Laboratory Tips

General
Burner Selection
Laboratory Conversion
Filter Paper Selection
Safe Temperature and Drying Times for Selected Materials
Syringe and Needle Cleaning

Glassware
Washing Glassware
Sterilizing Contaminated Glassware
Cleaning Volumetric Apparatus
Standard Taper
"To Contain", "To Deliver"

Environmental
Oil and Grease Determination by SPE

Water and Wastewater
Disposing of Bacteriological Tests
Collecting and Preserving Bacteriological Samples
Preparing Bacteriological Sample Containers

Challenging Application for pH
Tris, Sulfide and Proteins
Viscous Sample Common Problems: Difficulty Stirring, Sample Carryover and Electrode Breakage
Extreme pH or High Salt Content

pH Electrodes
Choosing the Right Measurement Technique
Maintenance and Storage of pH Electrodes
Long Term Storage of Electrodes
Electrodes Troubleshooting Checklist

Thermometers
Reuniting Separated Thermometer Mercury Columns
Computation of Emergent Stem Correction

Sterilizing Contaminated Glassware

Glassware which is contaminated with blood clots, such as serology tubes, culture media, petri dishes, etc. must be sterilized before cleaning. It can best be processed in the laboratory by placing it in a large bucket or boiler filled with water, to which 1-2 % soft soap or detergent has been added, and boiled for 30 minutes. The glassware can then be rinsed in tap water, scrubbed with detergent, and rinsed again.

You may autoclave glassware or sterilize in a large steam oven or similar apparatus. If viruses or spore-bearing bacteria are present autoclaving is absolutely necessary.

Standard Taper

Symbol used to designate interchangeable joints, stoppers and stopcocks that comply with the requirements of Commercial Standard CS-21 published by N.I.S.T.

Burner selection

While burners look similar, they are not interchangeable for different purposes. Different fuel gas molecules have different sizes. A specific volumnar flow of gas is necessary for proper operation. Each burner is stamped with the correct gas for operation. The BTU content of different fuel gases differs greatly. While Bunsen and Tirrill burners have similar flame characteristics, Tirrill burners have a much higher heat content.

Oil and Grease Determination by SPE

The Montréal Protocol on substances that deplete the ozone layer and the Clean Air Act of 1990 were established to control and eventually phase out the use of CFC's. Freon-113, otherwise known as 1,1,2 - trichloro - 1,2,2 - trifluoroethane, was included on this list. EPA Method 413.1 for Oil and Grease uses Freon-113 Liquid/Liquid Extraction and is soon to be replaced to comply with the above restrictions. The solvent determined by the EPA to provide analytical results most comparable to those achievable using Freon - 113 was n-hexane. A copy of the EPA Freon Replacement study can be obtained by contacting the National Center for Environmental Publications and Information at (513) 489-8190.

Laboratory Conversion

Filter Paper Size Selection

Standard 58° or 60° Funnel

Use this handy chart to select the appropriate size filter paper for your Funnel.

Safe Temperature and Drying Times for Selected Materials

*Total hours required are contingent on sample quantities and/or freeze dry system capabilities.

Disposing of Bacteriological Tests

Active bacterial cultures grown during incubation must be disposed of safely. This may be accomplished in one of two ways:

Bleach: Used test containers may be sterilized by using a 10% bleach solution. Add approximately 12 ml of bleach to each test container. Allow 10 to 15 minutes contact time with the bleach. Pour the liquid down the drain, and then dispose of the test containers in the normal waste.

Autoclave: Place used test containers in a contaminated-items bag or a biohazard bag and seal tightly. Test containers must be placed in a bag before autoclaving to prevent leakage into the autoclave. Autoclave used test containers in a bag at 121¡ C for 15 minutes at 15 pounds pressure. Once the test containers are sterile, they may be disposed of with the normal garbage. Place the bag of test containers in a separate garbage bag and tie tightly.

