General lab safety rules

The following are rules that relate to almost every laboratory and should be included in most safety
policies. They cover what you should know in the event of an emergency, proper signage, safety
equipment, safely using laboratory equipment, and basic common-sense rules.

1. Be sure to read all fire alarm and safety signs and follow the instructions in the event of an
accident or emergency.

2. Ensure you are fully aware of your facility's/building's evacuation procedures.

3. Make sure you know where your lab's safety equipment—including first aid kit(s), fire
extinguishers, eye wash stations, and safety showers—is located and how to properly use
it.

4. Know emergency phone numbers to use to call for help in case of an emergency.

5. Lab areas containing carcinogens, radioisotopes, biohazards, and lasers should be properly
marked with the appropriate warning signs.

6. Open flames should never be used in the laboratory unless you have permission from a
qualified supervisor.

7. Make sure you are aware of where your lab's exits and fire alarms are located.

8. An area of 36" diameter must be kept clear at all times around all fire sprinkler heads.

9. If there is a fire drill, be sure to turn off all electrical equipment and close all containers.

10. Always work in properly-ventilated areas.

11. Do not chew gum, drink, or eat while working in the lab.

12. Laboratory glassware should never be utilized as food or beverage containers.

13. Each time you use glassware, be sure to check it for chips and cracks. Notify your lab
supervisor of any damaged glassware so it can be properly disposed of.
14. Never use lab equipment that you are not approved or trained by your supervisor to
operate.

15. If an instrument or piece of equipment fails during use, or isn't operating properly, report
the issue to a technician right away. Never try to repair an equipment problem on your own.

16. If you are the last person to leave the lab, make sure to lock all the doors and turn off all
ignition sources.

17. Do not work alone in the lab.

18. Never leave an ongoing experiment unattended.

19. Never lift any glassware, solutions, or other types of apparatus above eye level.

20. Never smell or taste chemicals.

21. Do not pipette by mouth.

22. Make sure you always follow the proper procedures for disposing lab waste.

23. Report all injuries, accidents, and broken equipment or glass right away, even if the
incident seems small or unimportant.

24. If you have been injured, yell out immediately and as loud as you can to ensure you get
help.

25. In the event of a chemical splashing into your eye(s) or on your skin, immediately flush
the affected area(s) with running water for at least 20 minutes.

26. If you notice any unsafe conditions in the lab, let your supervisor know as soon as possible.
How to Clean Glass Apparatus:

Cleaning laboratory glassware isn't as simple as washing the dishes. Here's how to wash your
glassware so you won't ruin your chemical solution or laboratory experiment.

The Basics

It's generally easier to clean glassware if you do it right away. When a detergent is used, it's usually
one designed for lab glassware, such as Liquinox or Alconox. These detergents are preferable to
any dishwashing detergent that might be used on dishes at home.

Usually, detergent and tap water are neither required nor desirable. You can rinse the glassware
with the proper solvent, then finish up with a couple of rinses with distilled water, followed by
final rinses with deionized water.

Washing out Common Chemicals

 Water Soluble Solutions (e.g., sodium chloride or sucrose solutions): Rinse 3-4 times with
deionized water, then put the glassware away.
 Water Insoluble Solutions (e.g., solutions in hexane or chloroform): Rinse 2-3 times with ethanol
or acetone, rinse 3-4 times with deionized water, then put the glassware away. In some situations,
other solvents need to be used for the initial rinse.
 Strong Acids (e.g., concentrated HCl or H2SO4): Under the fume hood, carefully rinse the glassware
with copious volumes of tap water. Rinse 3-4 times with deionized water, then put the glassware
away.
 Strong Bases (e.g., 6M NaOH or concentrated NH4OH): Under the fume hood, carefully rinse the
glassware with copious volumes of tap water. Rinse 3-4 times with deionized water, then put the
glassware away.
 Weak Acids (e.g., acetic acid solutions or dilutions of strong acids such as 0.1M or 1M HCl or
H2SO4): Rinse 3-4 times with deionized water before putting the glassware away.
 Weak Bases (e.g., 0.1M and 1M NaOH and NH4OH): Rinse thoroughly with tap water to remove
the base, then rinse 3-4 times with deionized water before putting the glassware away.

