Methanogens

- Methane producing microbes are called Methanogens.
- Methane is the gas and metabolic byproduct of the organisms.
- Methanogens belongs to the archaea domain, they are obligate anaerobes.
- They are very sensitive to oxygen.
- They are commonly present in wetlands and digestive tracts of animals (ruminants, humans)

- Methanogens are cocci (spherical) or bacilli (rod) shaped bacteria. 
- They are chemoautotrophic organisms ( using inorganic energy source such as molecular hydrogen, hydrogen sulphide, elemental sulfur etc.) 

- They reduce the carbon dioxide to give methane (marsh gas) by using hydrogen as electron source: 

                     CO₂ + 4 H₂ →  CH₄ + 2H₂O

- Formation of methane from microbes called methanogenesis. 

 Examples of Methanogens :

-Methanobacterium 
-Methanobrevibacter
-Methanosarcinae
-Methanococcus
-Methanospirillum
-Methaothermobacter
-Methanothrix

Metabolic Pathway of Methanogens

Source: D Browne, Patrick & Cadillo-Quiroz, Hinsby. (2013). Contribution of Transcriptomics to Systems-Level Understanding of Methanogenic Archaea. Archaea (Vancouver, B.C.). 2013. 586369. 10.1155/2013/586369. 

Overview of the three major known methanogenic pathways in Archaea. Color coding indicates the steps common to all three types (black), unique to the methylotrophic pathway (green), unique to the hydrogenotrophic (or CO2 reducing) pathway (blue), unique to the aceticlastic pathway (red), and shared between hydrogenotrophic and aceticlastic methanogenesis (purple). 2e− represents reducing equivalents, produced or consumed during each reaction. MFR: methanofuran; H4MPT: tetrahydromethanopterin; CoM-SH: coenzyme M; CoB-SH: coenzyme B; CoA-SH: coenzyme A; CoM-S-S-CoB: heterodisulfide of coenzyme M and coenzyme B; ATP: adenosine triphosphate; R: ligand bound to methylated compound that serves as substrate for methylotrophic methanogenesis. *Tetrahydrosarcinapterin is a functional analogue of H4MPT found in the Methanosarcinales order of methanogens.

- Some times the marine methanogens produce methane form acetic acid. 

Measurement of Microbial Growth by Total Viable Count Method

Objective:

To find the generation time of microorganisms by total viable count method.

Principle:

The number of cells capable of dividing and forming the colonies on solid medium are called viable number cells. Total viable count method is more convenience because there is virtually no other reliable and virtual way to count living organisms. In this method, serial dilutions of a sample containing viable microorganisms are plated onto a suitable growth medium. The suspension is either spread onto the surface of agar plates (spread plate method), or is mixed with molten agar, poured into plates, and allowed to solidify (pour plate method). The plates are then incubated under conditions that permit microbial reproduction so that colonies develop that can be seen without the aid of a microscope. It is assumed that each bacterial colony arises from an individual cell that has undergone cell division. Therefore, by counting the number of colonies and accounting for the dilution factor, the number of bacteria in the original sample can be determined.

Materials required:

-Nutrient agar

- 10- 12 hrs old broth culture

-Sterile pipette  

-L –rod

-Turn table

-Petri dishes

Procedure:

-  The bacterial sample is serially diluting upto 10⁴ dilutions (add 0.5 ml of sample in 4.5 ml of saline).     

-Transfer the 0.1 ml from each dilution tubes to the corresponding agar plates by spread plate technique.

-  Incubate the plates  at 37 ͦ c  for 24 hours

- following the incubation number of cells will be counted.

- Repeat this process from initial time of inoculation (0^th hour) to 7-8 times for every 30 min.  

- Calculate the CFU each time and the results will be tabulated and plotted the graph.

- To determine the generation time by using the formula Gt = t2 - t1  (CFU2 -CFU1).

Result:

The generation time of ­­­_______cells found to ­­­­­________ min/hr.

