Practical No. Page
Assessment portfolio (submit electronically via Turnitin – hand-in deadline: 29/1/18)
Write-up practical No. 3 in the form of a scientific paper i.e. Title, Abstract (300 words), Introduction (300 words) Methods (300 words), Results (300 words [plus graphs where appropriate]), Discussion (300 words), Conclusion (bullet points) and References (Preferably Harvard style – you should aim to have at least 5-6 references). (NB word counts shown in brackets are the maximum amounts you are allowed per section.)
Bright-Field Light Microscopy (Bright Field)
Light rays from an illumination source are focused on the specimen to be examined by a condenser lens. The light rays leaving the specimen (mounted onto a slide, and placed onto the stage) are focused into a magnified image by two lenses placed at either end of a tube. The lens near to the specimen is called the objective lends and the one near the eye is called the ocular (or eyepiece) lens.
The extent to which a microscope can distinguish fine details in the specimen as separate, distinct image points is termed resolution.
In the light microscope:
Resolution (symbol: d) = 0.61 l
n sin a
l is the wavelength of the light used to illuminate the microscope
n is the refractive index of the transmitting medium surrounding the specimen/filling the space between specimen and objective lens.
a is the half angle of the cone of light entering the objective lens from the specimen
0.61 is a constant describing the degree to which image points can overlap and still be recognised as separate points by the observer.
Since resolution is a measure of the ability of a microscope to image fine details, the quantity ‘d’ becomes smaller as resolution improves. Therefore for best resolution ‘0.61 l’ should take on the smallest possible value, and ‘n sin a’ the largest possible value (eg. the value of n can be pushed to its maximum by placing a drop of immersion oil [refractive index 1.5] in the space between the objective lens and the specimen).
The best resolution possible with a light microscope is 0.2mm or 200nm because for visible light, l = 450nm. However, in a fluorescence microscope, resolution is enhanced to 0.1mm or 100nm, because ultraviolet light has a l of 250 nm.
Using a light microscope it is possible to accurately measure the sizes of objects using an eye piece graticule and a stage micrometer. The graticule fits inside the eye piece of the lens, and has a fine scale etched upon it; the eye piece graticule’s scale has to be calibrated using a stage micrometer slide (basically a microscope slide with a fine scale of 1mm, divided into 100 graticule units, imprinted on it).
In this practical session, we will use the calculations/conversions performed last week using an eye piece graticule and a stage micrometer in order to produce scale drawings of buccal epithelial cells obtained via a ‘cheek scrape’ protocol.
Consenting to Participate in this Practical
‘Cheek Scrape’ Protocol (wear gloves at all times):
Make scale-drawings of the samples you have prepared. (NB. If you have not succeeded in preparing samples, please note that an inventory of microscope slides is available; you can sign out slides, and use them as the basis for your scale-drawings.)
Draw your observations at x100 and x400 magnification. Indicate on your diagram cell sizes, and of the length of 2-3 nuclei – this should be used in order to calculate the mean size of a nucleus (NB. all measurements MUST be in microns (uM)).
Notes/Scale Drawings for Practical 1.
2) Observation of chromosomes undergoing mitosis in onion root tips
To put into practice what was covered before the break, we will now use the calculations/conversions performed previously (see above) using an eye piece graticule and a stage micrometer in order to produce scale drawings of chromosomes undergoing mitosis obtained via an ‘onion root tip’ protocol.
(NB. If you have not succeeded in preparing samples, please note that an inventory of microscope slides is available; you can sign out slides, and use them as the basis for your scale-drawings.)
Notes/Scale Drawings for Practical 1.
Within the cell, enzymes/multienzyme systems have characteristic intracellular locations. For example, in eukaryotic cells glycolysis occurs in the cytoplasm, the citric acid cycle occurs in the matrix of the mitochondrial membrane, and electron transport and oxidative phosphorylation occur on the inner mitochondrial membrane. However, this metabolic compartmentalisation does not necessarily mean that these systems act in complete independence, as transport mechanisms allow transport of metabolic intermediates between different cellular compartments.
The objective of this practical is to fractionate liver cells, and to determine the cellular locations of some important metabolic enzymes.
Practical 3, Session 1
Prac 1a. Preparation of Liver Homogenate and Cell Fractions
Theory – When liver is homogenised in isotonic media by mild procedures, the cell nuclei and mitochondria remain relatively intact. These subcellular structures can then be centrifuged out of the homogenate, leaving behind in the supernatant all the soluble components of the liver – including the cytoplasmic enzymes. (NB. However, mitochondria prepared in this way do not exhibit many of the properties demonstrable in more carefully isolated preparations. The membranes are ‘leaky’, with the result that many small molecules can permeate the mitochondrial membranes). Mitochondria may then be completely lysed by addition of the detergent Triton, which lyses the mitochondrial membranes and liberates all of the proteins present in the matrix of the mitochondria.
Store all fractions on ice.
Prac 1b. Determination of Lactate and Malate Dehydrogenase
Malate + NAD+ « NADH + H+
MDH activity can be assayed by measurement of the production of NADH. In this experiment, the NADH formed is used to reduce a dye which when reduced is red-coloured; hence NADH formation (and thus MDH activity) can be determined using a spectrophotometer. In fact two electron acceptors are actually employed – phenazine methosulphate (PMS) accepts electrons from NADH and transfers them to the electron acceptor iodophenyl nitrophebyl tetrazolium chloride (INT), which turns red on accepting these electrons.
NADH PMS Formazan (red colour)
NAD+ PMSH2 INT
Lactate + NAD+ « pyruvate + NADH + H+
The NADH formed in this reaction (and thus LDH activity) can also be determined spectrophotometrically via monitoring of generation of a red colour, using a PMS, INT cocktail.
(1: Lactate/Fraction A; 2: Lactate/Fraction B; 3: Lactate/Fraction C; 4: Malate/Fraction A; 5: Malate/Fraction B; 6: Malate/Fraction C; 7: Water/Fraction A; 8: Water/Fraction B; 9: Water/Fraction C).
Start the enzyme reaction by adding 250ml* of Fraction A to tubes 1, 4 and 7; 25ml* of Fraction B to tubes 2, 5 and 8; or 25ml* of Fraction C to tubes 3, 6 and 8. Mix well in all cases. [*Note: Depending on the enzyme activity in each of your fractions, you may need to alter the volume of fraction used, and re-run your assay (eg. if the absorbance at the end of the assay is very high, the INT has probably been used up before the end of the assay, and therefore the assay should be re-run using a smaller volume of fraction)].
If time allows, you should perform these enzyme assays in triplicate, and calculate mean values in each case.
Practical 3, Session 2
Estimation of Protein Concentration
Theory Polypeptide concentration may be estimated from the colour of a chelate formed at room temperature between copper in alkaline solution and the nitrogen atoms of peptide bonds. This is termed the Biuret reaction. Bovine serum albumin (BSA) is used to standardize the colour reaction. Each protein has a unique amino acid composition and a slightly varying colour yield will be given per unit mass of polypeptide. An assay of unknown protein by this method gives results which are really expressed in terms of the equivalent concentration of BSA.
Notes for Practical 3.
Discussion Topic Directions: Overview Early childhood is a time of exploration and discovery. Children are continually learning during this stage…