Washing Special Glassware

Rinse the glassware with the appropriate solvent. Use deionized water for water-soluble contents.
Use ethanol for ethanol-soluble contents, followed by rinses in deionized water. Rinse with other
solvents as needed, followed by ethanol, and, finally, deionized water. If the glassware requires
scrubbing, scrub with a brush using hot soapy water, rinse thoroughly with tap water, followed by
rinses with deionized water.

Burets

Wash with hot soapy water, rinse thoroughly with tap water, then rinse 3-4 times with deionized
water. Be sure the final rinses sheet off of the glass. Burets need to be thoroughly clean to be used
for quantitative labwork.

Pipets and Volumetric Flasks

In some cases, you may need to soak the glassware overnight in soapy water. Clean pipets and
volumetric flasks using warm soapy water. The glassware may require scrubbing with a brush.
Rinse with tap water followed by 3-4 rinses with deionized water.
Solution:

A solution is a homogenous mixture of two or more than two chemical substances having uniform
composition throughout. For example, a solution of orange juice.

A solution usually has two parts

Solvent: The substance which has the capacity to dissolve other chemical substances (solutes)

Solutes: A solute is a substance which has the property to dissolve in solvent and usually in smaller
quantity. For examples a solution of sugar containing water as a solvent and sugar is a solute.

Binary solution:

The solution which contains one solute and one solvent is called binary solution. For example
sucrose dissolves in water.

Multi solute Solution:

The solution which contains more than one solutes and one solvent is called multi solution. For
example Pepsi and 7 up drinks.

Concentrated solution:

The solution in which solvent is in small quantity as compared to the solute. For example rectified
spirit which contains 95% ethanol in which solute (ethanol) is in larger amount as compared to
distilled water acts as a solvent.

Dilute solution:

The solution in which solute is in small quantity as compared to the solvent. For example 5%
CuSO4 solution which contains 5g of CuSO4 solute which is in smaller amount as compared to
distilled water which acts as a solvent.
Use of the Analytical Balance

Preparing the balance for use

Before weighing anything on the analytical balance you must make sure that it is leveled and
zeroed.

To check the leveling on the balance, look at the leveling bubble on the floor of the weighing
chamber. If it is not centered, center it by turning the leveling screws on the bottom toward the
back of the balance.

Once the balance is leveled, close all the chamber doors and press the control bar on the front of
the balance. After a few seconds, a row of zeros will appear. This indicates that the balance is
zeroed and ready for use.

Weighing a liquid, powder, or granular substance

These substances must always be weighed using an appropriate weighing container.

Place the weighing container on the balance pan and close the doors.

Tare the container by briefly pressing the control bar. The readout will read zero with the
container sitting on the pan. This allows the mass of your sample to be read directly.

Add the substance to be weighed. Be careful not to spill chemicals on the balance. If need be,
you can remove the container from the weighing chamber while you add the sample provided
that noone presses the control bar before you weigh your sample.

With the sample and its container sitting on the pan, close the chamber doors and read the
display to find the mass of your sample.

Weighing a solid object directly on the balance

If the object you need to weigh is a solid object, you can weigh it directly on the pan. Be sure the
balance is zeroed. Open the chamber doors, carefully place the object on the balance pan, close
the doors, and read the mass of your object.
Cleaning up and shutting down the balance

When you are done with the balance, make sure you have properly cleaned up any chemicals that
may have spilled on the balance. At the end of the day, the balance can be turned off by lifting up
gently on the control bar.
Microscopy:

Microscopy is the technical field of using microscopes to view objects and areas of objects that
cannot be seen with the naked eye (objects that are not within the resolution range of the normal
eye). There are three well-known branches of microscopy: optical, electron, and scanning probe
microscopy, along with the emerging field of X-ray microscopy.

