Clinical Chemistry (Laboratory)

Glass and Plasticware

A. High Thermal Resistance Glass
Borosilicate with low alkaline content
In all purpose glassware (all types of beakers, flasks, etc.)
Can tolerate heating and sterilization for lengthy periods of time to 510C
Pyrex and Kimax, common brand name
Limitations:
a) Should not be cooled to quickly after heating
b) May cloud after use of strong alkali
c) Subject to scratching
Corex and Vycor brands

Aluminosilicate in nature
Corex brand, used for centrifuge tubes and thermometers
Strong and hard with temp. Stability to 672C, short term use to 850C
Resists scratching and subject to some acid or alkali attack at 100C
Vycor brand, used for ashing and ignition techniques
Can withstand very high temp. (900-1200C)
Most are alkali resistant

B. High Silica Glass

Contains 96% silica
Used for cuvets and thermometers
Radiation resistant, good optical qualities and temp. capabilities
Used for precision analytic work, for optical reflectors and mirrors
C. High Alkali Resistance Glass
Aluminosilicate
Can be used with strong alkali
Suffer minimal attack compared to borosilicate
D. Low Actinic Glass
High thermal resistance with red color added as an integral part of the glass
Give maximum protection to light-sensitive or photosensitive materials
Used for bilirubin, carotene, and vitamin A analysis
E. Soda Lime Glass
Mixture of the oxides of silicon, calcium and sodium
Lowest in cost
Poor resistance to high temp.

Plasticware (*sorry po di ko makita lahat ng nasa slide... nakaharang kasi si sir e >.<)
Resistance to corrosion and breakage
Inexpensive, disposable after each use
Types of Resins
a) Polystyrene
o Useful with water and aqueous salt solution
o Not recommended for use with acids, aldehydes, ketones, ethers,
hydrocarbons or essential oils
o Alcohols and bases can be used, storage beyond 24 hrs is discouraged
b) Polyethylene
o Excellent chemical resistance to most substances
o With the exception of aldehydes, amines, ethers, hydrocarbons, , essential oils
o Usage should be limited to 24 hrs at room temp
c) Polypropylene
o Same chemical resistant as linear polythylene
d) Teflon
o Excellent chemical resistant to almost all chemicals in the clin. Lab
e) Polycarbonate
o Very susceptible to damage by most chemicals
o Resistant to water, aqueous, salts, food, and inorganic acids
f) Polyvinylchloride
Glassware Problem
Routine Washing

Cleaning Technique
Dirty Glasswares: Immerse in soapy water or dilute bleach solution,
wash using detergent and rinse with tap water. Dry in an oven.
(Temp.<140C)

Use of Acid Dichromate:

Use of 20% Nitric Acid
*Wash with detergent and tap water
Blood Clots
10% NaOH
Dry Micropipets with acetone
New Pipets (SI Alkaline) Rinse with 5% Hypochloric Acid or 5% Nitric Acid. Wash with routine
procedure
Metal Ion Concentration Acid soak (20% Nitric Acid) for 12-24 hrs.
Rinse with distilled water 3-4 times
Grease
Soak with in an organic solvent
Dissolve 100g KOH in 100ml distilled water. Cool. Add 10% ethanol.

Permanganate Stains

Contrad 70 (commercially prepared)

50% HCl. Rinse with tap water. Wash. Dissolve 1% Ferrous sulphate
in 25% Sulfuric Acid

Pipettes

Glass or plastic tubes, usually open at both ends

Used to transfer specific amounts of liquid from one container to another
May be reusable or disposable
Used for volumes between 1 and 100 milliliters

Classification
I.

According to manner of calibration:

A. As to design
1. To deliver (TD)
o Water is used as calibrating fluid
o Deliver exact volume by allowing to flow by gravity
o Last drop should not be blown out
2. To contain (TC)
o Mercury is calibrating fluid (non-wetting liquid)
o Will not deliver the exact volume due to the tendency of the
fluid to cling to the glass surface
o Deliver exact volume by repeated filling and emptying with
the diluent
o Last drop is blown-out
B. As to drainage characteristics
1. Blow-out
o Exact volume is obtained when last drop is blown out
o Etched or frosted ring is seen near the mouthpiece
o The frosted band should not be confused with thicker colored
rings or colored dots, which are a manufacturers codefor the
maximum volume of the pipette.
Manufacturers code

Orange band 10 ml
Blue or red band 5 ml
Yellow or red band 1 ml
Two lines red color or white color 0.1 ml

2. Between two marks

o Exact volume is calibrated to fill the volume between two
calibrated points on the pipet
II.

