Practical of BSc 5th sem major (Microbial culture)

1. Preparation of liquid culture medium (LB) and inoculation

Aim of the experiment: To prepare liquid culture medium (LB) and inoculation of microbial sample

Principal: 

Microorganisms require nutrients for growth and multiplication. LB (Luria–Bertani) medium is a nutritionally rich liquid medium widely used for the cultivation of wide range of bacteria. It contains: Tryptone which provides amino acids and peptides, Yeast extract which supplies vitamins and growth factors and NaCl for maintaining osmotic balance. When bacteria is inoculated into sterile LB broth and incubated under suitable conditions (37°C), bacteria multiply exponentially which increases the turbidity (cloudiness) of the broth, indicating successful growth.

Materials Required:

·       LB broth powder (premixed)

·       Distilled water

·       Beaker, measuring cylinder, conical flask

·       Autoclave, Shaker Incubator, Laminar Air Flow Unit (LAFU)

·       Cotton plug, gloves, spatula

·       Inoculation loop, spirit lamp, 70% alcohol

Methodology/ Procedure:

1.     Weigh 1.25 gram of LB broth powder in a conical flask (size 250 ml) and add 100 ml of distilled water. Dissolve the media by gentle swirling.

2.     Sterilize by autoclaving at 121°C for 30 minutes at 15 psi pressure.

3.     Allow the sterilized LB Broth to cool up to 50°C at room temperature.

4.    Under LAFU, dip the inoculation loop in 70% alcohol and heat on the flame of spirit lamp till the loop becomes red hot. Allow it to cool for at least 2 minutes.

5.   Take a small number of bacteria from the bacterial sample and dip into the media. Swirl gently to mix the bacteria into the broth.

6.     Incubate the flask at 37°C for 24 hours with gentle shaking.

Results: 

The broth appeared to be turbid (cloudy) after incubation period, which indicates successful growth of bacteria in the LB broth media.  

Discussion: 

The quantitative bacterial growth can be observed by measuring the optical density (OD) of the media in a spectrophotometer. The degree of turbidity can be correlated with bacterial density, and optical density (OD) at 600 nm can be used for quantitative measurement.

2. Preparation of solid culture medium (LB) and growth of bacteria by spreading and streaking

Aim of the Experiment: To prepare solid LB (Luria–Bertani) agar medium and study the growth of bacteria on agar plates using the streak plate and spread plate methods.

Principle:

Microorganisms require nutrients and suitable physical conditions for growth. LB agar is a nutrient-rich solid medium that supports the growth of many bacterial species. The solidifying agent, agar, provides a firm surface for colony isolation. Two basic techniques are used for bacterial culture on solid medium:

• Streak Plate Method: This method is used to isolate individual colonies from a mixed or concentrated culture by spreading the inoculum in successive streaks with sterilized loop.
• Spread Plate Method: This method is used to distribute a diluted bacterial suspension evenly on the surface of the solid medium to obtain countable colonies.

Each bacterial cells give rise to a complete colony and thus represents pure culture.

Materials Required:

·       LB broth (premixed), Agar powder

·       Distilled water

·       Beaker, measuring cylinder, conical flask, sterile Petri plates

·       Autoclave, Incubator, Laminar Air Flow Unit (LAFU)

·       Cotton plug, gloves, spatula

·       Inoculation loop, spreader, spirit lamp, 70% alcohol

·       Bacterial culture

Methodology/Procedure:

A.    Preparation of LB Agar Medium

1.   Weigh 12.5 gram of LB broth powder in a conical flask (size 1000 ml) and add 1.2 gram of Agar powder. Adjust the volume to 500 ml of distilled water. Cover the flask with a tight cotton plug and dissolve the media by gentle swirling.

2.     Sterilize by autoclaving at 121°C for 30 minutes at 15 psi pressure. Allow the sterilized media to cool to around 50°C.

3.  Under LAFU, pour about 20-25 ml of media into sterile Petri plate and allow the plates to solidify at room temperature. 

B.    Inoculation by Streaking Method

4. Sterilize the inoculation loop in 70% alcohol and heat on the flame till red hot. Allow it to cool for at least 2 minutes.
5. Take a small portion of bacterial culture and streak it gently over the surface of the LB agar plate in three or four sectors, sterilizing the loop between each streak.
6. Incubate the plate in inverted position at 37°C for 18-24 hours.

C. Inoculation by Spreading Method

7.     Pipette out 100 µL of the liquid bacterial culture onto the surface of a solidified LB agar plate.

8.     Spread the bacteria evenly by rotating the plate in all directions using a sterile spreader (L-rod) sterilized in alcohol and flamed.

9.     Incubate the plate in inverted position at 37°C for 18-24 hours.

Results: 

1. In streak plate, discreate well separated colonies appear in later streaks, which represents pure bacterial colonies.

2. In spread plate, evenly distribute colonies appear over the surface, which can be assessed for quantitative growth by counting CFU/ml. 

3. Colony forming unit/ml (CFU/ml)= No. of colonies on plate× dilution factor. 

4. E. coli colonies are observed to be circular, creamy white and surface growing in nature