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Isolation and characterization of microbes

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Microbiology is a part of a field that deals with the study of various organisms that is, biology. We work on micro-organisms in this field to find out their strains, species and various helpful aspects. Micro-organisms are very helpful in nature. Majority of them are beneficial to the environment. Humans, animals et cetera. Being omnipresent in nature they are present everywhere. From air to land to sea, they can survive anywhere even in harsh conditions. They have such capabilities which can be harnessed by the application of microbiology. By doing so, we can apply such characters for the benefit of Mother Nature. Now the technicalities of this field are:

  1. A person should have the knowledge of various techniques and procedures applied in this field.
  2. A person should know how to handle instruments as they are very expensive as well as have high maintenance requirements.
  3. Being working with bacteria, there are always chances to get disease. So a person should always be aware of this fact and take proper precautions.


So as topic says Isolation and characterization of microbes, I took two samples for my isolation and characterization. My two samples were from water sources which are:

  1. 1. Tap water
  2. 2. Hand pump water.

To isolate various type of microbes present in both of these samples drawn. Both water sources are different so they both will have different microbes inhabiting the sources.


For the characterization of various microbes, first we will have to make nutrient media in which the colonies of various microbes will grow distinctly. Different microbes require different nutrients for growth, so various media are prepared. They are:

  • NA (nutrient agar) media
  • SCA
  • SDA
  • YPD
  • Rose Bengal


The sample was added on all the media through various techniques. Each media will have different colonies growing on them. All the techniques are performed in a sterile environment which is provided by laminar air flow. In this, a sterile environment is created by the help of UV radiation which kills the microbes inside and then a fan blows them out. Working area is cleaned by applying alcohol which kills the remaining microbes. While working in a LAF, one should sterilize hands first and keep them inside the LAF whole time during the experiment. Various techniques for growing colonies are:

  1. Pour plate method
  2. This is a technique which is used to culture the microbes on a nutrient media. In pour plate method, first the sample water is taken in two petri-dishes i.e. one with two three drops of tap water and other with two three drops of hand pump water. Now the freshly melted different nutrient media are poured in the dishes having two different water samples. After pouring, the dishes are allowed to cool down and are ready for incubation.

    (All of the above procedure is done in a laminar air flow which is an equipment discussed earlier.)

    In the laminar air flow, the petri plates are kept to cool and are sealed with paraffin seal to avoid any outside contamination. After the sealing is done the petri plates are kept in an incubator. Now the plates with nutrient agar are kept at the temperature of 37 degree Celsius for 24-48 hours as bacteria grow best at this optimum temperature. Bacteria grow well in nutrient agar that’s why they are kept at such temperature.

    Now all the other media poured into dishes are incubated at a temperature of around 30 degree Celsius for 72 hours as this much temperature and time is required for the growth of various other microbes such as fungi, algae, etc. They have a different incubator set as per their requirements.

  3. Streak plate method
  4. This technique is used to obtain a pure culture. You only get a single species of the microbe. In streak plate method, firstly the freshly melted media is poured and is allowed to solidify. Solid nature is due to the presence of agar. After the solidification a small portion of colonies grown in the nutrient agar media are scrapped lightly with a help of inoculation loop and spread in a zigzag form. In this technique, a pure line of micro-organisms is obtained after the sealing of dishes with paraffin seals and incubating them at 37 degree Celsius for 24-48 hours. The micro-organisms are bacteria as they were picked up from sample plates of nutrient agar media incubated earlier.


Now after the colonies of bacteria are obtained through above methods, we can use the gram staining to differentiate between gram positive and gram negative bacteria which we have isolated from the water sample via pour plate and streak plate methods. The gram staining is a very simple way to differentiate between bacteria. Here we use various stains to color the wall of the bacteria which makes it easy to differentiate between the colonies of these bacteria. Its procedure is as follows:-

  1. Prepare heat fix smear of the colonies on slides
  2. After fixing the slides, add crystal violet to the smear. Add only 2-3 drops of this stain.
  3. Now wait for about a minute then wash off the extra stain under running tap water.
  4. After washing add at least 3-4 drops of gram iodine. It has a special characteristic of forming a complex compound with the wall of gram positive bacteria.
  5. After adding gram iodine, wait for a minute and the wash it off.
  6. Now decolorize the smear be adding ninety-five percent (95%) ethanol to get rid of extra stain.
  7. Now air dry your sample and add second stain called safranine and just wait for half a minute and wash it off as it is taken up very fast by the cells.
  8. Now dry off the slide and observe under the microscopes i.e. perform microscopy.

Under the microscope, different colonies stained with crystal violet and safranine are visible. The difference clearly shows which ones are gram positive and which area gram negative bacteria.


From above test of gram staining we can easily identify the gram positive bacteria. For gram negative bacteria, a much specialized test is recommended which is known as MR-VP TEST or METHYL RED &


This MR-VP test is specially done for gram negative bacteria and in that only for the bacilli.


Methyl red is a pH indicator which is used to detect change in pH and especially the acidic range which is due to the acid produced when the organism carries out the glucose fermentation. This test was discovered by Clark and Lubs in the year 1915. They saw that different cultures of E.coli produced the red color when methyl red was added to them.

After incubation, the bacteria which were MR positive continued to produce the acid during fermentation whereas, MR negative bacteria do not impart any red color which clearly indicates that bacteria carry out the metabolism of fermentation products processed by decarboxylation.


This was observed when a red color was produced on the addition of potassium hydroxide to the bacterial cultures which were grown in their respective media. This test was modified by the addition of alpha napthol just before adding potassium hydroxide.

If there is the appearance of red color then the test is VP positive otherwise its VP negative.


This broth was developed to allow both the MR and VP test to take place in same medium of inoculation by distributing it in different test tubes.

Ingredients of MR-VP are:

3.5gm of dipeptone

2.5gm of dextrose

2.5gm of potassium hydroxide

500ml of deionized water

The pH should be maintained at 6.9 plus/minus 0.2 at the temperature of 25 degree Celsius.


  1. After the preparation of MR-VP broth, we distributed the broth in test tubes form inoculation from our water sample in nutrient agar plates.
  2. Now add the inoculums from nutrient agar plates to the provided test tubes.
  3. Incubate these test tubes for 72 hrs. at 37 degree Celsius
  4. Now take out the test tubes and add the reagents to it.


For MR test, we add methyl red to the given test tubes. And now observe the medium as it might give red color instantly.


Prior to VP test, reagents are prepared:-

VP1 made by adding 5% alpha napthol in absolute alcohol.

VP2 it is 40% potassium hydroxide solution

Now VP reagents are added simultaneously the other incubated test tubes and are left to settle for at least one hour but not more than that. Now, we have to wait for the appearance of red color if it is VP positive.

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