Culture Media and Isolation of Bacterial Colony

 

Looking ahead to solar systems, stars and Galaxies our own planet The Earth our world is so tiny, But all those luckiest Living or dwelling on the Surface of the earth or inside of  Water are in thousand and linked to each other, However this world is surrounded by unpredictable numbers of Organisms and from these the highest number is of Microorganisms, that we Humans can’t see through naked Eyes. But we are so developed that we can see these tiny creature, Observe these in lab, we can feed them and grow them for our purpose. The act of dealing with Microbial buddies is observed under Microbiology and This blog is all about Microbial feed termed as Culture Media. 

 

Introduction

In the World of Microbiology medium (media pl.) is the substance which provides nutrients for the growth of microorganisms. Microbiologist are using media to cultivate, transport and store microorganisms. The  nutrients on which microorganisms are cultivated and grown called culture medium (pl. culture media).

Microbes have been using  nutrients of special culture media as required food is their In vitro cultivation. Culture media are specific and vary in form and composition fulfilling requirement by the species to be cultivated. There is no general or single medium  which can actively support the growth of majority of microorganisms.

 

History

• Louis Pasteur was the first microbiologist to used simple broths made up of urine or meat extracts.
• Agar is an solidifying agent is obtained from red sea weed like Gelidium, and this is introduced  by Fannie Eilshemius Hesse, wife of Walther Hesse (who was an assistant to Robert Koch) that agar due to its unique properties was Used to solidify Broth culture media.
• Importance of solid media and used potato pieces to grow bacteria is realized by Robert Koch
• Before the use of agar in culture media, Microbiologist attempted to use gelatin as Solidifying agent. Gelatin had few inherent problems; it found as liquid at normal incubating Temperatures (35-37°C) and was digested by certain bacteria.

Types of Culture Media, On the basis of Consistency types of culture media.

1. Liquid culture medium is called broth. It is used for profuse growth, e.g. blood culture in liquid media. Broth can be solidified by mixing  agar-agar in the ratio of 1.5 – 2.0% for complete solid agar and less than 1% for semi-solid medium.
2. Solid Medium is  solidified media contains agar. Solid media is used for the isolation of bacteria as pure culture. Agar’ due to unique solidifying ability  is most commonly used to Prepare solid media.

Agar is muco-polysaccharide sulfonated  containing mainly D-galactose, D-glucuronic acid and 3,6 anhydrous  L-galactose. It is derived from red sea weed e.g., Gelidium and wasp. Agar’s no influence on bacterial growth as it is bacteriologically inert.

In solid media bacteria may be identified by studying the colony character, (b) Mixed Bacteria can be separated, (c) It remains solid at 37°C, and (d) It is transparent.

Based on chemical composition, media can be classified into.

1)    Natural Medium: Examples Diluted blood, vegetable juices, milk, urine, meat extracts, beef and tomato juice, blood etc.

2)    Semi-synthetic Medium: Examples Potato dextrose agar (PDA), Czapek-Dox agar, oat meal agar (OMA), corn meal agar (CMA), beef peptone agar and nutrient agar.

3)     Synthetic Medium: These media are important and useful in studying the physiology, metabolic nature and nutritional requirements of microbes. Examples- Mineral glucose medium, Richard's solution, Raulins medium etc.

Based on application or function, media can be classified as follows.

1. Selective media: Use to Isolate  selected species. Contains  nutrients that enhance the growth and predominance of particular Microorganism and don't enhance or may inhibit other types of organisms that may be present.

2. Differential media: It allows differentiation among morphologically and biochemically related group of organisms. It contains certain ingredients which are changed because of microbial metabolism and this change can be seen in form of change in opacity of agar, change in pH or change in color of media or colonies.

3. Assay media: Media of prescribed composition are used for the assay of vitamins, amino acids, antibiotics etc.

4. Enumeration media: Specific kinds of media are used for determining the bacterial population in milk, water, soil and food etc.

5. Maintenance media: It is used for satisfactory maintenance of viability and physiological characteristics of microorganism

 

Isolation of Bacterial Colony

This world is surrounded or mingled  by Microbial creatures, Unlikely there is no place with Microbes on Earth, Approximately there are  10,000 thousand named species and those which until unidentified thought to be about 100,000. Bacteria are largest in numbers than any other creature.

A single spoonful of soil can have 100 million individual bacteria. A scraping of your gums can yield 1 million bacteria per cm² the size of your little fingernail. The bacteria in and on our bodies makes up about 10% of our dry body weight.

 

Streaking Method and Its Type

Isolation of Pure Microbial Colony is a one of the Microbiological Technique known as Streaking.  By using the specific culture medium we Isolate a pure strain or colony of our desired Microorganism from single specie of microbe.

·         Streaking Techniques of isolation was first developed by Loeffler and Gaffky in Koch’s laboratory, that involves the dilution of bacteria by systematically streaking them over the exterior of the agar in a Petri dish to obtain isolated colonies which will then grow into the number of cells or isolated colonies.

Types of Streaking Method

There are several methods of streaking for isolation. The vast majority of our students have been most successful with the quadrant method of streaking which is described below:

Types of streaking, 

a. Quadratic streaking b. Continuous streaking c. Overlapped streaking

 

 

Need of Culture

• Taxonomic identification
• Diagnostics of pathogens
• Virulence and pathogenicity studies
• Elucidation of metabolic properties
• Testing sensitivity to antibiotics
• Genome sequencing
•  Proteomic studies

§  Strain deposition in microbial collections

Streaking for Isolation Procedure

1.    A well equipped and decontaminated laboratory is first and primary requirement for isolation of any bacterium sp.

2.    Label your plate with your name, date, section, and organism.

3. You can do the next step with your plate on the lab bench or holding it in your hand. You decide which works best. Lightly drag your loop back and forth across the surface of the Nutrient agar.

• The more you drag the more bacteria you deposit.
• The general idea is to decrease the bacterial concentration with each swipe.
• Four to five zigzags seems to work well.
• Experiment with your different plates. Be sure to keep track of what you did on each plate.

4.    Sterilize your loop. If you are using plastic loops, discard your used loop in the cavicide container and obtain a new sterile plastic loop.

5. Do not go back into the original broth tube.
6. Touch your loop to the agar surface against the far end of your first streak. Repeat by dragging back and forth.

o   Do not drag into the center of your plate.

o    You should be able to see the faint indentations of your streaking line on the agar surface.

7.    Using a sterile loop, repeat the procedure on your second streak.

8.    Using a sterile loop, repeat the procedure on your third streak. Zigzag the last part into the center of the plate.

o   You should end up with isolated colonies somewhere in your last streak.

9.    If you are using an incinerator, sterilize your loop. If you are using plastic loops, discard your used loop in the cavicide container.

10.  Replace the lid on your plate place your completed plates agar side up on the incubation rack on the front desk in the incubate section.

Place your completed plates agar side up on the incubation rack on the front desk in the incubate section.

 

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Notes

• It is absolutely essential that you sterilize your loop between each streaking, either by using the incinerator or by obtaining a new sterile plastic loop. This is the most common mistake students make.
• Don’t leave your plate open too long or extra bacteria from the environment will fall into your plate.
• Do not be disappointed if you do not get isolated colonies on your first try. This is a difficult procedure.
• Take Care of Sterilization and Contamination. 

By: Awais Maheshwari