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

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)

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

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.
Cows 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 cows 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 cows 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). Harpers 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