Collecting and Preserving Bacteriological Samples

Sampling should be properly carried out to ensure that seasonal variances are detected and that results are representative of the sample source. Collect a sufficient volume of sample for analysis. Standard Methods for the Examination of Water and Wastewater guidelines prescribe 100ml per sample. Sample contamination during collection must be avoided. No declorination is necessary if the sample is added directly to the medium on site. Otherwise, samples should be treated to destroy chlorine residual and transported for analysis immediately after collection. Sodium thiosulfate, which has been sterilized within the collection vessel, is generally used to destroy chlorine residual. Analyze as soon as possible after collection. The maximum time between collection and examination of samples should be 8 hours (maximum transit time 6 hours, maximum processing time 2 hours). If the time between collection and analysis will exceed 8 hours, maintain the sample at/or below 4¡C, but do not freeze. Maximum time between collection and analysis should not exceed 24 hours. Failure to properly collect and transport samples will cause inaccurate results. Collect at least 100ml of sample in pre-sterilized plastic bags or bottles, or in sterile glass or plastic bottles. Do not fill sample containers completely. Maintain at least 2.5cm of air space to allow adequate space for mixing the sample prior to analysis.

Handle the sample containers carefully. Open the sample containers just prior to collection, and close immediately following collection. Do not lay the lid or cap down and avoid contact near the mouths of the containers. Do not touch the inside of the containers. Do not rinse the containers.

Faucets, spigots, hydrants or pumps: Collect samples by allowing the water to run from a faucet, hydrant or pump at a moderate rate, without splashing, for two to three minutes before sampling. Do not adjust the rate of flow while the sample is being collected. Valves, spigots and faucets that swivel or lead, or those with attachments such as aerators and screens, should be avoided, or the attachments removed prior to the sample collection.

Rivers, lakes and reservoirs: When sampling a river, lake or reservoir, fill the sample container below the water surface. Do not sample near the edge or bank. Remove the cap, grasp the sample container near the bottom and plunge the container, mouth down, into the water. This technique excludes any surface scum. Fill the container by positioning the mouth into the current or, in non-flowing water, by tilting the bottle slightly and allowing it to fill slowly. Do not rinse.

Preparing Bacteriological Sample Containers

Care must be taken to prevent contamination when conducting bacterial tests. All materials used for containing or transferring samples must be sterile. Pre-sterilized plastic bags and bottles, or autoclavable glass and plastic bottles may be used to collect samples.

Pre-sterilized plastic bags and bottles Plastic bags and bottles are available with or without declorinating agent, commonly sodium thiosulfate. Use a declorinating agent when sampling potable or chlorinated water.

Autoclavable glass or plastic bottles: Glass or plastic bottles may be used instead of plastic bags. These containers should be prepared as follows:

1. Wash in hot water and detergent.

2. Thoroughly rinse with hot tap water, followed by a deionized water rinse to make sure that all detergent is removed.

3. If dechlorinating agent is needed, add agent to each container prior to autoclaving.

4. Steam sterilizes glass and autoclavable plastic containers at 121¡C for 15 minutes. Glass sample containers may be sterilized by hot air at 170¡C for one hour.

5. Store sterile containers tightly capped in a clean environment until needed.

Tris, Sulfide and Proteins

If an Ag/AgCl electrode is used, the junction may clog if the sample contains a species that complexes or precipitates silver. Tris, sulfide and proteins are common examples. An electrode containing a reference other than Ag/AgCl such as calomel, is recommended for these samples.

Maintenance and Storage of pH Electrodes

A regular maintenance schedule and proper storage of your pH electrode will maximize performance, help extend the life of your electrode and avoid the cost of frequent replacements.

Maintenance - On a weekly basis you should inspect your electrodes for scratches, cracks, salt crystal build-up and membrane/junction deposits. The reference chamber of refillable pH electrodes should be drained, flushed with fresh filling solution and refilled. To extend the life of your electrodes, pH Electrode Cleaning Kits are designed to clean deposits such as proteins, bacterial contaminants and oils and greases safely from your electrode.

