Reproductive Health ToxinsAcute ToxinsPhysical Health HazardsCommon routes of exposure in the

lab are inhalation and skin absorption, while ingestion is less common.

Carcinogens, reproductive toxins, target

organ

damage

Acute toxicity, fatality

Corrosives

Irritants, sensitizers, acutely toxicSlide40

Toxic Chemicals

Many chemicals commonly used in the lab are toxic.

Toxicology concerns the degree to which a chemical is hazardous to human health.How do toxicologists predict which chemicals will be toxic and determine their mechanism of action?Animal studies to determine a dose-response curve to predict a threshold level above which a chemical is toxic

Mechanistic studies to determine how a chemical will be toxic to animals and humansThe Ames test to assess DNA damage caused by carcinogens

Gene microarrays to determine target genesSlide41

Toxicity and Minimizing Exposure

Toxic reactions depend on the duration of exposure

Acute exposure – a single exposure, or multiple exposures over 1-2 daysChronic exposure – multiple exposures over a longer period of timeThe Department of Labor OSHA establishes legal permissible exposure limits (PELs) for the workplace

“However, these standards must not be taken to represent an absolute boundary between the positively safe and the positively unsafe.” – OSHA website

LD50 is a measure of acute toxicityLD50 is the lethal dose of a chemical required to kill 50% of a test animal population (measured in mg chemical per kg body weight).The lower a chemical’s LD50

, the more toxic it is.Slide42

Carcinogens

Genotoxic carcinogens

cause DNA damage directly (e.g., by forming a DNA adduct) and/or indirectly (e.g., by producing reactive oxygen species that inflict genomic damage).If unrepaired before replication, DNA damage results in a mutation.

Mutations can result in tumor initiation if they occur in genes related to cell division, programmed cell death, DNA repair, etc.Non-genotoxic

carcinogens promote carcinogenesis without damaging DNA For example, these chemicals might stimulate cell proliferation, tissue invasion, or angiogenesis by binding to a receptor.These carcinogens mostly cause tumor promotion.Slide43

Toxic Chemicals In Biochemistry

When working with toxic chemicals, extra attention should be paid to selection and use of PPE.

Protect yourself by using PPE properly and disposing of contaminated PPE.Protect others by not spreading the toxic chemical around the lab.Many chemicals commonly used in biochemistry are toxic. A few common examples are detailed on the following slides.Slide44

Ethidium Bromide

Ethidium

bromide is an intercalating agent commonly used as a fluorescent label in molecular biology laboratories for techniques such as

agarose gel electrophoresis.

Avoid working with the powder, which can be fatal when inhaled (instead work with solutions).

Handle in hoodMark areas of use and decontaminate frequentlyDo not heat agarose with ethidium bromide in it

Use gloves; absorbs through skinSlide45

Acrylamide

Acrylamide is the monomeric precursor to polyacrylamide used in SDS-PAGE.

Avoid working with the powder due to inhalation hazard (instead work with solutions)

Use gloves when handling

Polymerize excess solution for safer disposalMay cause cancer. May cause heritable genetic damage. Also toxic in contact with skin and if swallowed. Danger of serious damage to health by prolonged exposure through inhalation, in contact with skin or if swallowed. Slide46

Sodium Azide

Sodium

azide is commonly found in dilute solutions to prevent bacterial growth.

Do not allow sodium azide

to come into contact with heavy metals or their salts, because a reaction may form heavy metal azides, which are explosives.

Do not allow sodium azide to come into contact with aqueous acids, because reaction liberates highly toxic hydrazoic acid, which is a dangerous explosive.

 Containers should be stored in secondary containers in a cool, dry secured storage area separated from acids.  Avoid using metal spatulas



Do not dispose of solutions down the drain, as explosions could result.

The acute toxicity of sodium

azide

is highSlide47

Phenylmethanesulfonyl Fluoride

PMSF is used in solution to inhibit proteases

Wear gloves.

Take extra precautions when working with the powder. Do not leave any traces of spilled power on the bench, balance, etc., where it could endanger another lab user.

Toxic if swallowed. Causes severe skin burns and eye damage. Extremely destructive to tissues of mucous membranes and respiratory tract. Corrosive. Target Organs: Nerves, Heart, Blood, Eyes.Slide48

Toxic Chemicals in Other Branches of Chemistry

Many chemicals commonly used in other branches of chemistry

(e.g., synthetic chemistry) are also toxic.Some examples:

Halogenated aliphatic hydrocarbons (e.g., chloroform, carbon tetrachloride, etc.) – cause central nervous system depression, liver injury, kidney injury, and some degree of

cardiotoxicity. Many are carcinogenic.Aromatics (e.g., benzene, toluene, xylene) – cause central nervous system depression, skin irritation. Benzene causes bone marrow injury and is associated with leukemia.Slide49

Toxic Chemicals and Material Safety Data Sheets (MSDS)

Identify toxicity hazards for any chemical by consulting the MSDS. The Hazards section includes toxicity warnings:Slide50

Look for the health NFPA category rated from 0-4, and read warning statements.

