Types of Laboratory grade water

Water quality is considered by many laboratorians to be one of the most important preanalytical variables that impacts laboratory testing. Many people believe water to be a laboratory reagent, and it’s used to make buffers, blanks, calibrators, controls, mobile phase, reaction mixtures, and reagent reconstitution for a variety of lab tests. As a result, using clean water is essential for reliable laboratory testing.

Every clinical laboratory strives to provide accurate findings. Many reagents, buffers, and diluents used in clinical laboratory testing use purified water. It may also be used to wash and clean instruments and laboratory ware, generate autoclave steam, etc., making it an indirect component of testing. Inadequate contaminant removal in purified water is a major source of laboratory inaccuracy.

Water Quality and Related Contaminants

Water may include a variety of contaminants, which might impede and influence clinical test findings. To eliminate these impurities, many purification methods are currently available; each technique has advantages and drawbacks. To generate different levels of purified water that meet the requirements set for each technique, combined purification technologies are essential.

Many contaminants can be found in water, causing tests to fail and resulting in incorrect findings. Many laboratory tests are affected by such contamination.

Water Contaminants and Interference with Different Laboratory Assays

ContaminantInterference with Laboratory Assays
Particles and colloidsUsually, most laboratory assays and methodologies affected.
Particles might clog the nebulizer, making it impossible to spray the specimen solution into the Atomic Absorption assay flame efficiently.
Increased system back-pressure due to damage to the HPLC pump and injectors.
Bacteria and their endotoxins, enzymes, and nucleasesEndotoxin, RNAses, DNAses, and proteases catalyze DNA and RNA hydrolysis, rendering them unstable in PCR, RT-PCR, and microarrays.
Endotoxin inhibits cell cultureA high bacterial count raises the level of calcium-binding proteins, which lowers the actual level.
Cross-reaction can occur in EIA due to bacterial alkaline phosphates.
Bacteria and their metabolites alter the pH of the medium, contaminate pure cultures, inhibit cell growth, and limit IVF success.
Gases such as nitrogen, oxygen, chlorine, or carbon dioxideForm bubbles that obstruct optical sensors and fluid lines, interfering with accurate particle counting and spectrophotometric readings.
Chlorine in water can bleach histopathology slides’ H&E staining.
Organic contaminantsInterfere with spectrophotometric testing, particularly in the case of UV absorbance.
Calcium is used by organic components, which reduces the amount of calcium in the specimen.Increased background in fluorescence assays and HPLC, skewed peak morphologies, and drifting or noisy baselines.
Inhibit cell development by deactivating enzymatic processes.
Organics can influence DNA microarray hybridization, cleaning, analysis, and fluorescence detection.
Inorganic ionsIons act as catalystsContamination by the metal ions under analysis would result in a greater estimate and an increase in the blank signal in Atomic Absorption.
Heavy metals including lead, mercury, and zinc are harmful to different types of cells in cell cultures.Some ions absorb ultraviolet light, such as nitrates.

According to the initial NCCLS standards, water should be classified based on bacterial presence, ions, organic compounds (hence, organics), pH, silica, and particles. A three-tiered method was used to classify water quality originally (types I, II and III). The Clinical Laboratory Reagent Water (CLRW) were released by the CLSI and can be utilized in any job that previously required type I and II waters.

Grades of purified water

The CLSI recommendations specify pure water requirements; evaluating water purity often involves constant measurement and monitoring of contaminants. As a result, the CLSI classifies different types of water according to specified and purity criteria, for the proper use in laboratory test techniques. ASTM International (previously the American Society for Testing and Materials [ASTM]) has released requirements for reagent grade water types I, II, III, and IV.

Aditionally, water was classified as type A, type B, or type C depending on endotoxin and bacterial concentration.

NCCLS Water-Types Classification and Its Applications

Water TypeAssay Methods
Type IUltrapure water applications such as HPLC, GC, MS, AA, molecular-biology applications, cell culture, and IVF
Type IIGeneral laboratory applications such as biochemical assays, microbiological culture, media preparation, buffers, and pH solutions
Type IIINoncritical laboratory work, such as rinsing glassware, filling water baths, and autoclaving; used in filling or feeding type I lab water treatment system

The 2006 CLSI Specification for Reagent Laboratory Water

Water TypeCLSI SpecificationsApplication
CLRW (Clinical Laboratory Reagent Water)Maximum microbial content (CFU/ml) <10
Free of particulates >0.22 µm
Organic materials <500 ppb (parts per Billions)  
Most Laboratory procedures; similar to types I and II
Defined as minimum quality for routine biochemistry assays
SRW (Special Reagent water)Defined by lab for procedures that need different specifications than CLRWImmunoassay, molecular assays, HPLC, LC-MS, GC, microarray, IVF
IFW (Instrument feed water)IVD measurement systems allows IVD instrument manufacturers to clarify specifications for their particular methods.Autoclave use and washing

ASTM Specifications for Reagent-Grade Water (Guideline D1193-6-2011)

