LABORATORY SUPPLIES

Chemicals Used For Reagent Preparation

1. Analytical Reagent Grade (AR)
• This important for qualitative and quantitative analyses, and essential tor accuracy.
• Specifications were established by the American Chemical Society [ACS).
• Labels on these reagents either state the actual impurities for each chemical lot or
list the maximum allowable impurities (percentage of impurities)
• Uses: Trace metal analysis and preparation of standard solutions
2. Ultrapure Reagents
• These types of reagents have been put through additional purification steps.
• Examples: Spectrograde, nanograde, and HPLC pure
• Uses: Chromatography, atomic absorption, and immunoassays
3. United States Pharmacopeia (USP) and National Formulary (NF)
• It is approved for human consumption (not injurious to individuals) but may not
be applicable for laboratory analysis.
• Use: For drug manufacturing
4. Technical or Commercial Grade
• lt is used primarily in manufacturing
• It should never be used in clinical laboratory testing.
5. Chemically Pure (CP) or Pure Grade
• The impurity limitations of this type of chemicals are usually not stated.
• It also tails to reveal the tolerance limits of impurities.
• Preparation of these chemicals is not uniform.
• Purity is usually delivered by measurement of melting point or boiling point.
• It is not recommended for research and analytical chemistry unless further
purification or a reagent blank is included.

Types of Reagent Water

1. Type I Reagent water
• It is utilized for test methods requiring minimum interference.
• It is used for procedures that require maximum water purity for accuracy and
precision.
• It should be utilized immediately (storage is discouraged) after production.
• Uses: Flame Photometry, AAS, blood gases and pH, enzyme studies, electrolyte
testing, trace metal, iron studies, ultramicrochemical analyses, measurements or
hano8Td orSuonano8ram concentrations, tissue cell methods (microscopy), and
preparation of calibrators ana quality control materials
 • It is utilized for reagent preparations in hematology, microbiology, immunolog8Y,
and chemistry.
• It may also be considered for preparation of reagents for urinalysis, parasitology,
and histology.
• For procedures that require the use of specially prepared water such as heavy
metal testing or HPLC, type I water should not be used, instead consider the ultra-
pure water.
• It is advisable to use only type I water for most applications in the laboratory
[McPherson and Pincus, 2022).

2. Type II Reagent water

• It is used for washing glassware’s.
• Maintenance of the constant temperature in water bath is improved by filling it
with type ll (or type l) water.

Notes to Remember
• Chemicals must be stored according to their chemical properties and classification.
• According to the Clinical and Laboratory Standards Institute (CLSI), water should
be classified in terms of type instead of the method of preparation.
• The College of American Pathologists (CAP) recommends that laboratories
document culture growth, pH, ana specific water resistance on reagent grade
water.
• Filtration is the first step in preparing reagent grade water.
• Processes involved in the preparation of reagent grade water: filtration, distillation,
ion exchange, reverse osmosis, and UV oxidation.
• The CLS1/CAP requires that type I water have a resistivity greater than 10 Mlb-
cm.

Distilled Water
• It is the condensate collected from steam and created when water is boiled and
vaporized.
• It has been purified to remove almost all organic materials.

Deionized Water
• It is prepared by using deionizer (anion or cation), and it is free from mineral salts.
• Impurities are removed by ion exchange processes.
• It has some or all ions removed but organic material may still be present.
• It is purified from previously treated water such as pre-filtered or distilled water.
Reference Materials
• Calibration materials should meet the identity, labeling, and performance
requirements of CLSI (formerly NCCLS).
1) Primary Standard
a. It is a highly purified chemical that can be measured directly to produce a
substance of exact known concentration.
b. The International Union of Pure and Applied Chemistry (IUPAC) has
established the criteria for primary standard.
2) Secondary Standard
a. It is a substance of lower purity whose concentration is determined by
comparison with a primary standard.

Types of Solutions Used in the Clinical Laboratory:

1) Dilute solution: It has the presence of relatively little solute.
2) Concentrated solution: It has a large quantity of solute in solution.
3) Saturated solution: A solution in which there is an excess of undissolved solute
particles.
4) Super saturated solution: It has a greater concentration of undissolved solute
particles than does a saturated solution of the same substance.

Notes to Remember
• Water may be distilled more than once and each distillation cycle removes more
impurities.
• Water can also be purified by ultrafiltration, UV light, sterilization, or ozone
treatment.
• Detergent-contaminated water will have an alkaline pH.
• Hard water contains calcium, iron, and other dissolved elements.
• Gram-negative bacteria are the most common isolates in water after the
purification process is complete.
• Tests for water purity: Microbiological content, pH, resistivity, chemical oxygen
demand (COD), ammonia, ions, and metals.

