How to identify bacteria under a microscope?

Bactria are the prokaryotes that are the single celled organisms having not well defined nucleus and are directly submerged in the cytoplasm. They vary in shape, structure, size etc. For visualizing these distinguishing features we have to identify bacteria under microscope.

Microscopy

There are different microscopes that can be used for this, like digital, compound, binocular or trinocular. All microscopes work differently, however we suggest using compound microscope. Bacteria are firstly grown in a nutrient rich medium before seeing it in microscope. Microscopy provide assistance in division of cells in this way they increase in number. In microbiological labs, cultural medium is used and a few types are as follows:

• Basal media:
• Transport media:
• Selective media:
• Enriched media:

Basal media: This medium is used for bacteria like Staphylococcus and they do not specialized nutrient rich medium for the growth. It includes peptone water and nutrient broth.

Transport media: Such as amines media. This media is specifically used in preservation of sample. This media saves the bacteria from overgrowing, prior to deliver it to the labs etc.

Selective media: Like Lowenstein-Jensen media. Selective media is basically for selective growth and it forbid others.

Enriched media: For example with added blood.in this media such specific kind of constituents are used that support the growth of particular bacteria such are when blood is added in the specific media it favors the growth of Streptococci.

Preparation of slide:

Requirements:
 Bacterial sample cultured
 Distilled water
 Burner
 4Microscope
 Inoculating loop
 Dropper
 Wax marking pencil

Procedure:
The procedure is as follows:

Smear:
 Firstly make a spot for the smear by marking a circle in the center using a marking pencil on a clean slide.
 Either using inoculating loop by flaming it on the burner or by using dropper that must be clean, a drop of distilled water is placed on the spot that is marked on the slide. If we used broth media then there is not essential to use distilled water.
 The inoculating loop is again pass through the flam. Then it is cooled before spooning sample from petri dish or tube on the veneer of the cultural medium. The purpose for this is to prevent the sample from any kind of contamination and spoilage that’s why we pass the tips of tubes through flam prior to covering.
 Then the water droplet is mixed in order to from smear that is then dried in drying rack
 After this slide is passed through the flam before smear fixing that increases the stain perforation in cells.
Procedure for staining of bacteria:
Slides were placed on the staining rack and after this covered with the below mentioned gram stains just for 1 min.
1) Crystal violet
2) Methylene blue
3) Safranin
 Flow water on the slide gently for the removal of excessive stain
 By using absorbent paper extra water is removed from the corners and edges.
 After this the slide is viewed under microscope using low power. Immersion oil is used for high power.
Observation and discussion:
Note and also identify shape and size of bacteria and also observe either the stain is gram positive or gram negative type bacteria.

Morphology:

Based on their shape there are many kinds of bacteria:
• Coccus
• Bacillus
• Spirilla and vibrio shaped bacteria

Coccus

Cocci are commonly found bacteria having spherical or ovoid shape, they are apportioned into diplococcus, streptococcus, staphylococcus. Diplococcus is a pair of to cocci. Example is Neisseria spp. Streptococcus exists as a long chain or cell. For example, Streptococcus pneumniaea. Staphylococcus exists in the form of clusters such as Staphylococcus saprophyticus. It may also exists as tetrad or sarcina bacteria packet of eight to form a cube like structure.

Bacillus:

Bacilli are rod shaped bacteria. Bacilli all divide in one plane producing a bacillus is a single cell of bacteria. Streptobacillus is a chain of bacilli. When rod shaped bacteria occur in pairs then arrangement of cells is known as diplobacilli. Examples of rod shaped bacteria are Escherichia coli, Bacillus subtilis, Pseudomonas.

Spirilla and vibrio:

The spiral shaped bacteria are spirally coiled. Vibrio is curved or comma shaped rod.in contrast spirilla have flagella on its both end but vibrio have a flagellum at only one end for the movement. Example of spirilla is Campylobacter species and vibrio involves Vibrio harveyi.

Spirochetes :

They are flexible spiral bacteria. Such as Spirochaetales. They move in corkscrew like motion.
Gram positive and gram negative bacteria:
On the basis of how the bacteria stain there are two types: gram positive bacteria and gram negative bacteria.

Gram positive bacteria:

They are stained purple such as retain primary dye due to formation of CV-1 complex. They have thick layer of peptidoglycan they have no outer membrane, periplasmic space is present in some and they have more permeability as compared to gram negative type bacteria. By fixing with heat allows the stain to penetrate layer, which is then retained even when the cells are washed away using alcohol. For example: Staphylococcus epidermidis, Bacillus cereus and Clostridium botulinum etc.

Gram negative bacteria:

They are stained pink such as retain secondary dye in color. They have thin layer of peptidoglycan, they have outer membrane, periplasmic space is present in all and they have less permeability. Examples: Salmonella species, Escherichia coli etc.

Conclusion:

Bacteria the single celled prokaryotic cells that vary in shape, size and structure can be identified under a light microscope in various laboratories. For this various culture mediums are used. Different techniques are applied. By doing all this we can understand how to identify bacteria under a microscope.