Storage - Specially formulated pH Storage Solutions assist you in keeping your electrode in proper working order and avoid dilution of the electrode filling solution. To make storing your electrodes easier or to allow you to safely transport your electrodes in the field, use Electrode Storage Bottles.

Viscous Sample Common Problems: Difficulty stirring, sample carryover, Electrode breakage.

Difficulty stirring is a problem that cannot be avoided. If at all possible, stirring is recommended. However, if stirring is abandoned altogether, the pH buffers used for calibration should not be stirred. Note that the electrode response will be slower if the samples are not stirred.

Glass electrodes break easier in viscous samples. Using an epoxy bodied or rugged bulb electrode may lessen the occurrence of breakage.

Viscous samples "cling" to the electrode surface. Thorough rinsing with distilled water is recommended. Using a sleeve or Sure-Flow junction simplifies electrode cleaning and rinsing.

Extreme pH or High Salt Content

Samples of extreme pH or high salt content pose special problems for the reference portion of the electrode. Outside of normal conditions of a pH range of 2 to 12 or when the ionic strength of the sample is less than 1.0M, a liquid junction potential forms due to incompatibility of the sample and internal filling solutions. This causes drift and slow response. Using a reference or combination electrode with a double junction should alleviate any problems because the flow is more uniform.

For low-level ISE measurement, use an Erlenmeyer Flask to reduce atmospheric interferences

Choosing the Right Measurement Technique

Direct Measurement is a simple procedure for measuring a large number of samples. Only one-meter reading is required for each sample. Calibration is performed in a series of standards. The concentration of the samples is determined by comparison to the standards.

Low-Level Measurement is a similar method to Direct Measurement. This method is recommended when the expected sample concentration is within the non-linear response range of the electrode. A minimum three-point calibration is recommended to compensate for the electrode's non-linear response at these concentrations. Calibration is performed in one beaker, reducing the chance of cross contamination of the solutions.

Known Addition is a useful method for measuring samples, since calibration is not required. This method is recommended when measuring only a few samples, or when samples have a high (>0.1M) ionic strength, or a complicated background matrix. The electrodes are immersed in the sample solution and an aliquot of standard solution, containing the measured species, is added to the sample. The original sample concentration is determined from the change in potential before and after the addition. As in direct calibration, any convenient concentration unit can be used.

Analate Subtraction is also a useful method for measuring samples, since calibration is not required. The electrodes are immersed in a reagent solution that contains a species that the electrode senses, and that reacts with the sample. It is useful when sample size is small, for samples for which a stable standard is difficult to prepare, and for viscous or very concentrated samples. The method is not suited for very dilute samples. It is also necessary to know the stoichiometric ratio between standard and sample.

Titrations are quantitative analytical techniques for measuring the concentration of a species by incremental addition of a reagent (titration) that reacts with the sample species. Sensing electrodes can be used for determination of the titration end point. Ion selective electrodes are useful as end point detectors, because they are unaffected by sample color or turbidity. Titrations are approximately 10 times more precise than direct calibration, but are more time consuming.

Indicator Titration Methods are useful for measuring ionic species where an ion selective electrode does not exist. With these methods, the electrodes sense a reagent species that has been added to the sample before titration.

Long Term Storage of Electrodes

Ag/AgCl Reference Electrodes: Drain completely, rinse internally with distilled water and store dry with protective cap covering the junction.

Standard Line Ag/AgCl Electrodes: Fill the reference chamber with filling solution, and cover the fill hole. Put a few drops of storage solution in a storage bottle, or electrode protective cap, and cover the sensing element and reference junction.

Electrode Troubleshooting Checklist

The following lists the more common electrode symptoms and corrective actions.

Low slope:

• Clean and/or replace electrode(s).
• Condition electrode(s) properly.
• Use fresh buffers or standards and clean glassware. Prepare fresh standards using proper technique.
  