TYPES OF GLASSWARE
Borosilicate Glass
• It is used for heating and sterilization purposes; most commonly used
• It is characterized by a high degree of thermal resistance, has low alkali content,
and is free from the magnesium lime- zine group of elements.
• It does not contain heavy metals, arsenic, ad antimony.
• Examples: Pyrex and Kimax glassware
• Strain point: 515° C (Pyrex)
Boron-free Glassware/Soft Glass
• It has high resistance to alkali.
• Its thermal resistance is less as compared to borosilicate glass.

Corex (Corning)
• lt is a special alumina-silicate glass that has been strengthened chemically than
thermally; six times stronger than borosilicate.

Vycor (Corning)
• It is utilized for high thermal, drastic heat shock, and extreme chemical treatment
with acids (except hydrofluoric) and dilute alkali; it can be heated to 900°C.

Flint Glass
• It is made up of soda-lime glass and a mixture of calcium, silicon, and sodium
oxides.
• It is used to make disposable glassware.
• It has poor thermal resistance; easy to melt.

PIPETTE CLASSIFICATION
I. CALIBRATION MARKS/DESIGN
A. To Deliver (TD) Pipette
• It delivers the exact amount it holds into a container; should not be blown out.
• It is designed to meet the requirements of Class A-type pipettes.
• It is designed to drain by gravity, and the desired volume is obtained when draining
stops
• Proper usage: Hold vertically with the tip against the side of the container and
should not touch the accumulating liquid.
• Examples: Serologic, Mohr, and volumetric transfer pipettes
B. To Contain (TC) Pipette
• It holds a particular volume but does not dispense the exact volume.
• It is also known as the rinse-out pipette.
• It does not meet Class A certification criteria.
• Examples: Sahli hemoglobin and Long-Levy pipettes

II. DRAINAGE CHARACTERISTICS

A. Blow-out Pipette
• It has continuous etched rings on top or near the mouthpiece of the pipette and
exact volume is obtained when the last drop is blown out.
• This type of pipette is not rinsed out.
• Examples: Ostwald-Folin and serologic pipette
B. Self-draining Pipette
 • It allows the liquid to drain by gravity.
• It does not have etched or frosted rings.

III. TYPES OF PIPETTES

A. Transfer Pipette
• Volumetric Pipette - for non-viscous fluid; self-draining; small amount left in the
tip should not be blown out
• Ostwald Folin - for viscous fluid; with etched ring
• Pasteur Pipette transfers fluids without consideration of a specific volume
• Automatic macro- or micropipettes
B. Graduated or Measuring Pipette
• Serological Pipette with graduations to the tip; blow-Out pipette
• Mohr Pipette without graduations to the tip; calibrated between 2 marks; self-
draining Pipette
• Bacteriologic Pipette
• Ball, Kolmer, and Kahn Pipette
• TC pipettes - Glass Micropipettes (<1 mL)
o Sahli-Hellige Pipette
o Lang-Levy Pipette
o RBC and WBC Pipettes
o Kirk and Overflow Pipettes

MECHANICAL/AUTOMATIC PIPETTES
Air Displacement Pipette
• It relies on piston for suction creation to draw the sample into a disposable tip.
• The piston does not come in contact with the liquid.
• A disposable, one-time use polypropylene tip is attached to the pipette barrel.

Positive Displacement Pipette

• It operates by moving the piston in the pipette tip or barrel, much like a
hypodermic syringe.
• It does not require a different tip for each use.
• It utilizes a Teflon-tipped plunger that fits tightly inside the capillary which is either
a siliconized glass or plastic.
• It is useful if a reagent reacts to plastics.

Dispenser/Dilutor Pipette
• It obtains liquid from a common reservoir and dispenses it repeatedly.
• It combines sampling and dispensing functions.

For Calibration of Pipettes and Glassware:

 • Class A pipettes do not require recalibration. To prepare a piece of glassware for
calibration, thoroughly wash and dry 1t using appropriate cleaning procedures.
• Distilled water is the calibrating medium for TD pipettes, while mercury is for TC
pipettes.
• Gravimetric and spectrometric methods verify the accuracy and precision of pipette
volumes.
• 0.1% phenol red solution in distilled water is utilized to compare the reproducibility
of brands of pipette tips.
• Micropipettes must be checked for accuracy and precision before they are used.
• Volumetric flasks for the preparation of standards and other solutions must meet
Class A specifications as defined by the National Institute of Standards and
Technology (NIST).
• Any glassware that fails to fulfill the Class A tolerance should be discarded.

Maintenance Check of Automatic Pipettes

• Air displacement pipette has a fixed stroke length that requires regular preventive
maintenance monitoring.
• Air displacement pipette has a seal to prevent air leakage into the pipette when
the piston is moved, and these seals require periodic greasing to maintain their
integrity.
• Positive displacement pipettes need to have their spring checked and the Teflon
tip replaced periodically.
• A slide wire is used to quickly check the plunger setting of positive displacement
pipettes.

