Inoculation and Plating Methods

Inoculation – Definition and Methods

Inoculation is the process of inducing immunity against infectious diseases through artificial means.

The quantitative determination of bacterial populations can be done in various ways. Here, let’s look at the meaning of inoculation and the various inoculation techniques like the pour plate method in detail.

Table of Contents

• What is Inoculation? – Inoculation Definition
• Various Inoculation Methods Used in Bacteriology
• Streak Plate Method
• Pour Plate Method
• Agar stab technique
• Spread Plate Method
• Frequently Asked Questions

What is Inoculation? – Inoculation Definition

Meaning of Inoculation

Inoculation is the act of inducing immunity by introducing infectious agents into the body.

In the past, the process of inoculation meant introduction of the infectious substances into the absorptive surface of the skin. With advancements came vaccines, the process of inoculation included vaccination which required the introduction of an altered and weakened version of the infectious particles into the body.

Other definitions of Inoculation

• Inoculation can also be referred to the act of introduction of a microbe into a new medium like the inoculation of bacteria in the culture medium. Broadly, it is to introduce antigenic substances into tissues.

It has a precise definition for processes carried out in vitro which involves the transferring of microbes from and to lab apparatus in research laboratories. It also finds its usage in commercial applications such as baking, brewing, wine making, producing antibiotics and more. A good example is the production of blue cheese through the inoculation of ripening cheese with dedicated moulds of bacteria.

Inoculation in Microbiology

• In simple terms, inoculation in microbiology is the process of introducing microbes into a culture media so that it reproduces there. Commonly, it is used in the introduction of vaccines, serum or any antigenic substance in the body so as to boost immunity against a particular disease.

For instance, inoculation could mean the addition of a kind of chemical or nutrient in a suspension of bacteria. That is referred to as inoculating that suspension with that particular chemical or nutrient. In the introduced culture, the microbes can reproduce and grow. Mostly, it is used in research and laboratory practices wherein researchers aim to cultivate and study some species of bacteria. Bacteria and other microbes can be inoculated into a range of media where it thrives.

This process of Inoculation is carried out in different media such as – plate culture, slant culture, agar plates, stab culture, sweep plate method and so on.

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 loop from the inoculated area and distribute into a second part
• Extract the loop from the other section and disperse it to the 3rd section. Continue for the 3rd and 4th section. Make sure that sections 1 and 4 are not overlapping. Unload 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 and 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.

The major difference between the streak and pour plate method is that in the streak plate, the melted agar is added first, followed by a loop of bacteria from a slant, whereas in the pour plate, the bacterial broth is added first, followed by the agar.

Agar stab technique

It is used in the preparation of stab cultures, from a plate select single colonies.

• Select a well-isolated colony through 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 loosely during incubation enabling exchange of gases
• Incubation of this stabbed plate at the suitable temperature is carried out

Spread Plate Method

It is used for evenly spreading cells to ensure growth of the isolated separate colonies. Further, it can be used for serial dilutions. The spread plate method is used for enrichment, enumeration and screening and selection of microorganisms. 

• Onto the agar media, with the help of a sterile spreader, inoculate the clinical specimen where we spread the bacteria gently on the whole culture media surface. This is done by rotating the plate while spreading it backwards and forward. Refrain from allowing the spreader to touch the edges of the plate
• Substitute the lid and ensure the plate is standing in an upright position for drying (10-12 minutes)
• Now incubate the spread agar plate at the optimum temperature with the lid at the base (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. 

Also check: Bacterial Culture Methods

Inoculating loops and Inoculating needles

Inoculation loops (smear loops) or a inoculation rod are used by microbiologists to mainly transfer inoculum (some amount of sample of microbe culture). The apparatus comprises a fine handle with a tiny loop towards the terminal. The twisted wire loops have now been substituted by moulded plastic ones, commonly in use.

Inoculation needles are lab tools used in transferring inoculate living microbes. These can be re used or are disposable. A standard one is made from platinum or nichrome which is fixed with a metallic handle. Inoculation needles that can be disposed are usually made from plastic resins.

While inoculation needles are used to retrieve dense or solid media, an inoculation loop can retrieve liquid media.

Uses of Inoculating loops and Inoculating needles

Inoculation needles and loops are hand-held apparatus. They are of the following uses –

• Inoculation needles are functional to sample and transfer small quantities of inoculum, they are used to study fungi and bacteria on semi-solid media
• Inoculation handles are used in inoculation, sterile sampling, serial dilution, spreading and transferring microbial samples
• Inoculation needles works on isolation of highly defined areas of the cultures and requirements of minimum disturbance between two densely crowded colonies
• Inoculation needles can be used in streaking on streak plates, inoculation of stab cultures, fish tail inoculation of slant cultures
• Loop threads can be used to take large samples
• Straight needles are functional in taking single colony samples of microbes

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

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