Optical microscopy and electron microscopy involve the diffraction, reflection, or refraction of
electromagnetic radiation/electron beams interacting with the specimen, and the collection of the
scattered radiation or another signal in order to create an image. This process may be carried out
by wide-field irradiation of the sample (for example standard light microscopy and transmission
electron microscopy) or by scanning a fine beam over the sample (for example confocal laser
scanning microscopy and scanning electron microscopy). Scanning probe microscopy involves the
interaction of a scanning probe with the surface of the object of interest. The development of
microscopy revolutionized biology, gave rise to the field of histology and so remains an essential
technique in the life and physical sciences. X-ray microscopy is three-dimensional and non-
destructive, allowing for repeated imaging of the same sample for in situ or 4D studies, and
providing the ability to "see inside" the sample being studied before sacrificing it to higher
resolution techniques. A 3D X-ray microscope uses the technique of computed tomography
(microCT), rotating the sample 360 degrees and reconstructing the images. CT is typically carried
out with a flat panel display. A 3D X-ray microscope employs a range of objectives, e.g., from 4X
to 40X, and can also include a flat panel.

Two parameters are especially important in microscopy: magnification and resolution.

Magnification is a measure of how much larger a microscope (or set of lenses within a
microscope) causes an object to appear. For instance, the light microscopes typically used in high
schools and colleges magnify up to about 400 times actual size. So, something that was 1 mm wide
in real life would be 400 mm wide in the microscope image.
The resolution of a microscope or lens is the smallest distance by which two points can be
separated and still be distinguished as separate objects. The smaller this value, the higher the
resolving power of the microscope and the better the clarity and detail of the image. If two bacterial
cells were very close together on a slide, they might look like a single, blurry dot on a microscope
with low resolving power, but could be told apart as separate on a microscope with high resolving
power.
Spectrophotometer:

Spectrophotometry is a tool that hinges on the quantitative analysis of molecules depending on

how much light is absorbed by colored compounds. Spectrophotometry uses photometers, known
as spectrophotometers that can measure a light beam's intensity as a function of its color
(wavelength). Important features of spectrophotometers are spectral bandwidth (the range of colors
it can transmit through the test sample), the percentage of sample-transmission, the logarithmic
range of sample-absorption, and sometimes a percentage of reflectance measurement.

A spectrophotometer is commonly used for the measurement of transmittance or reflectance of

solutions, transparent or opaque solids, such as polished glass, or gases. Although many
biochemicals are colored, as in, they absorb visible light and therefore can be measured by
colorimetric procedures, even colorless biochemicals can often be converted to colored
compounds suitable for chromogenic color-forming reactions to yield compounds suitable for
colorimetric analysis. However, they can also be designed to measure the diffusivity on any of the
listed light ranges that usually cover around 200 nm - 2500 nm using different controls and
calibrations. Within these ranges of light, calibrations are needed on the machine using standards
that vary in type depending on the wavelength of the photometric determination.

An example of an experiment in which spectrophotometry is used is the determination of the

equilibrium constant of a solution. A certain chemical reaction within a solution may occur in a
forward and reverse direction, where reactants form products and products break down into
reactants. At some point, this chemical reaction will reach a point of balance called an equilibrium
point. In order to determine the respective concentrations of reactants and products at this point,
the light transmittance of the solution can be tested using spectrophotometry. The amount of light
that passes through the solution is indicative of the concentration of certain chemicals that do not
allow light to pass through.

The absorption of light is due to the interaction of light with the electronic and vibrational modes
of molecules. Each type of molecule has an individual set of energy levels associated with the
makeup of its chemical bonds and nuclei, and thus will absorb light of specific wavelengths, or
energies, resulting in unique spectral properties. This is based upon its specific and distinct
makeup.
The use of spectrophotometers spans various scientific fields, such as physics, materials science,
chemistry, biochemistry, Chemical Engineering, and molecular biology. They are widely used in
many industries including semiconductors, laser and optical manufacturing, printing and forensic
examination, as well in laboratories for the study of chemical substances. Spectrophotometry is
often used in measurements of enzyme activities, determinations of protein concentrations,
determinations of enzymatic kinetic constants, and measurements of ligand binding reactions.
Ultimately, a spectrophotometer is able to determine, depending on the control or calibration, what
substances are present in a target and exactly how much through calculations of observed
wavelengths.

