VI. WATER QUALITY
Water is critical to all biological research labs. Water is used (a) in safety showers and eyewashes, (b) for washing lab-ware (i.e., glassware and plasticware), and, most importantly, (c) as the principal ingredient of chemical/biochemical solutions used in experimental work. Living systems consist of >90% water by weight, and most cell/tissue activities take place in a watery intracellular or extracellular environment. Water is thus the natural solvent for most biomolecules, and watery media constitute the natural environment for laboratory studies of living cells.
Most biological research laboratories are therefore concerned with having a constant supply of water that meets certain criteria for purity. Water taken directly from the tap ( = "tap water") is contaminated with a variety of impurities which vary in amount depending upon where you are. These contaminants fall into the following general categories: (1) particulates, (2) organics, (3) minerals (ionized salts), (4) gases, (5) bacteria, and (6) endotoxins or pyrogens (endotoxins: complex substances produced by bacteria, containing phospholipid, protein, and carbohydrate, that are poisonous to higher organisms; pyrogens: endotoxins that produce a fever in the affected organism). Tap water can be used for purpose (a) and for initial steps of (b) (in paragraph above), but final labware rinsing steps as well as the making of solutions for experimental use require specially purified water. This assures that the results of experiments are not due to random contaminants, making experiments repeatable. Therefore, methods have been developed to remove the various contaminants listed above.
A. "TYPES" OF WATER
In order to standardize water quality, several professional organizations have defined three "Types" of "reagent grade" water in terms of the levels of impurities. These organizations include The College of American Pathologists (CAP), the National Committee for Clinical Laboratory Standards (NCCLS), and the American Society for Testing and Materials (ASTM). The three types are "Type I", "Type II", and "Type III", with Type I being the purest, and Type III the least pure.
Water purification is achieved by applying several techniques, as there is no single technique that can remove all types of impurities well. Particulates (particles, large aggregates, precipitates) and bacteria are removable by filtration, using coarse and very fine filters. Organics (organic compounds) can be removed by carbon adsorption (using a carbon-filled cylinder or cartridge), distillation (using a still, which is a device for boiling water and recondensing the steam into water), and several other methods. Minerals (ionized salts) and gases can be removed by distillation, deionization (using ion exchange resin cylinders), and "reverse osmosis" (using pressure to force water through a semi-permeable membrane in the "reverse" direction, i.e., from a more concentrated solution of impurities (lower purity water) to a less concentrated solution (higher purity water). Endotoxins (or pyrogens) can also be removed by distillation, reverse osmosis, and a few other methods.
The level of ionized contaminants in water can be determined by measuring the "resistivity", i.e., the resistance of the water to passage of an electric current through it at a specified temperature. The purest water has a resistivity of 18.3 megohm-cm at 25oC. The resistivity specifications for Types I, II, and III reagent grade water are, respectively, 10, 2, and 0.1 megohm-cm, minimum. The resistivity of water decreases when ionized impurities are present.
"Conductivity" is the reciprocal of resistivity, i.e., it is a measure of the ability to conduct an electric current. The conductivity of water increases when ionized impurities are present. The "conductivity" specifications for Types I, II, and III reagent grade water are, respectively, 0.1, 0.5, and 10 micromhos, maximum. (One micromho = one millionth of a mho; 1 mho = reciprocal of 1 ohm, i.e. 1/ 1 ohm). Even the purest water exhibits slight conductivity, due to the fact that water ionizes to a very small but measurable degree, as described by the following equation:
H2O + H2O <--> H3O+ + OH-
All three organizations that have classified water Types specify Type I water as the safest to use experimentally. However, in practice, water of such high purity is difficult to have available in constant supply. Type I water can rapidly degrade during storage, due to contamination with CO2, other gases, and bacteria.
As described above, "resistivity" is used to measure water purity in terms of level of contamination with ions. "Resistivity" can be measured using a water purity meter, in units of
megohm-cm. The water available in the Techniques Facility includes: (a) tap water, and (b) deionized distilled water. Obtain a 400-500 ml sample of tap water in a 500 ml beaker. Use the Barnstead "Puromatic" water purity meter to measure its resistivity.
Instructions for use of the water purity meter:
1. Obtain the box labeled "Water Purity", located on same shelf as the bottle of Tech facility deionized distilled water, above sink.
2. Remove the water purity meter, set meter dial to "OFF" if not already off, then plug meter in.
3. Immerse the meter probe into the water. Meter should read zero (pointer needle to far left).
4. Turn dial to "CHK" (= check); meter should read approximately 10 on the black scale. This is just a test to see if meter is working properly.
