Question

(a)You isolate a new pathogenic bacteria, from the sputum of a human patient, which you hypothesize was the cause of a severe respiratory infection. How could you use Koch’s postulates to prove your hypothesis? Which postulate would not be satisfied if the organism was a human-only pathogen? (b)What steps would you take to obtain a pure culture of the bacteria? What staining methods would you use to visualize the sample if you knew that the bacteria had a flagella? (c) What microscope would you use to visualize the microbe and why?

Answer 1

Robert Koch stated the following postulates:

(i) The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in the healthy organisms.

(ii) The microbe must be isolated from a diseased organism and must be grown in a pure culture.

(iii) The cultured organism must cause the disease when introduced into a healthy organism.

(iv) The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

In order to prove the new pathogenic bacteria to be the causative organism for the disease, the Koch's Postulate (iii) can be used which stated the isolation of the pathogen from diseased organism and introducing into a healthy organism and observe whether the diseased is caused in the healthy organism.

Koch's postulate (i) would not be satisfied if the organism were a human-only affecting microorganism.

For obtaining a pure culture of bacteria :

We can plate the isolated microorganism in blood agar containing petri-plate.

In general streak culture is a better method . Incubate the plates for 16-24 hr duration at 37^oC in the incubator to obtain colonies in streak.

There are two major methods for staining flagella:

One method invloves the use of Ryu staining (wet mount method)

Place a drop of sterile water over microscopic slide and dip a sterile inoculating loop in it.

Now touch the sterile loopful water on one colony of the obtained on 24 hour incubated culture in petri-plate.

Touch the loopful containing inoculum back to the slide containing water and cover the wet mount using a cover slip without formation of any air bubbles provided they are formed only at edges.

Examine the cells using the phase-contrast microscope at 40x or 50x magnification. If motility is seen then the cells possess flagella.

Move on to the staining procedure. Place 2 drops of Ryu stain from the edges of cover slip containing air bubbles which will spread to the wet mount by capillary action.

After 10 minutes observe the slide under 100x (with oil) magnification in the optimum stain region especially on the cells attached to cover slip which facilitates the visualization of flagella.

Another traditional method is the Leifson staining (Dry stain method) that consist of tannic acid, basic fuschin and ethanol

Staiining procedures are similar to the stain

The ethanol gets evaporated after staining and the flagella is examined as thick layer thatis due to the deposition of tannic acid.

The method has a drawback of the basic carbol used being unstable.

Phase contrast microscopy is used for the visualization of flagella since it is so thin to be viewed under light microscope, phase contrast works on the principle accentuating differences between bending of the light passing though different components of a specimen.

Or Electron microscopes are used for flagellar visualization.