CFU calculation:

No. of CFU / volume plated (ml) x total dilution used    to     No. of CFU/mL

Nutrient Agar Composition:

Peptone   5g

Beef extract   2g

Yeast extract    3g

Sodium chloride   5g

Agar   15 g

Distilled water   1000ml

Saline  Preparation: 

0.9 gm of Nacl in 100ml of water.

Measurement of Microbial growth by turbidometric method

Objective:

To find the generation time of given organism by turbidity method.

Principle:

Turbidometric method is the rapid, sensitive and useful technique to measure the growth of microbes. Turbidometry is based on the fact that microbial cells scatter light striking them. Since the microbial cells in a population are of roughly constant size, the amount of scattering is directly proportional to the biomass of cells and indirectly related to cell number. One visible characteristic of growing bacterial culture is the increase in turbidity (cloudiness) of the medium.  When the concentration of bacteria increasing, the medium appears slightly cloudy or turbid. Further increase in concentration results in greater turbidity. When a beam of light is passed through a turbid culture, the amount of light transmitted is measured, Greater the turbidity, lesser would be the transmission of light through medium. Thus, light will be transmitted in inverse proportion to the number of bacteria. Turbidity can be measured using spectrophotometer.

Materials required:

-Nutrient broth

- 10- 12 hrs old broth culture

-Spectrophotometer

-Cuvette

Procedure:

-  Prepare and take 50ml of nutrient broth in sterile flask.

- Then this broth is inoculated with 1 ml of overnight bacterial culture and places it in incubator.

- The 0^th hour reading take by using spectrophotometer and the culture is again keep it in incubator.

- The flask is removing from incubator for a particular interval time (30 min.) and the sample is transfer aseptically to the cuvette using sterile pipette for observe the reading.

- The optical density reading in spectrophotometer is at 620nm.

- The reading will be tabulated and plotted the graph by OD value in Y-axis and incubation time in X -axis.  

Determination of generation time:

-Select the 2 points on the optical density scale that represents doubling the turbidity.

 The generation time is to be determine by using the formula

 Gt = t (OD2) –t(OD1)

Result:

The generation time of ­­­_______cells found to ­­­­­________ min/hr.

Peptone Broth Composition:

Peptone    20g

Sodium chloride    5g

Distilled water   1000ml

Direct Microscopic count

Objective:

To determine the concentration of bacterial cell in a given sample by direct microscopic count method and calculating the generation time of the cell.

Principle:

Direct microscopic counts are performed by spreading a measured volume of sample over a known area of a slide, counting representative microscopic fields, and relating the averages back to the appropriate volume-area factors. Specially constructed counting chambers, such as the Petroff-Hauser chambers and Heamocytometer, simplify the direct counting procedure because they are made with depressions in which a known volume overlies an area that is ruled into squares. The ability to count a defined area and convert the numbers observed directly to volume makes the direct enumeration procedure relatively easy. Direct counting procedures are rapid but have the disadvantage that they do not discriminate between living and dead cells.

The ruled area of the hemocytometer consists of several large 1 x 1 mm (1mm² ) squares, which are subdivided in three ways; 0.25 x 0.25 mm (0.0625 mm²), 0.25 x 0.20 mm (0.05 mm²) and 0.20 x 0.20 mm (0.04 mm²). The central, 0.20 x 0.20 mm marked, 1 x 1 mm square is further subdivided into 0.05 x 0.05 mm (0.0025 mm²) squares. Hold the cover slip(  0.1 mm)  at the raised edges of hemocytometer, which gives each square a defined volume.

 

	Area	Volume at 0.1mm depth
1 x 1 mm	1 mm2	100 nl
0.25 x 0.25 mm (1/16)	0.0625 mm2	6.25 nl
0.25 x 0.20 mm (1/20)	0.05 mm2	5 nl
0.20 x 0.20 mm (1/25)	0.04 mm2	4 nl
0.05 x 0.05 mm (1/400)	0.0025 mm2	0.25 nl

                                            

Materials required:

-Uniform bacterial cell suspension in peptone/nutrient broth

-Haemocytometer

-Coverslip

-Micropipette

-Microscope

 Procedure:

-Preparation uniform cell suspension:  Take cell suspensions in test tube and pipette out up and down for several times to get accurate cell counting and uniform distribution of the cell.

