Inoculation

Definition: Inoculation in microbiology is the process of introducing microbes into a culture media so that they reproduce.

This process of Inoculation is carried out in different types of Culture Media such as –

1. Broth culture

   1. A broth culture is a liquid medium to cultivate microorganisms in a laboratory. It consists of a nutrient-rich solution that supports microbial growth. Broth cultures are an essential tool in microbiology to study microorganisms' growth, metabolism, and identification. Broth cultures are used in microbiology for the following purposes:

      • 1. To isolation of Microorganisms

      • 2. To study Microbial Growth

      • 3. To the preservation of Microorganisms

      • 4. To preparation of Inoculum:.

   Types of Broth Culture: There are three types of broth culture.

   • 1. General-Purpose Broth: It is used for a wide range of microorganisms.

   • 2. Selective Broth: It is used to isolate specific types of microorganisms.

   • 3. Enrichment Broth: It is used to increase the number of specific types of microorganisms,

2. Stab culture

   • Stab culture is a technique to isolate and maintain pure cultures of microorganisms using solid culture media. The needle loaded with the microorganism sample is inserted vertically into the solid culture medium. The needle creates a stab or line of inoculation. Stab culture media is used for the following purposes

     • 1. Isolation: Vertical needle insertion into a solid culture does not promote the horizontal spread of microorganisms. This promotes the development of microbial colonies in vertical lines. It will be easier to isolate the microorganism

     • 2. Maintenance: The deep penetration of the needle into the medium provides a low-oxygen environment. This can be beneficial for certain microorganisms. Stab cultures can be used to store microorganisms for a longer duration.

     • 3. Testing: It is used to assess the ability of microorganism motility in solid media.

   Types of Stab Cultures

   • 1. Straight stab: The needle is inserted vertically into the center of the medium.

   • 2. Slant stab: The needle is inserted at an angle into a slanted medium.

Various Inoculation Methods Used in Bacteriology

In Bacteriology, there are several techniques used for inoculating. Some of the most commonly used techniques are as discussed:

Streak Plate Method

This method is used to obtain completely isolated colonies from a culture or specimen inoculum through the creation of sections of increasing dilution on a single plate.

• Inoculate clinical specimens through the use of sterile inoculation loops into the agar media. Spread the specimen gently on a section of the culture media surface

• Extract the loop from the inoculated area and distribute it into a second part

• Extract the loop from the other section and disperse it to the 3rd section. Continue for the 3rd and 4th sections. Make sure that sections 1 and 4 are not overlapping. Unload the inoculation loop used into suitable containers

• Substitute the lid followed by incubating the streaked agar plate at the optimum temperature (inverted stance), so as to curb condensation

Pour Plate Method

The pour plate method is a laboratory technique for isolating and counting viable microorganisms like bacteria and fungi in a liquid sample that is added to a molten agar medium. In general, this technique counts the total number of CFUs (colony-forming units) on the surface of the solid medium.

• Serial dilution – If the sample is a liquid, it can be diluted serially with sterile broth or distilled water. If the sample is semisolid or solid, it must first be emulsified before being serially diluted to reduce the microbial load to the permissible limits.

• In the pour plate method, the sample is either added to the Petri plate or then poured with the molten agar medium, or the sample is mixed with the molten agar medium before pouring.

• Now the medium is allowed to solidify before being incubated at the appropriate temperature to grow the microbes present in the sample. The number of isolated colonies is counted after incubation.

Agar Stab Technique

• It is used to prepare stab cultures, from a plate to select single colonies.

  • Select a well-isolated colony through an aseptic technique using an inoculating stab needle (sterile) and stab it a few times via the agar to the base of the tube

  • Substitute the cap and secure it loosely during incubation enabling the exchange of gases

  • Incubation of this stabbed plate at a suitable temperature is carried out

Spread Plate Method

• It is used for evenly spreading cells to ensure the growth of the isolated separate colonies. The spread plate method is used for enrichment, enumeration and screening, and selection of microorganisms.

  • Inoculate the clinical specimen onto the agar media. Spread it gently with the help of a sterile spreader on the whole culture media surface. Also, rotate the plate backward and forward to spread the clinical specimen.

  • Substitute the lid and ensure the plate is standing in an upright position for drying (10-12 minutes)

  • Now incubate the agar plate at the optimum temperature with the lid (inverted)

• The biggest advantage of a spread plate method is that the morphology of the isolated bacteria can be seen vividly.

• The only disadvantage is that sometimes fungal colonies may grow.

• This was a brief on culture media used; some other media such as anaerobic culture techniques and liquid culture techniques also exist.

Aerobic and Anaerobic culture

Microorganisms are classified into two groups based on their oxygen requirements. These are aerobic and anaerobic microorganisms. Aerobic microorganisms require oxygen to grow. Anaerobic microorganisms can grow in the absence of oxygen.

• Aerobic Cultures: Aerobic culture contains aerobic microorganisms. These microorganisms require oxygen to generate energy through respiration. Aerobic cultures are grown in incubators with air or a mixture of oxygen-rich gases. Many common bacteria, such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, are aerobic.