According to Graduation

A. Graduated/Measuring
1. Mohr Pipettes
o The graduation on these always end before the tip
o Has a dead space
2. Serological pipettes
o The graduation marks continue to the tip
o Generally blow-out pipette
Specifications on a measuring pipette
Printed on the neck of the pipette are the specifications that indicate:

The maximum volume of liquid that can be transferred

The size of the divisions on the pipette
The temperature at which calibrations were made
If the pipette is a to deliver(TD) or to contain(TC) pipette
Ex.:
5 in 1/10 ml
TD 20C
Specifications on a pipette as shown above indicate that
the pipette is calibrated in 1/10 ml divisions and will deliver up to
5.0 ml within published tolerance levels at 20C.

B. Transfer dispense one volume without further subdivision

1. Volumetric pipettes
o Has the greatest degree of accuracy and precision
o Has a bulb like enlargement located midway between the
mouthpiece and the delivery tip
o Self-draining
o Designed to dispense or transfer aqueous solution
o Should be used when diluting standards, calibrators, or
quality control materials
Specifications on a volumetric pipette
On a volumetric pipette, the specifications indicate:
How much liquid will be transferred if the liquid is drawn up to
the calibration line on the neck
The temperature at which the calibration was made
Whether it is a TD or TC pipette
When emptying a volumetric pipette, the liquid is allowed to
drain out. It is NOT forced out
After it is emptied, the small amount of liquid which remains in
the tip should not be blown out

Volumetric pipettes are NOT blow-out pipettes

2. Ostwald-Folin
o Bulb like enlargement is closer to the short delivery tip
o Blow-out (etched or frosted ring)
o Used with biologic fluids having a viscosity greater than that
of water
o Used in measuring viscous fluids such as whole blood
3. Pasteur pipette
o No calibration
o For biologic fluid without specific volume
4. Micropipettes
o Calibrated to contain the stated volume rather than to
deliver it for accuracy
o Entire content must be emptied
o Used when small amount of blood or specimen is needed (less
than 1 ml)
5. Automatic pipettes
o Macro- > 1 ml > ; Micro- < 1 ml
o Most routinely used pipettes
o Use specifically designed precision-molded, non-wettable,
disposable plastic tip
Advantages:
a.
b.
c.
d.
e.

Eliminates pipette cleaning

Eliminates danger of cross-contamination
Eliminates error due to variation in technique
Permits rapid repetitive measurement
Delivery of equal volumes of specimens followed by
diluent at constant ratio to specimen

6. Air Displacement Pipettes

o Relies on piston to create suction to draw the sample
o The piston does not come in contact with the liquid
o Second stop blow-out stroke removes any remaining sample
from the tip
o Used for aqueous solutions
7. Positive Displacement Pipettes
o Operates by moving the piston in the pipet lip or barrel much
like a hypodermic syringe

o The piston enters into direct contact with the liquid

There is no air interference
o Enhance accuracy and precision
o Allows aspiration of volatile liquids without evaporation
o Prevents cavitation and aerosol formation
o blow-out stroke is used during aspiration
o Prevent carry over and sample to sample contamination
8. Dispenser Pipettes
o Obtain the liquid from a common reservoir and dispense it
repeatedly
o Maybe bottle top, motorized, hand-held, or a dilutor
o Dilutor combines sampling and dispensing functions
Handling and Disposing of Pipettes
Chipped and cracked pipettes should be replaced as they are unsafe and may affect
the accuracy of measurement
Never mouth pipette
Hold the pipette between the thumb and the forefinger
The pipet is held upright, not an angle
Draw up the fluid to a point slightly above the desires graduation line
Close the mouthpiece with the index finger
Wipe the outside of the pipet from any adhering liquid with tissue then bring the liquid
down the calibration mark
Mouth pipetting is strictly prohibited
All reading must be made with the eye level to the bottom meniscus
Use the smallest pipet which holds the desired volume

Dispose dirty pipettes by placing in soapy water solution tray. Place disposable pipettes
in a cardboard holder. Do not leave pipettes on counters or sinks. If you are working
with radioactive materials be sure to dispose pipettes in a properly marked container

Handling sterile pipettes

When using sterile pipettes, be sure to use proper sanitary techniques. If you have a
sterile package of disposable pipettes, tear only a small corner of the package open
and push one pipette out of this opening, then immediately close the package to
prevent contamination.
If you are using sterile pipettes in a pipette canister, place the canister on its side, slide
off the cover, pull out one pipette and replace the cover immediately.
Transferring a precise volume liquid
A pipette bulb is used to draw liquid up into the pipette. There are many types of
pipette bulbs.

