Macromolecules/ Large molecules

these are polymers with high molecular mass and are composed of relatively smaller units or building blocks. among these macromolecules include: polysaccharides, lipids, proteins and nucleic acid.

Macromolecules/ Large molecules

serve us sources of energy (except for nucleic acid) for organisms macromolecules must be converted to their corresponding monomeric units in order for them to be used as energy source through decomposition.

A. Liquefaction of Gelatin Gelatinase test detects the presence of the enzyme gelatinase. Medium used: Nutrient Gelatin

1. Beef extract and peptone- provides carbon,nitrogen, vitamins and amino acids.
2. Gelatin- the solidifying agent and substrate for detection of gelatinase.

A. Liquefaction of Gelatin

Gelatin -a soluble mixture of polypeptides derived from hydrolysis of collagen.

Gelling characteristics: -solid at temp. below 28C. -liquid at temp. above 28C.

A. Liquefaction of Gelatin

Principles:

o Gelatinase is a proteolytic enzyme that cleaves peptide bond linking the amino acids present in gelatin.

o Gel lattice is destroyed due to cleavage of the bonds. o Destruction of the lattice leads to liquefaction of gelatin, an irreversible reaction.

A. Liquefaction of Gelatin

Results:

(+) the medium remains in liquid form even after cooling in an ice bath (-) the medium is in solid state after cooling in an ice bath

A. Liquefaction of Gelatin

Control microorgansims: E. coli- gelatinase (-) B. subtilis- gelatinase (+)

B. Lipolytic activity

This test aims to differentiate bacteria based on their ability to hydrolyze lipid using the enzyme lipase. Lipids
-compounds with high molecular mass and are water insoluble but are soluble in non-polar solvents. -examples are triacylglycerols (triacylglyceride) and phospholipids - triglycerols are the simplest lipids and are composed of fatty acids linked to glycerol via ester bonds.

B. Lipolytic activity

Medium used: Sierra Medium with Tween 80 1. Peptone- source of carbon, nitrogen, vitamins and amino acids 2. Sodium chloride- maintains osmotic balance 3. Calcium chloride monohydrate- calcium source 4. Tween 80- lipid source

B. Lipolytic activity

Principle: -Lipases are able to hydrolyze Tween 80 present in the medium by breaking the ester bond linking fatty acids to glycerol.

B. Lipolytic activity

-The free fatty acid (oleic acid) reacts with calcium and sodium ions that are also present in SM.

B. Lipolytic activity

Result:
(+) formation of white zone of precipitate around the line of streak (-) no white zone around the colonies

C. Reactions in Litmus Milk

Litmus milk serves as a excellent medium for differentiation of microorganism on the basis of the substrate they are able to catabolize.

C. Reactions in Litmus Milk

Medium Used: Litmus Milk 1. Skim Milk - reduced fat (usually 0.5%) - lactose - casein, lactalbumin, lactoglobulin 2. Litmus- serve as pH indicator and as redox indicator. Pink/red: acidic (pH <4.5) Blue: alkaline (pH >8.2) Colorless: Reduced (gain electron) Pink/blue: oxidized (loss electron)

Reaction
1. Acid pH

Principle/cause
-Lactose is broken down by the enzyme -galactosidase. -lactic acid is released causing a decrease in pH.

-medium turns pink to red.

2. Acid curd -Production of lactic acid causes coagulation of casein. -lactic acid precipitates casein as calcium caseinate and form an insoluble clot in clear watery fluid (whey). -the curd remains immobile even when the tube is inverted. 3. Stormy clot -during lactose fermentation carbon dioxide and hydrogen gases are released.

C. Reactions in Litmus Milk

Reaction
4. Alkaline pH

Principle/cause
-proteolytic enzymes acting on protein components of the medium cause formation of basic amines and ammonia.
-pH is increases due to production of basic products

-litmus turns blue to purple

5. Rennet curd/ Alkaline coagulation -rennin-like enzyme causes conversion of casein to paracasein

-results to formation of soft, semi-solid clot that flows when the tube is tilted 6. Peptonization -proteases act on the proteins present in the milk -the reaction releases amino acids and ammonia - this results in the development of deep blue band in the upper portion of the tube with translucent, brown, whey-like medium.

Reaction
7. Reduction of litmus

Principle/cause
- due to removal of oxygen by the enzyme reductase -oxygen (the final e- acceptor) is limited inside screw-capped tube. The bacteria need to use alternate e- acceptor. -Litmus is a biological e- acceptor and will change color as it undergoes reduction (gain of electron). -oxidation of lactose is accompanied by removal of hydrogen and these hydrogen ions are accepted by e- acceptor so that redox reaction is possible and cells can manufacture energy.

C. Reactions in Litmus Milk

C. Reactions in Litmus Milk

Control organisms: E. coli- acid curd with gas production

P. fluorescens- alkaline with rennet curd

B. subtilis- alkaline

References:
Harley, J. P. (2004). Laboratory Exercises in Microbiology. McGraw-Hill.
Lehninger, A. L., Nelson, D. L., & Cox, M. M. (2008). Principles of Biochemistry. W.H. Freeman.

http://www.himedialabs.com/TD/M609.pdf http://web.clark.edu/tkibota/240/Unknowns/LitmusMilk.htm http://www.elmhurst.edu/~chm/vchembook/186indicator.html http://www.mesacc.edu/~johnson/labtools/Dbiochem/gel.html http://www.blinn.edu/natscience/phillips/micro%20pictures.htm#Litmus