• Anaerobic Cultures: Anerobic culture contains anaerobic microorganisms. These anaerobic microorganisms do not survive in oxygen. Anaerobic cultures require special techniques to create an oxygen-free environment. This can be achieved using:

  • 1. Anaerobic chambers: These are sealed chambers filled with a mixture of gases that do not contain oxygen.

  • 2. Anaerobic jars: These are sealed containers that use chemicals to absorb oxygen.

  • 3. Oxygen-free media: Some media, such as pre-reduced anaerobically sterilized (PRAS) media, are designed to create an anaerobic environment. Anaerobic bacteria, such as Clostridium botulinum and Bacteroides fragilis, are often associated with infections in deep tissues or environments with limited oxygen availability.

Isolation of pure cultures

Methods of Isolation of Pure Cultures

Pure culture means a culture media containing only one type of microorganism. Isolation of pure cultures is essential in microbiology to study the characteristics and properties of individual species.

1. Streak Plate Method

• Principle: This method involves diluting the microbial sample across the surface of an agar plate using a sterile loop. As the loop is streaked, the number of bacteria decreases. This leads to the formation of isolated colonies.

• Procedure:

  • i. Sterilize the loop.

  • ii. Collect a sample of the microbial culture using a sterilized loop.

  • iii. Streak the sample onto an agar plate in a zigzag pattern.

  • iv. Wash the loop. Sterilize the loop again and streak a new section of the plate.

  • v. Repeat the process several times.

  • vi. Incubate the plate and observe for the formation of isolated colonies.

2. Pour Plate Method

• Principle: This method involves mixing a diluted microbial sample with molten agar and pouring it into a sterile Petri dish. The bacteria are trapped in the medium during agar media solidification. This forms isolated colonies.

• Procedure:

  • i. Dilute the microbial sample.

  • ii. Mix the diluted sample with molten agar.

  • iii. Pour the mixture into a sterile Petri dish.

  • iv. Allow the agar to solidify.

  • v. Incubate the plate and observe for the formation of isolated colonies.

3. Spread Plate Method

• Principle: This method involves spreading a diluted microbial sample evenly over the surface of an agar plate using a glass spreader.

• Procedure:

  • Dilute the microbial sample.

  • Pour molten agar into a sterile Petri dish and allow it to solidify.

  • Pipette the diluted sample onto the surface of the agar plate.

  • Spread the sample evenly using a glass spreader.

  • Incubate the plate and observe for the formation of isolated colonies.

4. Enrichment Culture

• Principle: This method involves creating conditions that favor the growth of a particular microorganism while inhibiting the growth of others.

• Procedure:

  • Use a selective medium that contains ingredients that promote the growth of the desired microorganism while inhibiting the growth of others.

  • Inoculate the enrichment medium with the sample.

  • Incubate the medium and observe for the growth of the desired microorganism.

5. Selective Media

• Principle: Selective media contain substances that inhibit the growth of certain microorganisms while allowing others to grow.

• Examples:

  • MacConkey agar: Selects for lactose-fermenting bacteria.

  • Sabouraud dextrose agar: Selects for fungi.

  • Blood agar: Differentiates bacteria based on their ability to hemolyze red blood cells.

DISPOSAL OF CULTURES

The proper disposal of microbial cultures is essential to prevent the spread of pathogens and maintain laboratory safety. The following methods are used for the disposal of cultures:

1. Autoclaving

• Principle: Autoclaving uses high pressure and temperature to sterilize microbial cultures.

• Procedure:

  • i. Place the culture tubes or plates in a biohazard bag.

  • ii. Seal the bag and place it in the autoclave.

  • iii. Set the autoclave to a temperature of 121°C (250°F) and a pressure of 15 psi for at least 15 minutes.

  • iv. Allow the autoclave to cool before handling the sterilized materials

2. Incineration

• Principle: Incineration is burning the cultures to completely destroy them.

• Procedure:

  • Place the culture tubes or plates in a designated incineration chamber.

  • Ensure that the incineration is complete, leaving no residual material.

3. Chemical Disinfection

• Principle: Chemical disinfectants can be used to kill microorganisms in cultures.

• Procedure:

  • Immerse the culture tubes or plates in a disinfectant solution for the recommended contact time.

  • Dispose of the disinfected materials according to local regulations.

4. Specialized Waste Disposal Services

• Principle: For large quantities of cultures or highly hazardous materials, specialized waste disposal services may be required.

• Procedure:

  • i. Contact a licensed waste disposal company that handles biological materials.

  • ii. Follow their guidelines for packaging and transportation of the cultures.

Important Considerations:

1. Biohazard Labeling: All containers containing microbial cultures should be clearly labeled as biohazardous.

2. Personal Protective Equipment (PPE): Wear appropriate PPE, such as gloves, lab coat, and eye protection, when handling cultures.

3. Local Regulations: Follow all local regulations regarding the disposal of biohazardous materials.

4. Emergency Procedures: Have a plan in place for handling spills or accidental releases of cultures.

These guidelines ensure the safe and responsible disposal of microbial cultures. This protects both microbiologists and the environment.

Dr Pramila Singh