5. There are three scales on the meter: black (BL), green (GR), and red (RD). These differ from each other in sensitivity by a factor of 10. Look at the middle of each scale. On the black scale, it reads 6-8 megohm-cm, on the green scale 0.6-0.8 megohm-cm, and on the red scale 0.06-0.08 megohm-cm.
6. Turn dial to "BL"; see if you can obtain a reading from the black scale. Wait until needle stops moving. If it is all the way to the left, turn to the green scale and try to obtain a reading. If not, continue to the red scale.
7. Record reading in Tech facility Lab Notebook.
8. Turn meter to "OFF". Pour off the tap water and replace with deionized distilled water obtained from the Tech facility supply bottle. Make (and record) a resistivity measurement on the deionized distilled water
9. Now, to the same beaker of deionized distilled water, add a little NaCl from the NaCl "salt shaker" located in same box as meter. Stir with a plastic stirrer (in same box). What is the effect on resistivity? Record reading.
10. Turn off the meter and rinse the probe by immersing several times in deionized distilled water.
11. Repeat steps 8 and 9, except this time add a little sucrose from the "sugar shaker" in the box. Stir - what is the effect of sugar on resistivity? Record reading.
12. Return meter and tightly capped "salt shaker" and "sugar shaker" to box, and return box to original place on shelf. Discard stirrers in red lab waste bag.
13. Compare the four measurements with each other and with the specifications for the 3 "Types" of water as stated above.
14. When finished, completely submerge the beaker that you used in the cleaning solution in the labware wash bucket in the sink. Then, proceed to the next section.
B. WASHING LABWARE
Laboratory glassware and plasticware must be washed using stringent conditions, so that there is no residue of either the original labware contents (before washing) or of the detergent solution or other wash medium used. Such residues could affect experimental work in unknown, unpredictable, and unrepeatable ways. Therefore, procedures have been developed for both machine and hand washing that involve soaking in cleaning solutions followed by extensive rinsing with tap water and then purified water. The final rinse is normally done with water of approximately the same purity used for making experimental solutions.
For all of your work in the Tech Facility, you will be expected to wash any labware that you have used (unless it is designated as disposable and you have been instructed to dispose of it). Thus, there is a Tech Facility labware washing procedure to be followed, posted near the lab sink. It involves soaking used labware immediately in a bucket containing dilute "Micro" cleaning solution, rinsing in warm tap water until soap is not visible in the rinse water, rinsing 10 times more in warm tap water, and 3 times more in deionized distilled water (the same water used to make solutions). The rinses do not require complete filling of labware, but rather exposure of the complete inner surface to the rinse water each time. The procedure is posted near the lab sink.
Studies have shown that removal of traces of detergent (or cleaning acid, if used) requires 7 - 10 water rinses. Thus, the Tech Facility procedure has an extra "margin of safety" built in. It may differ from that used in a research lab in which you ultimately work.
SOAKING LABWARE PROPERLY:
Instructions for proper soaking? How difficult can that be? Surprisingly, beginning researchers, as well as experienced researchers who are in a hurry, frequently soak labware improperly! The objective is to fill the interior of the vessel (beaker, flask, etc.) completely with the wash solution, or to expose the entire working surface of, say, a spatula, to the wash solution. In practice, this takes a bit of "doing"! Inspection of labware wash buckets frequently reveals partially submerged volumetric flasks, or centrifuge tubes and bottles floating bottom-up with a large air bubble inside. Even graduated cylinders and beakers can be subject to this disease. The result: unclean labware. This is particularly bad technique in a biosciences research lab that deals with proteins or other macromolecules that can adhere to glass or plastic. They may dry down onto the air-filled interior surface of the labware while it is "soaking"!
So, for proper soaking: (1) fill the labware item completely with the wash solution as you submerge it in the wash bucket. In addition, (2) check that you are not putting a heavy item on top of a small thin one already in the bucket, and (3) in any event, lower things gently into the bucket, so as not to break other labware within.
PROBLEMS THAT CAN ARISE DURING LABWARE RINSING:
1. Contamination of the distilled/deionized water delivery tube.
Occasionally, as you are rinsing labware, the tip of the plastic tubing that delivers distilled/deionized water touches an unclean or soapy surface, or possibly even dips into the detergent suds in the wash bucket. What to do?? Use the following lab trick: first, rinse the tip of the tubing thoroughly under running tap water, just like labware. Then, point the tip upward (forming a "U" shape), and turn on the flow slightly. The distilled/deionized water will flow out and over the edges of the tip, rinsing itself off.
2. Contamination of the labware drying surface with soapy water from the outside of the labware.
Suppose that you did a very good job rinsing the detergent off of the inside of a beaker, but ignored the outside completely. You then invert the beaker and place it in a drying basket. What happens? The soapy water on the outside can run down into the paper lining the drying basket, contaminating it and any beakers placed there in the future. Therefore, you must rinse the outside of the labware just as well as the inside, at least when doing the tap water rinses. It is, however, good practice to also rinse the outside of labware with distilled water.