-Place a coverslip over the calibrated surface of the counting chamber.

- Using a pipette, transfer the suspension to the groove of the counting chamber to fill the chamber by capillary action.

-To observe the cells by using high magnification in the microscope, and count the number of cells in at least 5 of the small squares.

-Do the bacterial cell counting by using above methods, from 0^th hour upto several times for every 30 minutes to find out the generation time of cells.

-Take cell suspension for every time from culture flask which is incubated at 37 ͦ C

-Use the following formula to calculate the number of cells and plot the graph to find out the generation time of particular microbial cells

               Bacteria/mm³ = (bacteria/square) (25 squares) (50) (10³) 

Result:

The generation time of ­­­_______cells found to ­­­­­________ min/hr.

Peptone Broth Composition:

Peptone    20g

Sodium chloride    5g

Distilled water   1000ml

Nutrient Broth Composition:

Peptone   5g

Beef extract   2g

Yeast extract    3g

Sodium chloride   5g

Distilled water   1000ml

Carbohydrate Fermentation Test

Objective:

To determine the ability of microorganism to ferment the given carbohydrates with acid or acid and gas end products. 

Principle:

Fermentation is the metabolic process to breaking down or catabolism of the sugars under anaerobic conditions. The result of this process is production of energy source ATP by substrate level phosphorylation, in the absence of aerobic respiration electron transport chain. Microorganisms utilize the carbohydrates by their enzymatic reactions and produce organic acids, acid with gas and alcohol. The types and proportion of the products depend on bacterial species and carbohydrates.

 Formation of acids in sugar broths can be detected by using the pH indicator phenol red which is red at neutral pH and changes to yellow at acidic conditions, slight amount of acid can cause color change. Gas formation can be detected by using an inverted ‘Durham’s tube’.

Materials required:

-24 Hours old bacterial cultures (E.coli. Pseudomonas sp. Klebsiella sp. and Salmonella sp.)

-Peptone broth. Nutrient broth with pH indicator (Phenol red)

-Test tubes and Durham tube

Procedure:

- Prepare and sterilize the tubes containing 5 ml of peptone broth/nutrient broth for each sugar lactose, sucrose and glucose with pH indicator.

- Carefully insert the Durham tube in the broth tube in inverted position without air bubble.

- Incubate the tubes at 37°C for 24 - 48 hours

Result:

-E.coli and Klebsiella sp., shows both yellow color changes in the medium and bubble formation inside Durham’s tubes in all the sugars.

-Salmonella sp. shows yellow color changes in the medium and gas bubble formation in glucose, only yellow color change in sucrose and no reaction in lactose sugar.

-Pseudomonas sp. shows yellow color change in glucose, no reaction in sucrose and lactose.

Interpretation:

If the medium changes colorless to yellow color and gas bubble formation in Durham’s tube that indicates acid production and gas production. In some cases the gas does not evolved during fermentation. If no changes in the medium it indicates the sugars not degraded by the organisms.

Carbohydrate Fermentation Medium Composition:

Peptone     10.0 g

Sodium chloride   5.0 g

Sugar    1 g

Phenol red   5 ml

Distilled water    1000ml

pH   - 7.00

Sugar fermentation by different bacterial species

Urease Test

Objective:

To determine the ability of microorganisms to degrade urea by the enzyme urease.

