GELATIN HYDROLYSIS TEST

Material required: 

1. Gelatin Agar(Peptic digest of animal tissue 5 g/L, Beef extract 3 g/L, Gelatin 120 g/L, Final pH ( at 25°C) 6.8±0.2),
2. Boiling test tubes
3. Cotton Plug
4. Inoculation needle
5. Culture(Bacillus subtilis and Escherichia coli).

Theory:

Gelatin is a protein derived from the connective tissues of vertebrates, that is, collagen. It is produced when collagen is boiled in water. Gelatin hydrolysis detects the presence of gelatinases. Gelatinases are proteases secreted extracellularly by some bacteria which hydrolyze or digest gelatin. This process takes place in two sequential reactions.
In the first reaction, gelatinases degrade gelatin to polypeptides.

Then, the polypeptides are further converted into amino acids.

 
 FIG Reactions involved in gelatin hydrolysis. 

The bacterial cells can then take up these amino acids and use them in their metabolic processes.
Gelatin hydrolysis is detected using a nutrient gelatin medium. This medium contains peptic digest of animal tissue (peptone), beef extract, and gelatin. Gelatin serves as both solidifying agent and substrate for gelatinase activity. When nutrient gelatin tubes are stab-inoculated with a gelatinase-positive bacterium, the secreted gelatinases will hydrolyze the gelatin resulting in the liquefaction of the medium. Since gelatin is digested and is no longer able to gel, the medium will remain liquid when placed inside a refrigerator or in an ice bath. A nutrient gelatin medium inoculated with a gelatinase-negative bacterium will remain solid after the cold treatment. The medium can be inoculated with both aerobic and anaerobic bacteria and incubated as appropriate.

TABLE  List of common bacteria and their reactions to the gelatin hydrolysis test performed on nutrient gelatin

Species	Growth	Liquefaction
Bacillus subtilis	+	+
Clostridium perfringens	+	+
Escherichia coli	+	-
Proteus vulgaris	+	+
Serratia liquefaciens	+	+
Staphylococcus aureus	+	+

Procedure:

1. Prepared gelatine agar and distributed in three boiling tubes (two for two bacteria and one for control).
2. Autoclaved at 121 ˚C for 15 minutes.
3. Sterilize the needle in the blue flame of the Bunsen burner till red hot and then allowed to cool. 
4. Inoculate Bacillus subtilis and Escherichia coli in two nutrient gelatin tubes(Test) and keep one tube uninoculated as(control). 
5. Incubate at 37 ^˚C .
6. After incubation gently place the two tubes in the refrigerator for 15-30 minutes.   
7. After 15-30 minutes, if the gelatin remains LIQUID, the result is POSITIVE
8. If the gelatin is SOLID, the result is negative.   

 

EXPECTED RESULT:

CARBOHYDRATE FERMENTATION

Objective: 

• To find the ability of microorganisms to ferment the given Carbohydrate.
• To determine the ability of microorganism to produce gaseous end products in fermentation.

Principle:
A metabolic process performed by almost all types of bacteria is known as fermentation. This will result in the production of ATP, the ultimate energy source of the organism. This will happen either in the presence or absence of atmospheric oxygen. Bacteria utilize the nutrients in their environment to produce ATP for their biological processes such as growth and reproduction. The enzyme systems in bacteria allow them to oxidize environmental nutrient sources. Bacteria will use different energy sources in the medium depends on the specific enzymes of each bacteria. Many bacteria possess the enzymes system required for the oxidation and utilization of the simple sugar, glucose. Some bacteria have the ability to degrade complex carbohydrates like lactose, sucrose or even polysaccharides. Such bacterium should possess the enzymes that should cleave the glycosidic bonds between the sugar units and the resulting simple carbohydrate can be transported into the cell. Lactose is a disaccharide consisting of the glucose and galactose connected by glycosidic bond. The bacteria which produce the enzyme lactase will break this bond and thus release free glucose that can be easily utilized by the organism. The characteristics feature of the enzyme production in the bacteria enables them to use diverse carbohydrates and this will aid in the identification of unknown bacteria.