In astronomy, the term spectrophotometry refers to the measurement of the spectrum of a celestial
object in which the flux scale of the spectrum is calibrated as a function of wavelength, usually by
comparison with an observation of a spectrophotometric standard star, and corrected for the
absorption of light by the Earth's atmosphere.
UV Spectrophotometer:

Ultraviolet spectroscopy refers to absorption spectroscopy or reflectance spectroscopy in part of

the ultraviolet and the full, adjacent visible spectral regions. This means it uses light in the visible
and adjacent ranges. The absorption or reflectance in the visible range directly affects the perceived
color of the chemicals involved. In this region of the electromagnetic spectrum, atoms and
molecules undergo electronic transitions. Absorption spectroscopy is complementary to
fluorescence spectroscopy, in that fluorescence deals with transitions from the excited state to the
ground state, while absorption measures transitions from the ground state to the excited state.

Principle of ultraviolet absorption

Molecules containing bonding and non-bonding electrons (n-electrons) can absorb energy in the
form of ultraviolet or visible light to excite these electrons to higher anti-bonding molecular
orbitals.[2] The more easily excited the electrons (i.e. lower energy gap between the HOMO and
the LUMO), the longer the wavelength of light it can absorb. There are four possible types of
transitions (π–π*, n–π*, σ–σ*, and n–σ*), and they can be ordered as follows: σ–σ* > n–σ* > π–
π* > n–π*.
Atomic Absorption Spectrophotometer:

Atomic absorption spectroscopy (AAS) and atomic emission spectroscopy (AES) is a

spectroanalytical procedure for the quantitative determination of chemical elements using the
absorption of optical radiation (light) by free atoms in the gaseous state. Atomic absorption
spectroscopy is based on absorption of light by free metallic ions.

In analytical chemistry the technique is used for determining the concentration of a particular
element (the analyte) in a sample to be analyzed. AAS can be used to determine over 70 different
elements in solution, or directly in solid samples via electrothermal vaporization, and is used in
pharmacology, biophysics, archaeology and toxicology research.

Atomic emission spectroscopy was first used as an analytical technique, and the underlying
principles were established in the second half of the 19th century by Robert Wilhelm Bunsen and
Gustav Robert Kirchhoff, both professors at the University of Heidelberg, Germany.

The modern form of AAS was largely developed during the 1950s by a team of Australian
chemists. They were led by Sir Alan Walsh at the Commonwealth Scientific and Industrial
Research Organisation (CSIRO), Division of Chemical Physics, in Melbourne, Australia.

Atomic absorption spectrometry has many uses in different areas of chemistry such as clinical
analysis of metals in biological fluids and tissues such as whole blood, plasma, urine, saliva, brain
tissue, liver, hair, muscle tissue, semen, in some pharmaceutical manufacturing processes, minute
quantities of a catalyst that remain in the final drug product, and analyzing water for its metal
content.

Principle:

The technique makes use of the atomic absorption spectrum of a sample in order to assess the
concentration of specific analytes within it. It requires standards with known analyte content to
establish the relation between the measured absorbance and the analyte concentration and relies
therefore on the Beer-Lambert Law.
Standard Curve and Graphing Former:

A spectrophotometer measures light quantity. It tells you how much light is passing through
a solution (transmittance) or how much light is being absorbed by a solution (absorbance).

If you graph absorbance versus concentration for a series of known solutions, the line, or
standard curve, which fits to your points can be used to figure out the concentrations of an
unknown solution. Absorbance, the dependent variable, is placed on the y-axis (the vertical
axis). Concentration, the independent variable (because it was set by you when setting up
the experiment), is graphed on the x-axis. When you measure the absorbance of an unknown
sample, find that y-value on the standard curve. Then trace downward to see which
concentration matches up to it. Mouse over the graph below to see an example of this.