Principle:

 Urea is a nitrogen containing compound that is produced during decarboxylation of the amino acid arginine in the urea cycle. Urea is highly soluble in water and is therefore an efficient way for the human body to discharge excess nitrogen. This excess urea is then taken out of the body through the kidneys as a component of urine. Some bacteria have the ability to produce an enzyme urease as part of its metabolism to break down urea to ammonia and carbon dioxide.
 Many enteric bacteria have the ability to hydrolyze urea as part of their metabolism, members of the genus Proteus are considered rapid urease producers due their efficiency in carrying out this process. Therefore, this experiment is useful in distinguishing members of Proteus, a urinary tract pathogen, from other enterics based on their ability to rapidly hydrolyze urea. Many enterics can hydrolyze urea but only a few can degrade  it rapidly. These are commonly referred as "rapid urease-positive" organisms. Members of the genus Proteus have the ability to hydrolyze urea rapidly.

  

Urea Hydrolysis:  Urea is waste product excreted in urine by animals. Some enteric bacteria produce the enzyme urease, which splits the urea molecule into carbon dioxide and ammonia. 

Urease, which is produced by some micro organisms, is an enzyme that is especially helpful in the identification of Proteus vulgaris, although other organisms may produce urease, their action on the substrate urea tends to be slower than that seen with Proteus species. Therefore this test serves to rapidly distinguish members of this genus from other lactose non fermenting enteric microorganisms.Urea is unstable and is broken down at 15 psi or pressure. It cannot be added to the medium for autoclaving and is therefore filter sterilized and added to the medium after autoclaving.

 Materials Required:

24 hours broth cultures (Proteus sp. and E.coli). 

Urease both / Urease Slant

Procedure: 

Using a sterile technique, inoculate each experimental organism into its appropriately labeled tube by means of loop inoculation. Incubate cultures 24-48 hours at 37°C.

Urea Broth method: -Inoculate the urea broth with the inoculation loop containing the organism from the broth cultures.-Incubate for 24-48 hours at 37°C. –-Obtain the broths from the incubator and observe the colour.

Urea Slant method:-Inoculate the urea slant (slope) with the inoculation loop containing the organism from the broth cultures. Do not stab the butt.-Incubate for 24-48 hours at 37°C.-Obtain the broths from the incubator and observe the color.

Result: -Proteus sp. gives urease positive reaction  -E.coli gives urease negative reaction

 Interpretation: Proteus sp. produces urease enzyme to degrade the urea to release the ammonia, it leads to alkaline condition of the medium. The phenol red indicator in the medium turns red pink color in alkaline condition and gives positive result.  E.coli does not produces urease enzyme and giving negative reaction.

Urea Broth Composition:

Urea      20g

Dibasic Sodium phosphate    9.5 g

Monopotassium phosphate    9.1 g of

Phenol red      0.01 g

The pH is made to   6.8±0.2 at 25°c.

Distilled water   1000 ml

  

Urea Slant Composition:

Same as above composition, but add agar as one of the ingredient.

Hydrogen Sulphide (H2S) Production Test

Objective:

            To determine the ability of microorganism reduces the sulphur containing compounds to hydrogen sulphide during metabolism.

Principle:

            Some bacteria utilize sulphur containing amino acids such as cysteine from the proteins by enzyme desulferase; it loses the sulfur atom then reduced by addition of hydrogen atom from water to form hydrogen sulphide.  This organism also reduces the inorganic sulphur compounds such as sodium thio sulfate, sulfate and sulfite to gaseous hydrogen sulphide.

            The SIM (Silfide, Indole, and Motility) agar contains the peptone a source of cysteine and sodium thiosulfate as sulfur substrates, ferrous ammonium sulfate which behaves as the H₂S indicator. If H₂S produced it combines with ferrous ammonium sulfate, forming insoluble black ferrous sulfide precipitate.   

Materials required:

24 Hours old bacterial cultures (E.coli. Proteus sp., Klebsiella sp. and Salmonella sp.)

SIM agar medium

Procedure:

- Prepare and sterilize the SIM agar deep tubes, adjust the pH to 7.4

- Inoculate the bacterial cultures by straight stabbing to a depth of 2-3 cm.

- Incubate the tubes at 37°C for 24 - 48 hours

Result:

Salmonella sp. and Proteus sp. gives positive result.

