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
The phenomenon of producing immunity and vaccination technique consisting of introducing infectious agents into an absorptive surface of skin, in contrast to introducing the substance in tissues through injecting, is inoculation. 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.
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.
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.
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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Frequently Asked Questions
What are the different methods of inoculation?
Different methods of inoculation include stab culture, slant culture, agar culture and broth culture.
What is the spread plate and pour plate method?
Spread plate method is the method of inoculation where the microbial culture is spread onto already poured and solidified nutrient media. On the other hand, the pour plate method is pouring the bacterial inoculation first onto the plate and later pouring the nutrient media in it.
What is the purpose of streaking?
Streaking on a solidified agar plate is done to achieve isolated colonies for morphology and also sometimes to check contamination of a culture broth.