Lab Manual: Litmus Milk reactions
What is lactose?
A milk sugar
What are the 3 milk proteins?
1. Casein
2. Lactalbumin
3. Lactoglobulin
The major milk substrates capable of transformation are the
1. milk sugar- lactose
&
2. the 3 milk proteins: casein, lactalbumin & lactoglobulin.
Why is litmus incorporated into the medium?
To distinguish among the metabolic changes produced in milk.
Litmus is an
oxidation reduction indicator.
Litmus milk now forms an excellent differential medium in which microorganisms can
metabolize milk substrates depending on their enzymatic complement.
______ ____ now forms an excellent differential medium in which microorganisms can metabolize milk substrates depending on their enzymatic complement.
Litmus milk
~LACTOSE FERMENTATION~
Organisms capable of using __a____ as a carbon source for energy production utilize the inducible enzyme __________b__________.
a. lactose
b. ßglalctosidase
~LACTOSE FERMENTATION~
Organisms capable of using lactose as a carbon source for energy production utilize the inducible enzyme ß-galactosidase and degrade lactose as follows:
Lactose → ßgalactosidase→ GLUCOSE+ GALACTOSE
↓Embden-Meyerhof pathway
Pyruvic acid
↓
Lactic acid
~GAS FORMATION~
The end products of microbial fermentation of lactose are likely to include the gases
CO2↑(carbon dioxide)+H2↑(hydrogen peroxide).
~GAS FORMATION~
The presence of gas may be seen as
separations of the curd or by the development of tracks or fissures within the curd AS GAS RISES TO THE SURFACE.
~LITMUS REDUCTION~
_________ is an anaerobic process involving biooxidations that occur in the absence of molecular oxygen.
Fermentation
~LITMUS REDUCTION~
_______ ___ cannot exist in the free state, there
must be an immediate & concomitant electron acceptor available to bind these hydrogen ions, or else oxidation reduction reactions are not possible & cells cannot manufacture energy.
Hydrogen ions
~LITMUS REDUCTION~
In the litmus milk test, _____ acts as such an acceptor.
litmus
While in the oxidized state, the litmus is _____ (what color)
purple
When the litmus accepts hydrogen from a substrate, it will become REDUCED & turn ____ or ____ colored.
white / milk
Definition of oxidized
1. To combine with oxygen; make into an oxide.
2. To increase the positive charge or valence of (an element) by REMOVING electrons
Oxidation of lactose, which produces _____ ___, butyric acid CO2↑ & H2↑ is as follows:
Lactose→Glucose→Pyruvic acid→Lactic acid
→Butyric acid
→CO2 + H2
lactic acid
~CURD FORMATION~
There are 2 distinct types of curds. What are they
1- acid
2-rennet
~CURD FORMATION~
Acid curd
Lactic acid or other organic acids precipitation of milk protein caseinas CALCIUM CASEINATE to form an insoluble clot. THE CLOT IS HARD & WILL NOT RETRACT FROM THE WALL OF THE TEST TUBE.
Acid curds are
hard & will not retract from walls of test tube.
~CURD FORMATION~
Rennet curd
Some organisms produce rennin, an enzyme that acts on casein to form paracasein, which in the presence of calcium ions is converted to calcium paracaseinate & forms an insoluble clot. Unlike acid curd, THIS IS A SOFT SEMISOLID CLOT THAT WILL FLOW SLOWLY WHEN THE TUBE IS TILTED.
Which curd is known to be soft & semisolid and will flow slowly if tube is tilted?
Rennet curd
Which curd is known to be hard and not retract from tube walls?
Acid curd
~Proteolysis (Peptioniztion)~
The inability of some microorganisms to obtain their energy by way of lactose fermentation means they must use other nutritional sources such as ______ for this purpose.
proteins
~Proteolysis (Peptioniztion)~
By means of proteolytic enzymes, these organisms __a___ the milk protein, primarily casein, into their basic building blocks, namely __b_______.
a. hydrolyze
b. amino acids
~Proteolysis (Peptioniztion)~
Hydorlyizng milk proteins into amino acids is accompanied by the evolution of __________________, resulting in alkaline pH in the medium
large quantities of ammonia
~Proteolysis (Peptioniztion)~
Large quantities of ammonia resulting in alkaline pH in the medium turns the litmus to a _________ color in the upper portion of the tube, while the medium begins to lose body & produces a translucent, brown, wheylike appearance as the protein is hydrolyzed to amino acids.
deep purple
~ALKALINE REACTION~
An alkaline reaction is evident when the color of the medium
remains unchanged or changes to a deeper blue.
When the medium remains unchanged or turns deep blue, that reaction is indicative of the
partial degradation of CASEIN into SHORTER POLYPEPTIDE CHAINS.
Litmus milk reactions image
Control= uninoculated
The far right (very last one) is proteolysis