E.coli and Klebsiella sp. gives negative result.

Interpretation:

Salmonella sp. and Proteus sp.  shows blackening color due to the production of hydrogen sulphide and gives positive reaction. E.coli and Klebsiella sp. are not showing blackening color.

SIM Agar Medium Composition:

Beef extract    3.0 g

Peptone  30.0 g

Ferrous ammonium sulphate   0.2 g

Sodium thiosulphate 0.025 g

Agar   3.0g

Final pH ( at 25°C) 7.3±0.2

Distilled water    1000ml

Triple Sugar Iron (TSI) Test

Objective:

            To differentiate the enterobacteriaceae members according to their ability to ferment lactose, sucrose and glucose sugars and production of the hydrogen sulphide.

Principle:

            The fermentation of sugars will help to distinguish enteric bacteria from other gram negative bacilli. The TSI agar contains 1% concentrations of lactose and sucrose, 0.1 % of glucose. The phenol red is an acid base indicator is incorporated in this medium to detect acid production from carbohydrate fermentation.

            Acidification of medium is caused by intestinal organisms to attacking the sugars and it changes the phenol red to yellow color. This medium also contains sodium thio sulfate, the organisms reduces the sulfur to form hydrogen suphide gas and it is react with ferrous sulfate which is present in the medium to give black precipitation.

-0.1% Glucose: If only glucose is fermented, only enough acid is produced to turn the butt yellow.  The slant will remain red.

-1.0% lactose/1.0% sucrose:  a large amount of acid turns both butt and slant yellow, thus indicating the ability of the culture to ferment either lactose or sucrose.

-Sodium thio sulfate : Substrate for Production of Hydrogen sulphide (H₂S)

-Iron: Ferrous sulfate: Indicator of H₂S formation

-Phenol red: Indicator of acidification (It is yellow in acidic condition and red under alkaline conditions).

Materials required:

24 Hours old bacterial cultures (E.coli ., Proteus sp. and Klebsiella sp.)

Triple sugar iron agar

Procedure:

- Prepare TSI medium and adjust the pH to 7.4

- Sterilize the medium and pour into sterile test tubes and make slants

- Inoculate TSI agar slants by first stabbing in center of the medium and streaking on the surface of the slant

- Incubate the tubes at 37°C for 18 - 24 hours

Result:

-E.coli and Klebsiella sp., - shows yellow (acid) slant/ acid (yellow) butt with gas production.

-Proteus sp., shows acid butt, alkaline (red) slant with hydrogen sulphide (black precipitation) and gas production.

E.coli  & Klebsiella sp.             -   A/A with gas

Proteus sp.,                                -  K/A with H2S production

Interpretation:

From the above results klebsiella sp. and E.coli both are able to ferment all the sugars present in the TSI agar and produces acid and gas production indicate by bubble formation. Proteus sp. is ferment the glucose only, doesn’t ferment the remaining sugars, this organism also reduced the sulfur to hydrogen sulphide gas and it is observed as blackening precipitation.

Composition of TSI Agar

Beef extract   3.0 g

Yeast extract  3.0 g,

Peptone     15 g,

Protease peptone  5 g,

Lactose 10.0 g

Sucrose 10.0 g

Glucose 1.0g

Ferrous sulphate   0.2 g

Sodium chloride   5.0 g

Sodium thiosulphate   0.3 g,

Phenol red    0.024 g

Agar   12 g

Distilled water    1000ml

some examples of Triple Sugar Iron (TSI) Agar Reactions: 

Name of the organisms	Slant	Butt	Gas	H2S
Escherichia, Klebsiella, Enterobacter	Acid (A)	Acid (A)	Pos (+)	Neg (-)
Shigella, Serratia	Alkaline (K)	Acid (A)	Neg (-)	Neg (- )
Salmonella, Proteus	Alkaline (K)	Acid (A)	Pos (+)	Pos (+)
Pseudomonas	Alkaline (K)	Alkaline (K)	Neg (-)	Neg (-)