Below is a standard curve generated from absorbance data similar to what we generated in
class. Notice that as concentration increases, absorbance increases as well. While you can
estimate concentration of an unknown from just looking at the graph, a more accurate way
to determine concentration to actually use the equation of the line which fits to your data
points. This equation is given in the y-intercept form: y=mx+b
where m is the slope of the line and b is the y-intercept (where the line touches the y-axis).

The equation y=mx+b can be translated here as "absorbance equals slope times
concentration plus the y-intercept absorbance value." The slope and the y-intercept are
provided to you when the computer fits a line to your standard curve data. The absorbance
(or y) is what you measure from your unknown. So, all you have to do is pop those three
numbers into the equation and solve for x (concentration).

An example: your unknown's absorbance (y) is 6.00

Based on the curve below, slope (m) = 8 and b=0
if y=mx+b then 6.00 = 8*x + 0
and 6/8=x
and 0.75=x
so the concentration of the unknown would be 75% the original stock, which was 100
ug/ml. 75% of 100 ug/ml = 75 ug/ml concentration.
The units on the graph below are absorbance (y) versus the dilution factor (x) of each
solution used (0=water to 1=undiluted stock).
 Chromatography:
Chromatography is a laboratory technique for the separation of a mixture. The mixture is
dissolved in a fluid called the mobile phase, which carries it through a structure holding
another material called the stationary phase. The various constituents of the mixture travel
at different speeds, causing them to separate. The separation is based on differential
partitioning between the mobile and stationary phases. Subtle differences in a compound's
partition coefficient result in differential retention on the stationary phase and thus affect the
separation.
Chromatography may be preparative or analytical. The purpose of preparative
chromatography is to separate the components of a mixture for later use, and is thus a form
of purification. Analytical chromatography is done normally with smaller amounts of
material and is for establishing the presence or measuring the relative proportions of analytes
in a mixture. The two are not mutually exclusive.

Techniques by chromatographic bed shape

Column chromatography

Column chromatography is a separation technique in which the stationary bed is within a tube.
The particles of the solid stationary phase or the support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube
(open tubular column). Differences in rates of movement through the medium are calculated to
different retention times of the sample.

Planar chromatography

Planar chromatography is a separation technique in which the stationary phase is present as or on

a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary
bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate
(thin-layer chromatography). Different compounds in the sample mixture travel different distances
according to how strongly they interact with the stationary phase as compared to the mobile phase.
The specific Retention factor (Rf) of each chemical can be used to aid in the identification of an
unknown substance.
Paper chromatography

Paper chromatography is a technique that involves placing a small dot or line of sample solution
onto a strip of chromatography paper. The paper is placed in a container with a shallow layer of
solvent and sealed. As the solvent rises through the paper, it meets the sample mixture, which starts
to travel up the paper with the solvent. This paper is made of cellulose, a polar substance, and the
compounds within the mixture travel farther if they are non-polar. More polar substances bond
with the cellulose paper more quickly, and therefore do not travel as far.

Thin-layer chromatography (TLC)

Thin-layer chromatography (TLC) is a widely employed laboratory technique used to separate

different biochemicals on the basis of their relative attractions to the stationary and mobile phases.
It is similar to paper chromatography. However, instead of using a stationary phase of paper, it
involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a
flat, inert substrate. TLC is very versatile; multiple samples can be separated simultaneously on
the same layer, making it very useful for screening applications such as testing drug levels and
water purity. Possibility of cross-contamination is low since each separation is performed on a new
layer. Compared to paper, it has the advantage of faster runs, better separations, better quantitative
analysis, and the choice between different adsorbents. For even better resolution and faster
separation that utilizes less solvent, high-performance TLC can be used. An older popular use had
been to differentiate chromosomes by observing distance in gel (separation of was a separate step).

