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Exercise 9: Study of Storage Granules

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Armen Jheannie Barrameda

on 4 September 2013

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Transcript of Exercise 9: Study of Storage Granules

Exercise 9: Study of Storage Granules
Staining Intracellular Lipid
Done by preparing smear, fixing by heat, flooding with SUDAN BLACK STAIN for 15 minutes, rinsed with xylene and counterstained with safranin,
Staining Intracellular Lipid
Results
POSITIVE RESULT- Lipid inclusions would appear blue to black; otherwise, one would only see the red-colored cell.
Why such results?
The answer relies on the nature of the stains as well as the reagents used in staining.
Stains Used
Staining Glycogen and Starch Granules
Staining Glycogen and Starch Granules
This is done by mixing a loopful of culture to a slide with a drop of Lugol's iodine and observing it under OIO.
Results
In glycogen, a positive result would yield reddish brown structures.
In starch granules, a positive result would appear dark blue to violet.
Glycogen vs. Starch Granules
The colour of the complex depends of the 3D structure of the polysaccharide.
Glycogen is a branched molecule, so it turns red when iodine binds to it.
On the other hand, iodine stains starch as blue due to iodine's interaction with the coil structure of the polysaccharide in starch.
Staining Metachromatic Granules
Staining Metachromatic Granules
This is done by applying methylene blue to a heat-fixed slide of bacteria.

Metachromatic granules are a special type of storage granules in bacteria and are easily distinguished by its characteristic change in color.
Metachromasy
The phenomenon where one dye stains more than one color.
The phenomenon in which certain substances (termed chromotropes) develop a colour different to that of the dye used to stain them.
Results
Metachromatic granules have a dark blue color in contrast with the bacterial cells which would stain a lighter blue color.
Factors affecting the formation of Storage granules in bacteria
Presence or absence of certain nutritive material in the medium
Amount of water in medium and granule formation
Influence of metabolic products of the organism
Influence of temperature
Oxygen tension

References
http://www.ask.com/question/how-does-lugol-s-solution-detect-starch
http://stainsfile.info/StainsFile/theory/lysochrm.htm
http://web2.uwindsor.ca/courses/biology/fackrell/Methods/Fat_Stain.htm
http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/SUDANF.PDF
http://www.microbiologytext.com/index.php?module=book&func=displayarticle&art_id=58
Madigan, M.T., et.al. 2012. Brock Biology of Microorganisms. 13th ed. CA, USA: Pearson Education, Inc
Singleton, P. and D. Sainsbury. 2006. Dictionary of Microbiology and Molecular Biology. 3rd ed. United Kingdom: John Wiley & Sons, Co. Ltd.
Armen Jheannie D. Barrameda
MCB 101 F-2L
~end~
Safranin
served as a counterstain
which stains cells red
Xylene
nonpolar solvent which is soluble in lysochromes
Lysochromes-soluble dye used for biochemical staining of triglycerides, fatty acids, and lipoproteins; mostly insoluble in polar solvents.

Sudan Black B Stain
a dye that is insoluble in water but dissolves in fat.
slightly basic dye that would combine with the acidic groups in compound lipids.
Storage granules
Nutrients and reserves that may be stored in the cytoplasm in the form of glycogen, lipids, polyphosphate, or in some cases, sulfur or nitrogen.
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