ContaminantsParameter and Measurement UnitType IVType IIIType IIType I
IonsResistivity, minimum MΩ-cm (25°C)
Organic materialsTOC, max (µg/L)NS2005050
pHpH units5-8Not SpecifiedNot SpecifiedNot Specified
Chloride, maximum (µg/L) 501051
Sodium, maximum (µg/L) 501051
ColloidsSilica (µg/l)Not Specified50033

Classification for water quality based on bacterial and endotoxin content

ContaminantsParameters UnitType AType BType C
EndotoxinEndotoxin unit/mL<0.030.25Not Specified
BacteriaCFU/100 mL1101000

Methods of Water Purification

There are different types of water purification processes that can assist laboratories in producing high-quality purified water appropriate for laboratory testing processes. Despite the fact that there are various approaches, none of them are sufficient to meet the CLSI criteria. Many laboratories, on the other hand, combine these approaches to enhance the purification process and, as a result, create highly pure water.


Distillation is the process of vaporizing and condensing a liquid to purify or concentrate it, or to separate it from less volatile components. It is the earliest technique of purifying water. Carryover of volatile contaminants and entrapped water droplets that may contain impurities into the purified water are issues with distillation for producing reagent water. This causes volatiles, Na, potassium, manganese, carbonates, and sulfates to contaminate the distillate. As a result, water treated solely by distillation does not fulfill the type I water’s particular conductivity requirement.

Ion Exchange

Ion exchange is a method of removing ions from water to generate deionized water that is mineral-free. Commercial equipment, ranging in size from small, disposable cartridges to huge, resin-containing tanks, is the most convenient way to produce this type of water. Deionization is achieved by passing feed water through insoluble resin polymers that exchange hydrogen (H+) and oxygen hydrogen (OH) ions for the contaminants present in the ionized state of the water. Cation exchangers, anion exchangers, or a mixed-bed resin exchanger, which combines cation and anion exchange resins in a single container, may be found in the columns.

A single-bed deionizer can often produce water with a specific resistance of more than 1 MΩ/cm. Mixed-bed deionizers generate water with a specific resistance greater than 10 MΩ/cm when linked in series.

Reverse Osmosis

Reverse osmosis is a molecular filtering technique in which water is pushed through a semipermeable membrane. Organic molecules, bacteria, other particulate debris, and 90 % to 97% of all ionized and dissolved minerals are removed by the membrane, while gaseous contaminants are left behind. Although this approach is insufficient for generating laboratory reagent quality water, it can be used as a first purification procedure.

Illustration of Reverse Osmosis process

Ultraviolet photooxidation

Breaking the bonds between carbon, nitrogen, and hydrogen atoms at wavelengths of 185 nm and 254 nm disrupts the DNA of live bacteria. At these wavelengths, photooxidation is used as a germicidal and disinfection method for water. In addition, photooxidation of organic molecules lowers the total organic content (TOC) below 5 parts per billion. UV photo-oxidation, on the other hand, has the drawback of producing free radicals, which might enhance water conductivity.

Water-Purification Methods; Pros and Cons

DistillationSimple, removes many impurities such as bacteria, ions, dissolved gases and organic materialsHigh maintenance cost, low flow rates, and storage reservoir needed. Some contaminants such as silica and sodium are not removed.
Reverse OsmosisRemoves most types of contaminants. Minimal maintenance cost and easy to monitor operative parameters.Limited flow rate. RO membrane damaged by scaling, fouling or piercing
Ion exchangeSoftener technology reduces water hardness before RO processing. Remove ions effectively, easy to use and relatively inexpensiveRequires high feed-water quality to prevented plugging. Limited capacity if resin is occupied.
UltrafiltrationRemove small contaminants (20-30 kDa) that are not removed by filtration.Clogging possible with large contaminants.
UV-oxidationReduces organic contamination Limited energy neededCarbon dioxide produced may decrease water resistivity UV light does not affect ions or particles.

Storage of Lab Grade Water

Ions, gases, bacteria, endotoxins, silica, and particles escaping from containers, inner liners, plasticizers, and pipework may readily contaminate water while it is being stored. Ultrapure water needs the use of special glassware to maintain its purity.

Commercial bottled water does not meet the standards for type I water because contaminants are often introduced during storage. The environment and water containers may contribute to the formation of ionic and organic pollutants. Type I water should be stored in glassware designed specifically for this purpose by the manufacturers.

Type II water can be kept in a properly built storage reservoir and in glassware that fulfills the manufacturer’s standards for a short time.

Type III water is commonly used for glassware washing and rinsing, with no storage requirements. Type III water is nearly identical to filtered tap water in most laboratories. To avoid extra environmental pollution that could interfere with test methods, proper water storage is crucial.


  1. Clinical Laboratory Standard Institute (CLSI) GP40-A4-AMD
  2. Basic Clinical Laboratory Techniques, by Estridge BH
  3. Tietz Textbook of clinical chemistry and molecular diagnostics
  4. Lab Med Fall 2014, 45:e159-e165: Review of the impact of water quality on reliable laboratory testing and correlation with purification techniques.

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