CELL BIOLOGY PRACTICALS
Aseptic techniques and measurements in cell biology
Introduction
Aseptic techniques
These are procedures that are performed under sterile conditions. This includes medical and
laboratory techniques, such as with microbiological cultures. These include techniques like flame
sterilization, washing with 70% alcohol, working under the fume hood cabinet etc. The main
objective is to prevent contamination of the specimen. Aseptic techniques are also practiced in
cell culture in plant experiments, animal surgical operations and microbiological cultures in
industry or laboratories. The techniques can be learnt and be mastered over time. It takes one’s
change of attitude and interests in the subject to quickly master the concepts and perform cell
biology experiments aseptically and efficiently.
Accuracy and precision of measurements
In the field of science, the accuracy of a measurement system is the degree of closeness of
measurements of a quantity to that quantity's actual (true) value. The precision of a measurement
system, also called reproducibility or repeatability, is the degree to which repeated measurements
under unchanged conditions show the same results. Although the two words reproducibility and
repeatability can be synonymous in colloquial use, they are deliberately contrasted in the context
of the scientific method.
Accuracy indicates proximity of measurement results to the true value, precision to the
repeatability or reproducibility of the measurement. A measurement system can be accurate but
not precise, precise but not accurate, neither, or both. A measurement system is designated valid
if it is both accurate and precise.
Precision and accuracy: These four sets of rifle shots illustrate the distinction that surveyors
make between the terms “accuracy” and “precision” as applied to surveying measurements and
observations
Practical 1: Simple Pipetting and weighing
Background
The correct use of a balance is a fundamental requirement for laboratory procedures. You will
learn to operate different balance scales by weighing tubes. You will learn the correct use of
different types of pipettes and assess the precision of your techniques by doing repeated
measurements. Comparing the observed values with expected ones helps to assess accuracy of
the measurements.
Co-efficiency of Variation (CV) = SDX100
_______
Mean
The lower the CV the more precise the method or instrument.
Inaccuracy = O-E X100
Where O is the observed value and E is the expected or true value
Procedure
Exercise 1
1. Weigh a 5ml tube
2. Remove from the weighing pan and return the scale to zero
3. Weigh the tube 9 more times to make a total of 10 readings for the same tube
4. Repeat the steps 1-3 on two other balances
5. Tabulate your results and imprecision of the measurements
Exercise 2
1. Weigh 10 different capped 5ml tubes.
2. Pipette 2ml water into each tube using either a 1ml graduated pipette or 1000ul
micropipette and reweigh.
3. Tabulate your results and Assess imprecision and inaccuracy of your pipetting technique.
Additional Information on how to write the lab report
Results: Describe your most important results in words. Tabulate your results. Give each table or
figure a title with explanatory notes
Discussion: comment on the reliability and accuracy of the balance that you used. Comment on
the sources of error when taking measurements.
Conclusion: How do your results relate to the stated objectives?
Practical 2: Practising aseptic techniques in cell biology
Exercise 1
A. GENERAL LABORATORY PROCEDURES AND BIOSAFETY
A primary feature of the cell and microbiology laboratories is that living organisms are employed
as part of the experiment. Most of the organisms are harmless; however, whether they are
non-pathogenic or pathogenic, the microorganisms are treated with the same respect to assure
that personal safety in the laboratory is maintained. Careful attention to technique is essential at
all times. Care must always be taken to prevent the contamination of the environment from the
cultures used in the exercises and to prevent the possibility of the people working in the
laboratory from becoming contaminated.
Objectives
● To illustrate the potential for contamination from the environment that is always present
when working with cells.
● To become familiar with using aseptic techniques to handle cells and microorganisms.
Materials
● Sterile swabs
● Sterile water
● Plate Count Agar
● Nutrient Agar
● Mackonkey agar
● Manitol Salt agar
● Petri dishes
Procedure
Work in pairs to complete the following exercise:
1) Label each of the plates with the name of the surface to be tested and the media type.
Label the half of the plates for each of the surfaces as “after disinfection”.
2) Moisten the swabs provided with a small amount of sterile water. Brush the surface
to be tested with the swab, and then use the swab to inoculate each of your two plates
in 1.
3) Disinfect the surface, moisten another swab, and repeat step 2 and inoculate into
plates labelled ‘after disinfection’.