Volume Measurements
• 1 lambda =1 microliter 0.001 mL)
• 1 microliter = 1.0 milligram

Plastic Materials for Laboratory Use:

1) Polycarbonate - tubes for centrifugation, graduated cylinders, and flasks
2) Polyethylene - test tubes, bottles, stoppers, disposable transfer pipettes,
volumetric flasks, and graduated tubes
3) Teflon - stirring bars, tubing, cryogenic vials, and bottle cap liners; almost
chemically inert; suitable for extreme temperature treatment (-70 °C to 200° C);
resistant to strong chemicals (acids, bases, alcohol, and hydrocarbons)

Notes to Remember:
• Plastic pipette tips are made primarily of polypropylene.
• Polypropylene plastic is chemically resistant and autoclavable.
 • A special type of polypropylene is utilized for cryogenic procedures and can
withstand temperatures down to -190
• As the fluid is allowed to drain into the receiving vessel, pipettes should be held in
a vertical position with the tips against the side of the receptacle.
• For volumetric TD pipette, it should not Ot be De shaken or hit against the wall o
of the container during draining because any disruption of the free-flowing liquid
may result in an inaccurate delivery of the liquid.
• Imperfect wetting or the presence of discreet droplets of water indicates that the
pipette is not sufficiently clean.
• Acid dichromate is a cleaning solution for glassware.
• When using mechanical pipettes, the forward mode is considered as the routine
and standard technique of pipetting
• The reverse mode can only be applied with air-displacement pipettes especially
when handing slightly viscous sample.
• Whenever an automatic pipette is used, an air cushion is possible, that is, the
amount of air between the lower part the pipette piston and surface level of the
sample.
• The NIST states that there is no fixed calibration interval for pipettes and
glassware,

Calibration of Analytical Balance and Thermometer

Analytical Balances
• Laboratory balance require calibration at regular intervals calibration intervals
should coincide with the requirements of the laboratory's licensing and accrediting
organizations.
• The new mass standards and test weight accuracy classes appropriate for
laboratory balances include the American Society for Testing and Materials (ASTM)
classes 1 and 2.
• NIST class 1 weights (extra-fine accuracy) up to 250 mg may be used for high-
precision (e.g, single-pan and electronic) balances that are precise to four decimal
places while the class 2 balances may be in excess of 1000 g.
• The operator must avoid direct contact with the weights by using clean gloves or
special lifting tools (e.g., forceps). Hand contact with the weights can cause
corrosion.

Thermometer
• Non-certified thermometers can be calibrated by using an NIST SRM934
thermometer or an NIST SRM 1968 gallium melting point cell.
• Temperature -monitoring devices should be verified for accuracy at 6- or 12-month
intervals.
• Types of thermometers:
 o Total immersion (freezers and refrigerators)
o Partial immersion (water baths and heating blocks)

Desiccators and Desiccant

• Many compounds combine with water molecules to form loose chemical crystals.
• Substances that take up water on exposure to atmospheric conditions are called
hygroscopic.
• Materials that are very hygroscopic can remove moisture from the air as well as
from other materials.
• Desiccants are most effective when placed in a closed, airtight chamber called a
desiccator.
• In the laboratory, desiccants are primarily used to prevent moisture absorption by
chemicals, gases, and instrument components.

Centrifuge
• Centrifugation is a process in which centrifugal force is used to separate solid
matter from a liquid suspension – carried out by centrifuge.
• It consists of a head or rotor, carriers, or shields.
• Always has a lid and an on/off switch and sometimes with built in tachometer.
• Centrifugal force depends on three variables: mass, speed, and radius.
• The speed is expressed in revolutions per minute (rpm), and the centrifugal force
generated is expressed in terms of relative centrifugal force (RCF) or gravities (g).
• The speed of the centrifuge is related to the RCF by the following equation:
• RCF = 1.118 x 10 - 5 x r x (rpm)2
• rpm (revolution per minute) refers to the speed of centrifugation and determined
by tachometer.
• Calibrator: Tachometer & Speedometer or Strobe light
• Calibrated: Monthly Basis: Every 3 mons.
• Disinfection: Weekly Basis: Every 2 weeks
• Cleaning: Daily Basis
• Types of Centrifuges:
o Horizontal-head Centrifuge (Swinging-bucket): Tubes are in
horizontal position when rotating. Produces a tightly packed, flat sediment
surface. Recommended for serum separator tubes.
o Angle-head Centrifuge: tubes are at fixed angle (25-40-degree angle)
when rotating. Capable of higher speeds. Produces slanted sediment
surface that is not tightly packed. Decantation is not recommended.
o Ultracentrifuge: High-speed, capable of 100,000 rpm. Refrigerated to
reduce heat.