3. Contamination because of incompletely rinsed magnetic stirring bars.
The best way to rinse a magnetic stirring bar is to place it into a small beaker (or flask), i.e., one bar per beaker. It is then rinsed at the same time as the beaker is rinsed. Caution: two magnetic stirring bars will bind tightly together if placed in the same beaker. The narrow region of contact does not get rinsed as well as the freely exposed surfaces. This is why the stirring bars should be rinsed individually.
4. Contamination from hands.
Imagine that you have just done a superb job washing a beaker, and are about to place it (inverted) into the drying basket. You peel off your gloves, and grab the beaker by pinching the edge between thumb and forefinger. What has happened? One of your fingers was pressed against the inside of the beaker. What was on that finger? Grease from lunch? Bacteria? Proteins? The point is, you probably won't know! When the beaker is subsequently filled, those unknown contaminants may ruin an experiment. Therefore, use common sense when washing labware; let nothing touch any surface that will be or could be in contact with experimental materials or solutions.
After washing and rinsing labware properly, it must be put in a clean place to dry. This can be a shelf or basket lined with clean absorbent paper, or a rack from which certain kinds of labware can be hung. Labware must be inverted so that dust/dirt from the air does not contaminate it during drying. The "peg-board" type vertical racks typically found near lab sinks are NOT to be used for drying labware UNLESS YOU ARE SPECIFICALLY INSTRUCTED OR GIVEN PERMISSION TO DO SO BY YOUR RESEARCH SPONSOR. The reasons for this are that (a) these peg boards are almost never washed, (b) they frequently are used for hanging up lab "odds and ends" that are not clean (like work gloves), (c) the older wooden ones may contain unknown contaminants deposited by others years ago, and (d) if too close to the sink, they can get splashed with cleaning solutions.
Look for the peg board in the Tech Facility, then examine it closely. What is hanging on it?? Are all of these items as clean as the inside of freshly washed labware would be?
DO NOT use the Tech Facility peg board for hanging up labware to dry. Instead, dry labware in the baskets on the table or cart near the sink, or, in the case of graduated cylinders and volumetric flasks, by hanging them from the wooden rack over the sink. The baskets are lined with absorbent paper, which is plastic coated on the underside, and absorbent on the exposed side. The lining of the baskets is replaced from a roll of this paper periodically, to keep it clean. Note that the rack has slots of different width to accommodate flasks and cylinders of different sizes.
PROBLEMS THAT CAN ARISE DURING LABWARE DRYING AND STORAGE:
1. Dirty bottoms vs. clean tops: Beakers, erlenmeyer flasks, and other types of labware are normally left to dry upside down. Only the open or "business" end of any container should be allowed to be in contact with the drying surface (e.g., beaker tops, cylinder tops, flask tops). After they are dry, their open end can be covered with Parafilm (or some other laboratory film, or plastic wrap) to keep them clean during storage on a lab shelf or in a cabinet. For beakers, the alternative is to store them upside down on a clean (lined) shelf or other surface. If this is done, other types of labware, such as flasks, should not be stored bottom-down on the same surface! The reason is that their bottoms are really not very clean due to handling and possible placement on bench tops. Dirty bottoms can contaminate the clean tops of beakers or other labware subsequently placed upside-down in the same spot.
2. Proper use of "the rack": You must be sure to use the rack slot of correct size, so that the hanging cylinder or flask cannot fall out. The cleanliness of the rack is not a critical consideration because only the bottoms of the flasks or cylinders touch it, not their "business end".
Wash the beaker that you used in the exercise on measuring resistivity. Before doing so, put on a pair of protective eyeware and rubber or plastic gloves, as the detergent solution can irritate eyes or skin. Then follow labware washing instructions posted above sink.
The Hunter College Department of Biological Sciences maintains a special labware washing facility known as the "Washeteria". It is located on the 8th floor of Hunter North, room 825. Many of our research laboratories use this facility for washing labware, and for certain other purposes including sterilization of media. The Washeteria contains several labware washing machines into which labware is put on special racks. It is then washed and rinsed automatically according to a sequence of instructions programmed into the machines. The final rinses are done with water that has been purified by removal of "organics" and by deionization and distillation.
EXERCISE #4 - "FIELD TRIP":
In this exercise, you are to venture forth from the cozy confines of the Tech Facility, going around the corner to the Washeteria. The supervisor's name and work hours are on the door. If open, look for him and explain that you are on your Tech Facility "field trip" and would appreciate it if he could tell you a little bit about the operation of the Washeteria. After this adventure, return to the Tech Facility and proceed with the next module.