4) Incubate at 30 degrees for 24-hours,
5) Evaluate your plate results and record the number of colonies present. Make
observations of colony morphology.
Questions
What differences in colony morphology and number were observed on the two types of media?
Why?
Does disinfection of work surfaces completely eliminate all microbial organisms?
What evidence do you have?
Using the Light microscope to visualize cells
Introduction
A microscope consists of one lens close to the specimen
(objective) and one lens close to the eye (eyepiece). The magnification of a microscope is the
product of the factors of both lenses. A 40x objective and a 10x eyepiece, for example, provide a
400x magnification. Magnification is achieved by the refraction of light travelling though lenses,
transparent devices with curved surfaces. In general, the degree of refraction, and hence,
magnification, is determined by the degree of curvature. However, rather than using a single,
severely-curved biconvex lens such as that of Leeuwenhoek's simple microscopes, Hooke
determined that image clarity was improved through the use of a compound microscope,
involving two (or more) separate lenses.
It is not only the magnification but also the resolution that indicates the performance capacity of
a microscope. Resolution is the ability to differentiate between
two closely adjacent points. According to the Rayleigh criterion, the minimum distance between
two points able to be separately imaged corresponds to approximately one-half the wavelength of
the light.
To make the microscopic resolution detectable to the eye, the image appears in the eyepiece with
corresponding magnification. The resolution and magnification are always directly inter
dependent. An objective with low magnification has a low numerical aperture and thus a low
resolution. Immersing the specimen in oil increases resolution.
Practical 3: The Microscopic Examination of prokaryotic cells
Simple Gramm Staining of cells
Background
Prior to viewing bacteria, two procedures must be performed: 1) fixation and 2) staining.
Fixation performs 2 functions: (i) immobilises (kills) the bacteria; and (ii) affixes them to the
slide. The most common fixation procedure for bacteria is heat fixation, whereby the slide
containing a drop or smear of bacterial culture is passed rapidly once or twice through the heat of
a Bunsen flame.
Bacteria are almost transparent and hence, unstained bacteria are not readily visible without
special techniques such as phase contrast microscopy or dark-field microscopy, which is also
referred to as negative staining. Any procedure that results in the staining of whole cells or cell
parts is referred to as positive staining. Most positive stains used involve basic dyes where basic
means that they owe their coloured properties to a cation (positively charged molecule). When all
that is required is a general bacterial stain to show morphology, basic stains such as methylene
blue or carbol fuchsin result in the staining of the entire bacterial cell.
Objectives
● To perform specialised staining procedures in order to examine different properties of
cells.
● To reinforce proper techniques for handling of cells.
Materials
● Stains; Crystal violet; Safranin; 5% Malachite green; Carbol fuchsin ; Methylene blue ;
Gram’s iodine
● 20% Sulfuric acid;
● 95% ethanol ;
● Bacteria culture,
● microscopes
Procedure
Exercise 1: Preparation of Films for Staining
You are required to make specimen slides from the following cultures Bacillus thuringiensis
Escherichia coli and Staphylococcus epidermidis.
● Obtain a clean slide and draw a circle on it approximately 1.5 cm in diameter. Turn the slide
over.
● Using a loop add a drop of water to the circle on the slide. Aseptically remove a small
quantity of culture from the plates provided and mix with the water to make a smooth
suspension.
● Allow the suspension to air dry. When dry, the film should be only faintly visible; a thick
opaque film is useless.
● The only fixation required is to pass the slide several times (maximum 10) through the
Bunsen burner flame until the slide is warm but not too hot.
Exercise 2: Gram Staining
To each of the slides prepared in exercise 1 above, perform the following steps
● Flood the smear with crystal violet solution for 1 min.
● Gently wash with tap water for 2-3 seconds and remove the water by tapping the slide gently
on paper towel.
● Add Gram’s iodine solution to the slide for 1 min. Wash gently with tap water and remove as
above.
● Decolourise with 95% ethanol by dripping ethanol on surface of slide until no more colour is
removed.
● Rinse gently with water. If too much alcohol is added, the Gram-positive organisms may
become Gram-negative.
● Remove the water after the last wash.
● Counterstain the slide with safranin for 30 seconds - 1 minute.