Using the common rubber bulb

Hold the pipette about 8cm below the mouthpiece with one hand. Then with your
other hand, squeeze the bulb and touch the opening to the mouth of the pipette.
Insert no more than one-half cm of the pipette into the bulb.
Place the tip into the colored liquid and slowly release the pressure on the bulb
The liquid will be drawn up into the pipette and will form a curved surface against the
glass.
This surface is called the meniscus. Pull the bottom of the meniscus up about 1cm past
the desired level.
Then quickly, but carefully, remove the bulb as you slip your free index finger over the
tip of the mouthpiece hold.
Never use your thumb, your index finger will allow you better control and will also
enable you to hold other items with your free fingers when necessary
Then with you finger still on the end of the pipette, gently lift the pipette out of the
soln
Then raise your finger just enough to allow the bottom of the meniscus to line up with
the desired graduation mark. You should observe the meniscus at eye-level while doing
this.
When the meniscus is at the desired level, touch the tip of the pipette to the inside of
the container holding the colored water, to remove any drops of liquid on the end of
the pipette.
Use of safety pipette filter (propigette)
With a gentle twisting motion, insert into the safety pipette filter about cm.
To produce a vacuum for aspiration, squeeze valve A with your thumb and index
finger on one hand while using your other hand to squeeze the bulb. A stands for air or
aspirate
To pull the liquid up into the pipette place the pipette into the liquid and squeeze the
S or suction valve until the liquid reaches the desired level.
Touch the tip of the pipette to the side of the vessel containing the liquid to remove
any adhering drops
To expel the liquid squeeze the E valve. This will allow the liquid to flow out of the
pipette except for the last drop
Pipetting Aids
The pipetting aid has a plunger at one end and a rubber ring at the other end into
which the pipette is inserted. The plungers are color coded depending on the volume of
liquid they can pull up
Insert the pipette into pipetting aid. Place you index finger over the hold in the plunger
and push it down completely and hold it there
Place the tip of the pipette into a beaker containing colored water and allow the
plunger to rise all the way up, keeping your finger firmly on the hole in the plunger.
Adjust the level of the meniscus by lifting your finger. If the meniscus level isnt high
enough, depress the plunger completely and allow it to rise again.

Other pipette bulbs

Other pipette bulbs that are often used include Vadosa pipette filter and the pipette
pumper
LAB SAFETY
OSHA
Occupational exposure to bloodborne pathogens standard
Universal standard precaution
o Require that all human body fluids, body fluids, and tissues be treated as if they
were infectious.
o Blood, semen, vaginal secretions, CSF, synovial fluid, Pleural fluid, etc.
General safety practices
Handwashing
o Soap and water
o Alcohol hand gels (62%)
Eating, drinking, smoking and applying cosmetics or lip balm are strictly prohibited in
the lab work area.
Hands, pens and other formites must be kept away from the workers mouth and all
mucous membranes
Food and drink must not be kept in the same refrigerator as lab specimens or reagents
Personal protective clothing and equipment must be provided to the worker
Wear closed shoes
Pin long hair
Do not horseplay
Do not insert or remove contact lenses
Do not wear chains, bracelets, necklaces
Do not use bare hands to pick up broken glasses
Do not allow visitors into the work area
Mouth pipetting must be prohibited
Needles and other sharp objects contaminated with blood should not be manipulated
in any way
Contaminated sharps
o Puncture resistant container labeled w/ universal biohazard symbol
Wear PPEs
o Lab gown: washed inside the lab
o Gloves
o Eyewear
o Masks
Housekeeping
o Use Na Hypochlorite (1:10) or 10%
1 part bleach + 9 parts water
Expiration date: 24 hours
HEB B Vaccination

