Experiment 2A: Animal Dehydrogenase Methodology: Three clean, dry test tubes were prepared. These were labelled as 1, 2 and 3, respectively. To each test tube, 5.0 mL of sterilized milk was added. The milk in test tube 1 was then boiled and was allowed to cool after. 1.0 mL of 0.02% methylene blue solution was added to each test tube. To test tubes 1 and 2, 1.0 mL of 0.4% formaldehyde solution was added. Both test tubes were mixed gently. After this, 1.0 mL of paraffin oil was added to each test tube. A water bath was prepared with a temperature maintained at 40 oC. All the test tubes were placed in the water bath for 30 minutes. The results were recorded after. Results: Test Tube Numbe r
Composition
Results (after heating in water bath)
Theoretical Results
1
5 mL milk(boiled) + 1 mL 0.02% methylene blue solution + 1 mL 0.4% formaldehyde solution + 1 mL paraffin oil
3 layers
Light blue solution (distinct methylene blue)
2
5 mL milk + 1 mL 0.02% methylene blue solution + 1 mL 0.4% formaldehyde solution + 1 mL paraffin oil
2 layers (clear and white)
White solution (indistinct methylene blue) Milk curdles at the bottom of the test tube
3
5 mL milk + 1 mL 0.02% methylene blue solution + 1 mL paraffin oil
2 layers (clear and white)
White solution (indistinct methylene blue) Milk curdles at the middle portion Milk powder at the bottom of the test tube
Suspended milk powder at the bottom of the test tube
DISCUSSION Animal Dehydrogenase Methylene Blue is a heterocyclic aromatic chemical compound with the molecular formula C16H18N3SCl. It is widely used as a redox indicator in chemical experiments. Solutions of this substance are blue in an oxidizing environment, but will turn colorless if exposed to a reducing agent. The theory of Barthel shows that the disappearance of methylene blue in milk takes place in two stages. First, the removal of the dissolved oxygen by bacteria and second, the reduction of the dye by constituents of the milk.
Methylene blue is used in the experiment to stain the milk and to check the amount of dissolved gasses present in the milk and presence of bacteria. Methylene blue will undergo reduction due to hydrogen donators present in the milk like aldehydes, citrates, succinates and a few more. The amount of dissolved gasses, like Oxygen compete with methylene blue in the reduction since they also have affinity to the hydrogen present. Thus, the more oxygen in the milk, the more intense the blue coloration will be. The addition of formaldehyde acts as a hydrogen donator w/c enhances the reduction of both methylene blue and oxygen present. Also it decreases bacterial contamination and prolong its keeping quality. Boiling of the milk was done to create a force to let all dissolved gas rise to the surface of the milk but will not get released due to the fatty layer on the surface of the milk. The bubbles formed during boiling will not burst easily due to the difference in pressure where in the bubble created has less pressure inside compared to outside the bubble. Also, heating of the milk may cause death of the bacteria present and denaturation of enzymes present. In addition, increased temperature in milk increases enzyme activity and decreases the time for each reaction. Paraffin oil is a non-polar alkane that has a density of around 0.8 g/cm3. Paraffin oil was added to all samples to prevent diffusion of atmospheric oxygen to the test tube and to prevent gas exchange from the atmosphere and the milk thus, preventing autoxodiation. Cow’s milk In the experiment, we can see that cow milk had a blue coloration in test tube 1, this may have been due to the fact that the milk in this test tube was boiled causing all the dissolved gasses to rise to the surface that when the formaldehyde was added to the mixture. Formaldehyde is an aldehyde with the formula CH2O. It acts as a proton donor, and can act as the reducing agent. According to a study by Booth (1938), formaldehyde can destroy the xanthine oxidase enzyme in solution, leading to a decreased rate of reaction. The oxygen immediately reacted with the hydrogen being released by formaldehyde which caused less reduction of the methylene blue. In the second test tube of cow’s milk, the color intensity was not that intense due to the dissolved gasses were spread throughout the mixture unlike in the first test tube where in the milk was boiled causing all the gas to rise. Since the intensity of dissolved gas was not that intense in the surface, the hydrogen released by the formaldehyde was able to react with most of the methylene blue which resulted in a less intense coloration.
For the third test tube of cow’s milk, the color intensity was similar with test tube 2 since it was not heated as well and methylene blue was allowed to react normally with the hydrogen released or present in the milk. Furthermore, curdling was seen in test tubes 2 and 3. Curdling occurs when acidification of milk happens. Acidification of milk occurs when, lactose turns into lactic acid. Increased acidity of casein milk proteins cause these proteins to tangle into solid masses, or curds. This process is due to the presence of proton donators such as aldehydes. In the Animal Dehydrogenase experiment, test tubes added with formaldehyde showed a short reduction as evidenced by the slow scattering of methylene blue in the milk solution as compared to test tube 3 (breast milk) which showed almost complete diffusion of methylene blue. Paraffin oil was also added to avoid influence of atmospheric oxygen. Putting the test tubes in a water bath increases the temperature thus increasing the enzymatic reaction.
REFERENCEs Murray,R. K, Granner, D. K., Rodwell, V. W. (2006). Harper’s Illustrated Biochemistry 27th Edition. Singapore: McGraw-Hill Companies, Inc. Fay, A. C. The detection of formaldehyde in milk by means of methylene blue reduction test. Retrieved from http://jds.fass.org/cgi/reprint/18 /5/327.pdf Thorrnton, H.R. and Hastings, E. G. Studies on oxidation-reduction in milk: The methylene blue reduction test. Retrieved from http://jds.fass.org/cgi/reprint/13/3/221 Whitehead, H. R. The reduction of methylene blue in milk: The influence of light. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1254496/pdf/biochemj01126-0005.pdf http://www.itisacqui.it/sitob/formagette/curdling.htm http://www.biochemj.org/bj/032/0503/0320503.pdf