Toxic Chemicals and Material Safety Data Sheets (MSDS)

0 – Hazard no greater than ordinary material

1 – May cause irritation; minimal residual injury

2 – Intense or prolonged exposure may cause incapacitation; residual injury may occur if not treated

3 – Exposure could cause serious injury even if treated

4 – Exposure may cause deathSlide51

Separate sections indicate toxicological data on the chemical:

Toxic Chemicals and Material Safety Data Sheets (MSDS)Slide52

Biohazard SafetySlide53

Biological Hazards

Biological hazards are potential sources of infectious agents that could be harmful to human health.

Bacterial, fungal, parasitic, viral, and prion agents.Sources may include animals, tissues, cells, blood, and nucleic acid samples, including recombinant DNA.Slide54

Biohazard Classification

The National Institutes of Health (NIH) has determined a classification system for

biohazardous agents based on Risk Groups (RG).

Risk Group 1 (RG1

)

Agents that are not associated with disease in healthy adult humans Risk Group 2 (RG2)

Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available

Risk Group 3 (RG3)

Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk)

Risk Group 4 (RG4)

Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk)Slide55

Biological Hazards

Sterilization techniques are generally effective in destroying biohazard agents (except prions and spores)

Autoclave

Bleach

AlcoholBiohazards are another reason (in addition to avoiding ingestion of toxic chemicals) for good lab hygieneGlove use and proper gloves hygieneHand washingNo food or drink in labSlide56

Other Biochemistry Safety ConcernsSlide57

Keep the -80° freezer closed!

Ultracold

(-80°C) Freezer Use

Many biological samples and chemicals need to be preserved at temperatures below room temperature

Always consult the label: 4°C (refrigerator), -20°C (conventional freezer), -80°C (

ultracold freezer), -196°C (liquid nitrogen)

Cold storage can slow cell death rate, preserve enzyme activity, inhibit contaminating bacterial growth, and prevent degradation.Use insulated gloves to handle ultracold materials.Handle glass dewars with caution – danger of exploding glass if they are knocked over and broken. Slide58

High Voltage Techniques

DNA and protein gel electrophoresis combine high voltage with the use of aqueous solutions

Take precautions to avoid electrocution.

Modern gel boxes have electrodes positioned on the lids to drastically reduce the risk of electrocution.

Always secure the gel box lid before turning on the voltage. Turn off the voltage before removing the lid to a gel box.Match the red and black electrodes to the corresponding red and black outlets on the power supply.Slide59

GHS SymbolsSlide60

Liquid Nitrogen Safety

Liquid nitrogen (LN2) is commonly used to rapidly freeze proteins and bacteria

LN2 rapidly evaporates and can displace air in enclosed spaces causing suffocation

LN2 can cause death of human tissue from extreme coldMinor contact can cause “burns”

Evacuated glass dewars can sometimes burst unexpectedlyLN2 can condense liquid oxygenSlide61

Liquid Nitrogen DON’T’s

DON’T use in confined space

DON’T freeze items in centrifuge tubes with snap caps

DON’T transport LN2 in a closed automobile DON’T transport LN2 in a passenger elevator

DON’T allow a storage dewar to tip overDON’T leave cold fingers on a vacuum line in LN2 overnightDON’T use without PPE!

NO!Slide62

Liquid Nitrogen Do’s

DO use or dispense LN2 only in well ventilated areas

DO ensure glass dewars

are taped or wrappedDO use approved containers only such as a dewar

or threaded cryovials for storageDO make sure any vessel with LN2 is VENTEDDO secure storage dewars against spilling

DO use appropriate PPE which includes:Face shield (or minimally goggles)Long thermal glovesApron or lab coat

Closed toed ShoesLong pants

YES!

NO!

YES!Slide63

Special Note on LN2 transport for Chemistry Magic Shows

Use only a sturdy LN2

dewar

Keep windows of car openDo not put LN2 next to any passenger in the car

Secure the LN2 such that it cannot tip over (use seatbelt, other heavy, bulky objects to block it in)Be prepared to leave the vehicle if a spill occursDo NOT allow K-12 students to touch LN2 Do NOT allow K-12 students to approach your glass

dewar without goggles