o Laboratorians should receive vaccination before or within 10 days after

beginning work in the lab
o If exposure accident happens, post-exposure evaluation and ff-up (prophylaxis,
medical consultation) should be available with no cost.
Proper waste disposal
Black: dry, non-infectious
Green: wet, non-infectious
Yellow: infectious, pathological and pharmaceutical waste
Orange: radioactive waste
Accidents
Basic causes of accidents
o Failure to follow lab instructions
o Careless work habits
o Failure to use protective equipment
o Fatigue
o Defective lab engineering design of the laboratory
Types of accidents
o Physical injury
Falling on wet floor
Cutting oneself with broken glass pieces
Getting long hair caught in equipment
Needlestick injury
o Electric shock
o Exposure to dangerous chemicals
o Exposure to radioactive chemicals
o Fire and explosion
o Biologic hazards
Most common causes of electrical hazard accdg to WHO:
Wet or moist surface near electrical equipment
Long flexible electrical connecting cables
Poor and perished insulation on cables
Overloading of circuits by use of adapters
Sparking equipment near flammable substances and vapors
Electrical equipment left switched on and unattended
Electrical Hazard
Electric wirings inside the lab should be inspected regularly
Use of extension cords should be avoided
Use of cheater adapters and gang plugs should be prohibited
All new instrument should be thoroughly inspected first before being used out for
service

Electrical equipment should not be placed in areas where ignitable vapors might
accumulate
Plug (and not the cord) should be pulled
If electrical equipment fails to function properly, disconnect the apparatus
In case of electric fire, use only carbon dioxide extinguisher. Never throw water

Chemical Hazard
All chemicals in the laboratory must be considered as poison
Label all chemicals properly
Follow all handling and storage requirements for the chemical. Refer to MSDS
(Material Safety and Data Sheet) when you have problems regarding a chemical
The wearing of contact lens should not be permitted when an employee is working
with xylene, acetone, alcohols, formaldehyde and other solvents
Flammable liquid = stored 5 feet away from the heat source
Do not store inflammable chemicals in refrigerators
Strong acids and alkalis are corrosive compounds. Always store them near the floor
with warning sign on the bottle
Adequate ventilation
Use bottle carriers (if more than 500mL)
PPE must be used
Biohazard and safety precautions
All clinical specimens are potential sources of infection
To prevent infections:
Wear the appropriate protective equipment and clearly label containers with essential
information. The outside of the container should be wiped with an alcohol swab.
Technicians must wear disposable gloves all the time
Mouth pipetting is totally prohibited
Never dispose biohazard waste with regular trash
Radiation Hazard
Ionizing Radiation
o Can be one of the most toxic substances of exposure to personnel in the lab
Pregnant women must not work with radioactive substances
Fire Hazard
Common causes of fire in the lab
o Electrical overloading
o Poor electrical maintenance
o Excessive long gas tubing and electricity
o Equipment left switched on unnecessarily
o Naked flames
o Deteriorated gas tubing
o Misuse of matches
o Carelessness with flammable materials

o Flammable and explosive chemicals stored in ordinary refrigerators

Four Classes
o Class A ordinary combustibles such as wood, paper and cloth
Use: pressurized water and dry-chemical extinguishers
o Class B flammable liquids, gases or grease
o Class C electrical equipment, motors and switches
Use: dry chemical and carbon dioxide extinguishers
o Class D flammable metals such as magnesium
Extinguishment is left to trained firefighters using special dry chemical
extinguishers

Fire Extinguisher
Pull
Aim
Squeeze
Sweep
Use of specialized Equipment
Centrifuge is a common source of mechanical hazard
Hair and clothing can become entangled in the machine if it is allowed to operate with
its cover open
Tubes and glassware allowed to be centrifuged without covering the machine can be
dangerous
Spinning rotors must never be slowed or stopped manually
Make sure the tubes are balances inside the machine. Breakage of centrifuge tubes
within centrifuge head is a source of biohazard
Use of biosafety cabinets is mandatory for the microbiology section of the laboratory. It
must not be used until it is working properly.
Apparatus and materials in the cabinet must be kept to a minimum
Air circulation at the rear plenum must not be blocked
Materials should be surface decontaminated before placing them inside the working
area of the cabinet
Bunsen burners must not be used in the cabinet
Traffic behind the operator must be minimized
The operator should not disturb the airflow by repeated removal and reintroduction of
his/her arms
The surface of the BSC should be wiped using an appropriate disinfectant after
completion of work and at the end of the day
Care for the microscope
How to focus
How to use
How to clean
Prevention is better than cure