 Phenol red broth is a general purpose fermentation media comprising of trypticase, sodium chloride, phenol red and a carbohydrate. The trypticase provides amino acids, vitamins, minerals and other nitrogenous substances making it a nutritious medium for a variety of organisms.  Sodium chloride helps in maintaining the osmotic balance and provides the essential electrolytes for the transport into the cell while the carbohydrate acts as the energy source. The phenol red is the pH indicator and is initially neutral (pH 7). It supports the growth of most organisms whether they are able to ferment sugar or not.  When the bacterium is inoculated into the tube, the bacterium which ferments the sugar will result in the production of acid that will change the color of phenol red. Fermentation reactions often begin with glycolysis. Glucose acts as an electron donor in the fermentation reaction, pyruvate, and metabolic product of glucose act as an electron acceptor. The other disaccharides and polysaccharides are hydrolyzed into glucose or converted into glucose and then the fermentation reaction will occur. Finally the reaction will result in the end products such as acid, ethanol, Hydrogen and Carbon dioxide and other compounds. This depends on the species of bacteria. Phenol red broth is a test is differential for gram negative bacteria.
When the organism ferments carbohydrates, acidic organic by products (Lactic acid, formic acid or acetic acid) is accumulated which turns the medium into yellow color with reduction in the pH (acidic). The inverted Durham tubes will detect the presence of gas. The degradation of peptones in the broth may result in the production of alkaline end products, which will change the broth color to pink often at the top of the tube.

 

Materials Required:

1. Phenol Red Carbohydrate Fermentation Broth(Phenol red dextrose broth, Phenol red sucrose broth, Phenol red glucose b).
2. Bacterial culture (Staphylococcus aureus, Escherichia coli and Alcaligenes faecalis).
3. Inoculation loop.
4. Incubator(37⁰ C).
5. Boiling test tubes.
6. Durham’s tubes.

Procedure:

I. Preparation of Carbohydrate Fermentation Broth

1. Prepare desired phenol red carbohydrate broth (Trypticase/peptone: 10 g/L, Carbohydrate;Dextrose, Sucrose, lactose : 5 g/L, Sodium Chloride: 5 g/L, Phenol red :  0.0189 g/L) in a conical flash.

1. Transfer 40-50 ml of desired phenol red carbohydrate broth(s) to boiling test tubes and label these with respect to the sugar added.
2. Insert inverted Durham tubes into all tubes, the Durham tubes should be fully filled with broth.
3. Autoclave at 115⁰ C for 15 minutes.

Important: Do not overheat the Phenol red Carbohydrate fermentation broth. The overheating will result in breaking down of the molecules and form compounds with a characteristic color and flavour. The process is known as caramelisation of sugar (the browning of sugar).

II. Inoculation of Bacterial Culture into the Phenol Red Carbohydrate Broth

1. Aseptically inoculate each labeled carbohydrate broth with bacterial culture(s) in laminar air flow hood.(keep uninoculated tubes as control tubes).
2. Incubate the tubes at 24 hours at 37^oC.
3. Observe the changes in colour of the medium and accumulation of gas in the durham’s tube with respect to the control fermentation tube.

Precautions: 

1. During aseptic inoculation of bacterial culture, never shake the inoculation loop in the sugar tubes in order to eliminate the chance of gas entrapment in the durham’s tube
2. After inoculation into a particular sugar, sterilize the loop in order to avoid cross contamination of the tube with other sugars.
3. Keep uninoculated sugar tubes as control tubes.
4. Do not use the tubes with Durham tubes that are partially filled or with bubbles.
5. Over incubation will help the bacteria to degrade proteins and will result give false positive results.

Expected Results:

1. Acid production: Changes the medium into yellow color- organism ferments the given carbohydrate and produce organic acids there by reducing the ph of the medium into acidic.
2. Acid and Gas production: Changes the medium into yellow color-organism ferments the given Carbohydrate and produce organic acids and gas. Gas production can be etected by the presence of small bubbles in the inverted durham tubes.
3. Absence of fermentation: The broth retains the red color. The organism cannot utilize the carbohydrate but the organism continues to grow in the medium using other energy sources in the medium.

BACTERIAL SPECIES	CARBOHYDRATES
	GLUCOSE	SUCROSE	LACTOSE
Escherichia coli	AG	A	AG
Staphylococcus aureus	A	A	A
Alcaligenes aureus
Control
NOTE: A: Acid only i.e Sugar broth turns yellow. AG: Acid and gas production i.e sugar broth turns yellow and gas/air bubble accumulation in the Durham’s tube. : No change : Variation reaction