• Use recommended ionic strength adjuster for the selected ISE electrode.

High Slope: Clean and/or replace electrode(s).

• Use fresh buffers or standards and clean glassware. Prepare fresh standards using proper technique.
• Calibrate with buffers or standards at the same temperature.

Drift:

• Verify correct filling solution(s) and compatibility with the sample.
• Calibrate and measure at the same temperature. Use an ATC probe for pH calibrations and measurements.
• Clean and/or replace electrode(s).
• Refer to the sample requirements and interferences sections in instruction manuals.

Erratic Response:

• Verify correct filling solution(s) and compatibility with the sample.
• Use recommended ionic strength adjuster for the selected ISE electrode.
• Clean and/or replace electrode(s).
• Perform meter checkout. Verify proper grounding and power.
  

Off-Scale and Over-Range Reading:

• Verify electrode connection. Immerse electrode(s) in solution.
• Remove air bubbles by gently tapping the side of the electrode.
• Clean and/or replace electrode(s).
• Condition electrode(s) properly.

"Wrong Answer":

• Verify correct filling solution(s) and compatibility with the sample.
• Use fresh buffers or standards and clean glassware.
• Prepare fresh standards using proper technique.
• Refer to the sample requirements and interferences sections in instruction manuals.

"To Contain," "To Deliver"

Blow Out," "To Deliver" - What's the Difference?

A "To Contain" pipet holds the exact volume of liquid specified. A "To Deliver/Blow Out" pipet must be allowed to drain, then the drop that remains in the tip must be blown out and added to the original delivery to equal the exact volume. A "To Deliver" pipet must be held vertically with the tip against the side of the receiving vessel and allowed to drain completely. The stated volume is obtained when the draining stops. The "Blow-Out" technique should not be used with this type of pipet.

Cleaning Volumetric Apparatus

Good laboratory technique demands clean glassware to eliminate error. In all instances, glassware must be physically clean; it must be chemically clean; and in many cases, it must be bacteriological clean.

Certain contaminants, especially grease, adhering to the walls prevent them from being uniformly wetted, and there is a tendency for water to collect into drops. Imperfect wetting causes irregularities in capacity of volumetric glassware by distorting the meniscus, and also by affecting the volume of the residue adhering to the walls after emptying glassware calibrated to deliver the indicated volume.

Glassware should be cleaned as soon as possible after use to avoid setting and caking of residues. Pipets may be placed in a convenient jar containing a weak antiseptic solution, immediately after use. Commercial cleaners (see Cleaners, page 265) can be used to remove surface contaminants such as silicone and other organic and biological residues.

Rinsing is an important step in the cleaning process. Some cleaning materials used may leave trace residue unless the rinsing process is carried out thoroughly. When glassware is to be calibrated, the final rinsing must be with distilled water.

Glassware marked "to contain" should be dried after cleaning. ACS Grade ethyl alcohol or acetone may be used. Drying may be hastened by blowing clean, dry air into the vessel.

Syringe and Needle Cleaning

The life of your syringe is directly related to its cleanliness.

It is best to use solvents known to be effective in solvating the sample and which are non-alkaline, non-phosphate and non-detergent based.

Adhesives used to cement needles and other terminations are the most chemically resistant adhesives available in today's technology. However, use of some solvents may deteriorate the adhesives resulting in frozen plungers and plugged needles. Rinse the syringe thoroughly after use with deionized water, acetone or another solvent compatible with the sample. To clean the plunger, remove it from the syringe barrel and gently wipe with a lint-free tissue. Reinsert the plunger into the barrel and pump deionized water or acetone through the needle and syringe. When reinserting a Teflon-tipped plunger into a syringe barrel, wet (lubricate) the tip with deionized water or another solvent compatible with the sample.

Washing Glassware

When washing, soap, detergent, or cleaning powder (with or without an abrasive) may be used such as alconox. The water should be hot. For glassware that is exceptionally dirty, a cleaning powder with a mild abrasive will give more satisfactory results.