Techniques by physical state of mobile phase

Gas chromatography

Gas chromatography (GC), also sometimes known as gas-liquid chromatography, (GLC), is a

separation technique in which the mobile phase is a gas. Gas chromatographic separation is always
carried out in a column, which is typically "packed" or "capillary". Packed columns are the routine
work horses of gas chromatography, being cheaper and easier to use and often giving adequate
performance. Capillary columns generally give far superior resolution and although more
expensive are becoming widely used, especially for complex mixtures. Both types of column are
made from non-adsorbent and chemically inert materials. Stainless steel and glass are the usual
materials for packed columns and quartz or fused silica for capillary columns.
Gas chromatography is based on a partition equilibrium of analyte between a solid or viscous
liquid stationary phase (often a liquid silicone-based material) and a mobile gas (most often
helium). The stationary phase is adhered to the inside of a small-diameter (commonly 0.53 –
0.18mm inside diameter) glass or fused-silica tube (a capillary column) or a solid matrix inside a
larger metal tube (a packed column). It is widely used in analytical chemistry; though the high
temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins
(heat denatures them), frequently encountered in biochemistry, it is well suited for use in the
petrochemical, environmental monitoring and remediation, and industrial chemical fields. It is also
used extensively in chemistry research.

Liquid chromatography

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. It can
be carried out either in a column or a plane. Present day liquid chromatography that generally
utilizes very small packing particles and a relatively high pressure is referred to as high-
performance liquid chromatography (HPLC).

In HPLC the sample is forced by a liquid at high pressure (the mobile phase) through a column
that is packed with a stationary phase composed of irregularly or spherically shaped particles, a
porous monolithic layer, or a porous membrane. HPLC is historically divided into two different
sub-classes based on the polarity of the mobile and stationary phases. Methods in which the
stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase, silica as
the stationary phase) are termed normal phase liquid chromatography (NPLC) and the opposite
(e.g., water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase)
is termed reversed phase liquid chromatography (RPLC).
Centrifugation:

A centrifuge is a device for separating particles from a solution according to their size, shape,
density, viscosity of the medium and rotor speed.

In a solution, particles whose density is higher than that of the solvent sink (sediment), and particles
that are lighter than it float to the top. The greater the difference in density, the faster they move.
If there is no difference in density (isopycnic conditions), the particles stay steady.

To take advantage of even tiny differences in density to sepa-rate various particles in a solution,
gravity can be replaced with the much more powerful “centrifu­gal force” provided by a centrifuge.
This technique plays crucial role in biochemistry or biotechno-logy as it is non-dispensable part of
one or the other step in every method involved in biological study right from the separation of cell
organelles to complex experiments involving separation of sub-cellular fractions.

Types of Centrifuges:

There are four major types of centrifuges. They are:

1. Small Bench Centrifuges:

They are used to collect small amount of material that rap-idly sediment like yeast cells,
erythrocytes etc. They have maxi-mum relative centrifugal field of 3000-7000 g.

2. Large Capacity Refrigerated Centrifuges:

They have refrigerated rotor chamber and have capacity to change rotor chambers for varying size.
They can go up to maximum of 6500 g and use to sediment or collect the substances that sediment
rapidly like erythrocytes, yeast cell, nuclei and chloroplast.

3. High Speed Refrigerated Centrifuges:

They can generate speed of about 60000g and are used to collect micro-organism, cellular debris,
larger cellular organelles and proteins precipitated by ammonium sulphate.

4. Ultra Centrifuges:

(a) Preparative ultracentrifuge:

It can produce relative centrifugal force of about 600000g and its chamber is refriger-ated, sealed
and evacuated. It is employed for separation of macromolecules/ligand binding kinetic studies,
separation of various lipoprotein fractions from plasma and deprotonisation of physiological fluids
for amino acid ananlysis.

(b) Analytical ultracentrifuge:

It is capable of operating at 500000 g. Three kinds of optical systems are available in analytical
ultracentrifuges: a light absorption system, and the alternative Schlieren system and Rayleigh
interferometric system, both of which detect changes in the refractive index of the solution.
Sampling and Preparation of Samples:

Sampling plays a critical role in plant analysis. When analyzing the nutrient status of plants, it is
essential to select the plant part for chemical analysis that reflects the status of the particular
element of interest. Four samplings during a growing season are usually sufficient to characterize
seasonal nutritional patterns. One sampling should be early in the growing season, two in mid-
season and the last one just prior to harvest. Four samples should be collected from each field or
management unit. Each sample should contain material from at least 20 plants to ensure adequate,
representative material for analytical testing. Separate samples should be taken from areas that
appear different from the rest of the field.

