Culturing Microorganisms

If you want to grow bacteria, you need to provide them with the right conditions to thrive. This is where culture media comes in. It contains carbohydrates, minerals, proteins, and vitamins that bacteria need to grow. You can use a solid form of agar jelly or a solution of nutrient broth as the culture medium. When bacteria are grown on agar plates, they form visible colonies on the surface or spread out evenly. However, it's important to note that dangerous pathogens are more likely to grow above 25°C. That's why microorganism cultures are not kept in school labs at temperatures higher than this. In industrial settings, cultures are incubated at higher temperatures to speed up the growth process.

Agar plate

To grow microorganisms, you need to place hot agar jelly in shallow, spherical Petri dishes to create agar plates. Once the jelly has cooled and set, you can transfer the microorganisms to the culture media using inoculating loops. Alternatively, you can use a spreader and sterile dropping pipette to evenly distribute the germs. After that, the microbes can grow and develop. 

Required practical – Investigating the effect of antibiotics on bacterial growth

The steps listed below can be used to assess howantibiotics affect bacterial cultures:

1.      On an agar plate with an even layer of bacteria, place paper discs soaked in several antibiotic kinds or concentrations. Disks should be separated from one another.

2.      In the agar jelly, the antibioticshould diffuse (soak). On the agar around the paper discs, antibiotic-resistantbacteria will keep growing while non-resistant strains would perish. Where thebacteria have died, a clean space known as an inhibition zone will remain.

3.      Becareful to employ a control. This paper disc is the original, untreatedversion. Soak it in sterile water instead. Then, you can be sure that anychange in the growth of the bacteria around a disc containing an antibioticcompared to a disc containing a control disc is caused by the antibiotic alone(and not, for example, by something strange in the paper).

4.      Leave theplate at 25 °C for 48 hours.

5.      Theinhibition zone will be greater the more potent the antibiotic is against thebacteria.

Replace the paperdiscs soaked in antibiotics with discs soaked in the solutions you'reinterested in to examine the impact of antiseptics (or disinfectants) onbacterial growth. Alternately, you might soak the discs in variousconcentrations of the same solution to examine the impact of antisepticconcentration. 

Preventing contamination

Your results will be impacted and pathogens may developas a result of undesired microbial contamination. Follow these guidelines toprevent this:

·      To get rid of any unwelcomebacteria that might be hiding on the Petri plates and culture medium, they mustbe sterilized (for example, by heating to a high temperature) before use.

·      If an inoculating loop is employedto introduce the bacteria into the culture media, it must first be sterilizedby passing it over a hot flame.

·      To prevent airborne microorganisms,the Petri dish cover should be lightly taped on after the bacteria have beentransferred.

·      To prevent condensation fromfalling onto the agar surface, the Petri dish should be stored upside down.

Calculating inhibition zones

To determine the effectiveness of antibiotics or antiseptics against germs, you can compare the sizes of the inhibition zones around a disc. The larger the zone, the more efficient the antibiotic is against the bacteria. While you can eyeball the sizes of the zones, it's better to measure them with a ruler to get more precise results. You can use the diameter of the zone to calculate its area using a simple equation. This equation can also be used to calculate the area of a colony. 

Culturing Microorganisms

The diagram below shows the inhibition zones produced by antibiotics A and B. Use the areas of the inhibition zones to compare the effectiveness of the antibiotics.

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314 mm squared is just over twice 154 mm squared, so youcould say that:

The inhibition zone of antibiotic B is roughly twice thesize of the inhibition zone of antibiotic A.

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What is the equation used to calculate inhibition zones?

Area = pie x radius squared

What are the steps in the requiredpractical?

1.      On an agar plate with an even layerof bacteria, place paper discs soaked in several antibiotic kinds or concentrations.Disks should be separated from one another.

2.      In the agar jelly, the antibioticshould diffuse (soak). On the agar around the paper discs, antibiotic-resistantbacteria will keep growing while non-resistant strains would perish. Where thebacteria have died, a clean space known as an inhibition zone will remain.

3.      Becareful to employ a control. This paper disc is the original, untreatedversion. Soak it in sterile water instead. Then, you can be sure that anychange in the growth of the bacteria around a disc containing an antibioticcompared to a disc containing a control disc is caused by the antibiotic alone(and not, for example, by something strange in the paper).

4.      Leave theplate at 25 °C for 48 hours.

5.      Theinhibition zone will be greater the more potent the antibiotic is against thebacteria.

How do you growmicroorganisms on an agar plate?

To make an agar plate, hot agar jelly is poured intoshallow round plastic dishes called Petri dishes. When the jelly’s cooled andset, inoculating loops (wire loops) can be used to transfer microorganisms tothe culture medium. Alternatively, a sterile dropping pipette and spreader canbe used to get an even covering of bacteria. The microorganisms then multiply.

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