Observational & Differentiation Methodologies [1]

Observation & Differentiation Methodologies :

A) Bacteria : Bacterial growth observation & differentiatiom which includes various aspect to observe & differentiate them by the following methodologies.

i) Role of Media

ii) Incubation

iii) Appearance on Media

iv) Gram Staining 

v) Motility Test

vi) Biochemical Test

i) Role Of Media :

Growth medium play epic role in observation & differentiation of micro-organisms i.e.Bacteria 

Its a plateform to oberve them & differentuate them by the growth medium.

How ?

Let me elaborate ,

Growth Medium is the food for bacteria which provides the all essential nutrients for the growth of Bacteria.

Once the growth occurs then it will appears on the surface of media.Well growth it self is an indication which can be observed on the surface of the medium which helps in differentiate them because different organisms produces different colonies which are totally dufferent to each other.

Medium can support different types of microbes which having different growth & varies in appearance those appearance can be observed by Microbiologist.

                         Role of Media 

Every organism have its own existence & various properties/specialities towards the medium utilization by which microbes produces physical apperance on the surface of medium by changing its color, showing optimal growth on it, various colony types, shapes & sizes basically they support directly or indirectly to understand the colony characters except Gram Nature & Motility Test right ...?

Beacuse these are descriptive as well as practical based technique unless & untill do cant reach to there.Base on microbial nutritional requirements supply medium can be Selective, Differential & Enriched.

These media used because they have speciality & specificity which organism always required such media supports only desired/targeted organism such media helps a lot in observation & differentiation of microbes.Liquid Media known as "Broth" & Solid Media known as "Agar".

Example : 

Liquid - Luria Broth & Nutrient Btoth etc.

Solid - Nutrient Agar & MacConkey's Agar etc.

ii) Incubation :

Incubation which means providing organism suitable environment for the growth which are Temperature, humidity & time.Where  each & every aspect of it has must have condition level conditions because without these factors growth cannot be optimal growth cant occurs and if growth disturbed & confusionous results will be obtained.

If the growth obtained successfully then and then only observation will be meaningful for the Microbiologist otherwise requires to repeat the process but if incubation sucessful then observation & differentiation have at the scientific level thus incubation has its own value also has great importance incubation period it self shows that time requesting process and microorganisms.

Different type of incubation period most of them need 24 hour incubation time or over night incubation can be enough for them but as depending upon growth affecting factor or growth rate can be varies 24/36/48/72 hours these are the different incubation time period for obtain the optimal growth.

Incubation derived from latin "incubationem" from "incubare" means to sit on eggs so as to hatch by Warmth of the body.

To incubate and obtain the optimal growth & best results a device used for it called as "Incubator" which provides favourable conditions to the micro-organisms for the growth.

iii) Growth & Appearance :

In the growth & appearance of micro-organisms in the forms of colony where that can be seen on the surface of agar plate by naked eyes beacuse the various media have various appearance behalf of growth on media.

a) E.Coli - MacConkey's Agar

Gram -ve bacilli lactose fermenter β-hemolytic on Blood Agar shows yellowish green colonies surrounded by an intense yellow green zone.

b) S.aureus - TSA/Blood/Chocolate/ Agar

Generally produces yellow pigments on Tryptic Soy Agar.

TSA - Yellow Color 

MacCokney - Pink/red

EMB Agar - Blue-Black with metalic sheen.

c) Vibrio Cholerae - On TCBS Agar Yellow color because it ferments Sucrose.

d) Pseudomonas aeroginosa - Cetrimide Agar blue-green color colony.

e) Clostridium botulinum - Nutrient Agar/ Sheep Blood Agar

On Sheep Blood Agar it shows Grey color colonies while on Nutrient Agar shows whitish grey color.

iv) Gram's Nature :

When ever term like this come meaning is clear that is Gram Staining might be there to know the Gram's Nature Bacteria.

Gram Staining is the standard staining procedure which provides the information about the cell wall of the Bacteria/Organism for observation & differentiation its a very important value huge help to understand cell wall structure Thin/Thick.

After performing it by SOP( Standar Operating Procedure) Nature of Organisms can be conclude conclusion have two type of Nature 

"Gram Negative & Gram Positive"

Gram Negative - Think wall peptidoglycan & Pink in color.

Gram Positive - Thick wall peptidoglycan & Purple in color.

This how Gram's Nature of Bacteria helps in Observation & Differentiation Methodologies which provide cell wall structure & its nature.

v) Motility Test :

Motility Test is the also one of main procedure which is known as "Hanging Drop Technique".This is used to observe the Motility/Movement of the Bacteria.

Now , How this can be possible right ?

Let me explain it _

Bacteria which can move from one place to another one this process is known as "Motility" this all process or called locomotion possible with the help of Flagella- microscopic hair like structure in the locomotion of the bacterial cell which help in motility flagella means 'whip' it propaling cell through the liquid flagellum is singular flagella  plural form.

Now how to observe them ?

With the help of Hanging Drop Technique its observable at microscopic level only 

In this technique a drop of culture is placed on a cover slip that is enriched with petroleum jelly.

The cover slip & drop are then inverted over the well of cavity slide.

The drop is hangs from cover slip & petroleum jelly forms a seal that prevents evaporation this preparation gives good views of microbial motility.

Observe the slide through eye piece & adjust the fine adjustment until the edge of the drop can seen thick its look line usually dark line observe cell morphology & motility.

vi) Biochemical Test :

Biochemical Test helps in the identification & confirmational manner of bacterial Genus & Species.It determines nutritional & enzymatic capabilities & combination of test confirmation of the bacteria genus the inhibitors can be presence (e.g. Salt, Surfectanta, toxins & antibiotics).

Biochemical tests are design to measure the presence of single enzyme test as well as complete enzyme pathways.

A.Single Enzyme Test 

-Assays for metabolic pathways : carbohydrate oxidation & fermentation

-Amino acid degradation

-Single substrate utilization

B.Establishing Inhibitor Profile 

C.Other More Specific Test

▪︎Commonly Single Enzyme Test -

Catalase test 

Coagulase test 

Pyrase test

Oxidase test

Indole & Urease test etc.

Nitrate Reduction test