23) A contaminated food sample contains several different species of bacteria. A

Question

23) A contaminated food sample contains several different species of bacteria. A food microbiologist is interested in studying just one of these species. Describe the sequence of procedures that the microbiologist must perform in order to obtain a pure culture of the bacterial species of interest from this food sample. Detail all necessary media and equipment.

24) Explain how and why immersion oil increases resolution but not magnification when using the 100x objective lens.

25) Describe the steps of the Gram stain, and explain how it can be an important diagnostic tool for infections.

Explanation / Answer

23)In order to isolate a single species of bacteria from.a contaminated food sample,first you should take a small quantity of sample from it to test it.

-The next step is to dilute the sample in water or normal saline (.85%).To prepare serial dilutions take 11 test tubes and label them as Neat,10^-1,10^-2 to 10^-10.

-Then take 9ml water/saline in test tube labelled as Neat and 10ml water/saline in test tubes labelled 10^-1 to 10^-10.

-Weigh 1gm of food sample and add it into the Neat test tube.Dissolve it.

-With the help of pipette, take 1ml from Neat and transfer it into 10^-1.

-Dissolve it, change the tip of pipette and take 1ml from 10^-1 and transfer it to 10^-2.

-Repeat the process till you have to add 1ml to last tube.Dissolve it fully.Now, discard 1ml from this into the discard jar.

-The total volume in each test tube will be now 9ml.

-Now, you have to plate all these dilutions in Nutrient agar plates.Nutrient agar medium is a fully supplemented medium in which all of the bacteria can grow easily.

-For plating , take one tube and mix it fully , and with the help of a pipette , take 100microlitre and add it as drops on to the surface of medium.Now, with the help of a glass spreader, spread the drops so that it gets absorbed on the surface fully in the entire area.

-Repeat this process for all the dilutions.

-Now , keep the plates in the incubator set at 37°c for overnight incubation.

-Next day , observe the plates with type of growth.

-Now select some colonies that you see are appearing to be different from each other. Pick these colonies and perform streaking in fresh plates.

-Incubate them in the incubator at same conditions.

-Now, observe the growth, if you have been able to got isolated colonies, then congrats you have been able to reach to a single bacterium.If not, you can again perform streaking and eventually you will get a single bacterial colony.

24) See in a normal microscope operates , the 10x and 40x objective lens are air based.The lens works in presence of air through which light rays pass between the slide and objective lens.

Due to air as a medium , the light gets refracted and eventually lost, because the refractive index of air is different from glass and when light passes through this air-glass interface, it gets refracted to larger degree.

And according to Abbe's equation , the limit of resolution is inversely proportional to numerical aperture.

Numerical aperture = refractive index of medium * angular aperture

It is also true that resolving power of microscope is inversrly related to limit of resolution.Therefore, in order to increase resolving powerm, numerical aperture should be increased.

Now. in a 100x microscope the distance between the slide and lens is small, which makes the angular aperture to be 90° therefore sin90= 1.

It implies that in order to further increase the numerical aperture, we need to increase the refractive index so we use oil whose refractive index is more than water or air.

As a result, numerical aperture increases due to increase in refractive index and which in all increases the resolving power.

25.Steps of gram staining-

-Take a clean slide and put a drop of water in the middle in case the culture is to be taken from the plate , or else take a loopful of culture directly from the broth.

- If the culture is from plate, just take a loopful of culture from a inoculating loop and mix it well with the drop of water.

-let it air dry.

-Now heat fix it on mild flame so that the bacteria gets fixed on the slide and doesnt swim when you observe it under microscope.

-Now flood the slide with primary stain crystal violet and keep it for 1minute.

-Wash the slide with water.

-Now, flood the slide with mordant Iodine and keep it for 1 minute.

-Wash it again.

-Now take the decoloriser alcohol and add it dropwise, You will see blue color coming off, wash it with water.Now repeat the process of intermittent adding alcohol and washing with water until no blue color comes off.

-Now stain it with counter stain Saffranin.Keep it for 1 minute and wash it again.Let the slide dry.

-Observe your slide under the microscope now.

Gram stain can act as a primary diagnostic tool when you suspect a infection. The infection that you have caught can be due to bacteria, virus or fungus and there is a different treatment plan for each one of them.

With a Gram stain, a doctor can get to know if the infection is bacterial and after knowing the results he can proceed with the treatment that if you nedd anitibiotics etc.

Gram stain can help to know if the bacterium is Gram positive or negative.So Gram stain is a differential stain. Then he can peform further tests to know the specific causative organism.

So Gram stain is used as a preliminary test in diagnostics.

Hope it helps, Good luck !!!

Please do press like !!! :)

Related Questions

Navigate

• Browse (All)
• Browse 2
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.