The abrasive should not scratch the glass. During the washing, all parts of the glassware should be thoroughly scrubbed with a brush. This means that a full set of brushes must be at hand - brushes to fit large and small test tubes, burets, funnels, graduates and various sizes of flasks and bottles. Motor driven revolving brushes are valuable when a large number of tubes or bottles are processed.

Do not use cleaning brushes that are so worn that the spine hits the glass. Serious scratches may result. Scratched glass is more prone to break during experiments. Any mark in the uniform surface of the glassware is a potential breaking point, especially when the piece is heated. Do not allow acid to come in contact with a piece of glassware before detergent (or soap) is thoroughly removed. If this happens, a film of grease may be formed.

Reuniting Separated Thermometer Mercury Columns

The largest single cause for failure of precision thermometers in the lab is due to separated mercury columns occurring in transit or in the lab. The life of the instrument can be greatly extended if the following procedures are rigidly employed. Other methods may damage the thermometer.

Immersion Types

Liquid-in-glass thermometers are manufactured to be used in one of three ways: partial immersion, total immersion and complete immersion. It is essential to understand these terms in order to obtain true temperatures. Immersion definitions are listed below.

Partial Immersion: Thermometer is designed to indicate temperature correctly when the bulb and a portion of the stem are immersed to the immersion line on the thermometer stem.

Total Immersion. Thermometer is designed to indicate temperature correctly when the bulb and the entire liquid column (not the entire thermometer) are exposed to the medium being measured.

Complete Immersion. Thermometer is designed to indicate temperature correctly when the entire thermometer is exposed to the medium being measured.

COOLING METHOD

With thermometer in an upright position, gradually immerse ONLY the bulb in a solution of solid CO2 (dry ice) and alcohol so the mercury column retreats slowly into the bulb. Do not cool the stem or mercury column. Keep the bulb in the solution until the main column, as well as the separated portion, retreats into bulb. Remove and swing thermometer in short arc forcing all the mercury into the bulb. Most mercury thermometers can be reunited using this method regardless of range (with the exception of deep immersion thermometers) provided ONLY THE BULB is immersed in the CO2.

Cautions

1. Do not touch the bulb until it has warmed sufficiently for the mercury to emerge from the bulb into the capillary.

2. Never subject the stem or mercury column to the CO2 solution as it will freeze the mercury column in the capillary and may cause the bulb to fracture.

HEATING METHOD

This method applies to thermometers with a maximum range of 260¡C or 500¡F, equipped with expansion chambers large enough to accommodate the separation plus a portion of the main column. Immerse as much of bulb AND STEM as possible in a large beaker containing a liquid whose flash point is well above the highest indication of the thermometer being reunited. Heat the beaker stirring the liquid with the thermometer until the separation and a portion of the main column enter the chamber. Tap thermometer in palm of gloved hand reuniting mercury. Allow to cool slowly.

Cautions

1. Never use open flame on bulb.

2. Never fill expansion chamber more than 2/3 full.

3. Make certain flash point of liquid is well above highest temperature indicated on the thermometer.

Computation of Emergent Stem Correction

When total immersion thermometers are used only partially immersed, a stem correction must be calculated and applied to the reading for precision results. To compute an emergent stem correction, the following variables must be defined:

T = the reading of the thermometer in situ

N = the number of degrees on the thermometer scale between the liquid surface and the top of the mercury column.

A= the average temperature of the emergent mercury column. To find value A, suspend alongside the subject thermometer a secondary or auxiliary thermometer with its bulb centered between the liquid level and the temperature indicated on the subject thermometer. The temperature indicated on this auxiliary thermometer will be value A.

Find the stem correction (SC) by computation from the following formula:

SC = 0.00016 x (N x (T-A)) for Celsius temperatures, or

SC = 0.00009 x (N x (T-A)) for Fahrenheit temperatures.