● Wash the slides with tap water, air dry on paper towel and examine under oil emmersion
Practical 4: Microscopic examination of plant and animal cells
Materials
● Sample specimen slides ; (microbiological, blood cells, onion cells)
● Scalpel, slides, cover slips, iodine solution, methylene blue
● Pipette droppers or fillers
● Onion bulb, sterile tooth picks/ earbuds
● microscopes
Exercise 1: Observing the structure of Onion cells
Tissue from an onion is a good first exercise in using the microscope and viewing plant cells.
The cells are easily visible under a microscope and the preparation of a thin section is straight
forward. An onion is made of layers, each separated by a thin skin or membrane. In this exercise
you will make a wet mount on a microscope slide and look at the cells of the onion membrane
magnified by the high power, compound microscope.
Procedure
1. Take a small piece of onion and using forceps (tweezers), peel off the membrane from the
underside (the rough side).
2. Lay the membrane flat on the surface of a clean glass slide, and then add one drop of
water or iodine.
3. Using a pin, lower a thin glass cover slip or cover glass onto the slide. Make sure there are
no air bubbles.
4. Make sure the lowest power objective lens (the shortest lens if there are several present) is
in line with the optical tube, and the microscope light is turned on. Then place the
prepared slide onto the stage of the microscope.
5. Looking from the side (NOT through the eyepiece), lower the tube using the coarse focus
knob until the end of the objective lens is just above the cover glass. Do this carefully so
as not to crack the cover glass (and possibly damage the objective lens).
6. Now look through the eyepiece and turn ONLY the smaller, fine focusing knob to move
the optical tube upwards until an image comes into focus. The cells should look something
like lizard skin.
7. Swap the objective lens for a higher powered one so that you can see the cells at greater
magnification. You should be able to make out a nucleus in each cell. Draw what you see
8. Repeat the process after adding a dye solution (iodine or methylene blue). Be very careful;
these dyes can stain your skin and clothes.
Exercise 2: Observing the structure of cheek cells
Procedure
Cells from the inside lining of your cheek are good for learning how to stain. They can be easily
scraped but can easily regenerate.
1. Gently scrap the inside of your mouth with the flat side of a toothpick/sterile ear bud.
This scraping will collect some of your cheek cells.
2. Place the cells on a flat slide and make a wet mount as you did in 2&3 above.
3. Now repeat this procedure so that you have two wet mounts of cells from the inside of
your cheek.
4. Stain one slide with methylene blue. To add stain, put a drop of the stain next to the cover
slip on the slide and then draw it under the cover slip by placing a piece of paper towel
against the other side of the cover slip. The paper towel will soak up the water, drawing
the stain under the cover slip around the cell. Drawing the stain under the cell is called
"wicking."
5. Follow steps 4-7 above to Observe the stained slide under the microscope using low and
high magnification. Draw what you see.
6. Repeat step 5 with the unstained slide. Is it different? Draw what you see.
GENERAL LAB INSTRUCTIONS
Proper use of the microscope
Intended to prevent damage to the objective lenses. The following techniques should be
followed:
● Never use the coarse focus knob while looking through the eyepiece. The point of focus will
be very near the cover glass. Looking from the side, lower the optical tube until the objective
lens is as close as you can get it to the cover glass without actually touching it. Starting with
the low power objective lens is the fastest way to achieve proper focus.
● Initially, slowly focus back (turn the fine focus knob to raise the optical tube) while looking
through the eye piece. Once the specimen comes into focus, you can make fine adjustments
up or down with the fine focus knob without fear of damaging the slide or the microscope.
● If the specimen does not come into view (does not focus), raise the tube a little with the
coarse focus knob and attempt to focus again with the fine focus knob. Once the object is in
focus, switching objective lenses (to a higher power) should be possible without any further
coarse adjustments
Preparation of scientific drawings
● Use a sharpened pencil; never ink. The lead should be hard.
● Place drawing to one side, usually the left, leaving room for labels to the right.
● Try to draw with one continuous line and do not retrace your lines. Do not shade.
● Place label lines horizontally (use a ruler), with no crossed lines.
● Objects labelled should be singular unless label line branches to multiple objects.
● Label only what you see, not what you think should be seen.
● Below the figure you should add:
a) The title of the diagram
b) The magnification of the drawing (see below)
● Magnification is defined as: size of drawing
actual size of sp