A young mature leaf is generally selected for analysis. The sample can be subdivided into blade
and petiole. The status of Cl, NO3-N, NH4-N, extractable K and P, in the form of PO4-P (2%
acetic acid) are generally determined through analysis of the petiole. Blades are used when
evaluating the status of K, Ca, Mg, Na, Fe, Mn, Zn, Cu, B, Mo, SO4-S and total-N in plants. For
diagnostic purposes, only leaves that have recently developed symptoms should be collected for
chemical analysis.

Drying Recommendations

After collection, plant material should be washed to remove any residual soil or dust. Fresh
samples or those suspected to be moist should be placed into paper bags (with adequate room for
air movement within the bag) and dried in a forced air oven at 55-60°C. In general, adequate drying
time is approximately 12 hours or until the material snaps or breaks easily. All samples, except
freeze drying material, should be turned every 24 hours.
Gas Exchange in Plants

In order to carry on photosynthesis, green plants need a supply of carbon dioxide and a means of
disposing of oxygen. In order to carry on cellular respiration, plant cells need oxygen and a means
of disposing of carbon dioxide (just as animal cells do).

Unlike animals, plants have no specialized organs for gas exchange (with the few inevitable
exceptions!). The are several reasons they can get along without them:

Each part of the plant takes care of its own gas exchange needs. Although plants have an
elaborate liquid transport system, it does not participate in gas transport.

Roots, stems, and leaves respire at rates much lower than are characteristic of animals. Only
during photosynthesis are large volumes of gases exchanged, and each leaf is well adapted to take
care of its own needs.

The distance that gases must diffuse in even a large plant is not great. Each living cell in the
plant is located close to the surface. While obvious for leaves, it is also true for stems. The only
living cells in the stem are organized in thin layers just beneath the bark. The cells in the interior
are dead and serve only to provide mechanical support.

Most of the living cells in a plant have at least part of their surface exposed to air. The loose
packing of parenchyma cells in leaves, stems, and roots provides an interconnecting system of air
spaces. Gases diffuse through air several thousand times faster than through water. Once oxygen
and carbon dioxide reach the network of intercellular air spaces (arrows), they diffuse rapidly
through them.

Oxygen and carbon dioxide also pass through the cell wall and plasma membrane of the cell by
diffusion. The diffusion of carbon dioxide may be aided by aquaporin channels inserted in the
plasma membrane.

Leaves

The exchange of oxygen and carbon dioxide in the leaf (as well as the loss of water vapor in
transpiration) occurs through pores called stomata (singular = stoma).

Normally stomata open when the light strikes the leaf in the morning and close during the night.
The immediate cause is a change in the turgor of the guard cells. The inner wall of each guard cell
is thick and elastic. When turgor develops within the two guard cells flanking each stoma, the thin
outer walls bulge out and force the inner walls into a crescent shape. This opens the stoma. When
the guard cells lose turgor, the elastic inner walls regain their original shape and the stoma closes.

Opening stomata

The increase in osmotic pressure in the guard cells is caused by an uptake of potassium ions (K+).
The concentration of K+ in open guard cells far exceeds that in the surrounding cells. This is how
it accumulates:

Blue light is absorbed by phototropin which activates

a proton pump (an H+-ATPase) in the plasma membrane of the guard cell.

ATP, generated by the light reactions of photosynthesis, drives the pump.

As protons (H+) are pumped out of the cell, its interior becomes increasingly negative.

This attracts additional potassium ions into the cell, raising its osmotic pressure.

Closing stomata

Although open stomata are essential for photosynthesis, they also expose the plant to the risk of
losing water through transpiration. Some 90% of the water taken up by a plant is lost in
transpiration.

In angiosperms and gymnosperms (but not in ferns and lycopsids), Abscisic acid (ABA) is the
hormone that triggers closing of the stomata when soil water is insufficient to keep up with
transpiration (which often occurs around mid-day).