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Isolation Of Living Organisms
Objective:

 To identify the bacterial unknowns in a mixed culture by morphological and biochemical methods.

Principle:

The identification of bacteria is a careful and systematic process that uses many different techniques to narrow down the types of bacteria that are present in an unknown bacterial culture. It produces benefits for many aspects of the research of microorganisms and helps physicians correctly treat patients. Multiple tests were performed to provide the fermentation abilities, presence of certain enzymes, and certain biochemical reactions. Qualitative observations were made on the tests, which were compared to unknown bacteria identification key to aid with the identification process. 

Various steps involved in the identification of unknown bacteria are:

Isolation: The importance of this step is to isolate pure colonies of bacteria. The streak plate is a qualitative isolation method; quadrant streaking is mostly done to obtain pure colonies. The inoculation of the culture is made on the agar surface by back and forth streaking with the inoculation loop over the solid agar surface. This will make a dilution gradient across the agar plate. Upon incubation, individual colonies will arise from the biomass.

The characteristics features of the colonies on solid agar media are then noted. This include

Shape : circular, irregular or rhizoid.

Size: small, medium, large( or in millimetres).

Elevation: elevated, convex, concave, umbonate/umbilicate.

Surface: Smooth, wavy, rough, granular, papillate or glistening.

Edges: entire, undulate, crenated, fimbriate or curled.

Colour: Yelow, green etc.( Note the colour of the colony).

Structure: opaque, translucent or transparent.

Degree of growth : scanty, moderate or profuse.

Nature: discrete or confluent, filiform, spreading or rhizoid.

In order to obtain the pure culture of organism, the isolated colonies are aseptically transferred on to different nutrient agar slant tubes and incubated overnight at 37 degree Celsius. It is then stored for future purpose.

 Staining Reactions:



 Staining is a simple basic technique that is used to identify microorganisms. Simple staining is used to study the morphology of all microorganisms (Fig 1). The simple stain uses the basic dyes such as Methylene blue or basic fuschin. The strong negative charge of the bacterial cell will strongly bind with the positive charged basic dyes and will impart its colour to all bacteria.

Fig 1: Simple staining of cocci



Gram staining is a differential staining technique that imparts different colours to different bacteria or bacterial structures. Usually it differentiates bacteria into two groups; gram positive and gram negative. The primary stain Crystal violet and mordent Iodine form a strong CVI complex all bacteria. Gram positive cells due to their thick peptidoglycan layer will retain the CVI complex even after it is subjected to decolourization with acetone or alcohol. Hence the counter stain Safranin has no action on gram positive cells. But in the case of gram negative, the thin peptidoglycan layer and more lipid contents in the cell wall will easily make them susceptible to the action of decolorizer and hence CVI complex is easily washed out and hence the gram negative cells will the colour of counter stain Safranin.  Hence after the gram staining, the gram positive cells appear as purple and gram negative cells appear as pink (Fig 2). The study of morphological features  and staining characteristics help in the preliminary identification of the isolate.

 


 Fig 2: Gram positive bacteria  Fig 2:  Gram negative bacteria

Biochemical reactions:

Gram negative enteric bacilli play an important role in the contamination of food. Hence they are the main causative agents of intestinal infection. Gram negative family includes Shigella, Salmonella, Proteus, Klebsiella,Escherichia,Enterobacter etc. Usually four tests are used for differentiation of the various members of Enterobactericeae. They are Indole test,Methyl red test, Voges proskauer test and Citrate test; collectively known as IMViC series of reactions.


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