Culture Media PART 3 - Classification Based on Chemical Constituents Part II - DR C R Meera

 

Based on the chemical constituents used for the preparation, media can be classified into simple media, complex media, defined or synthetic media and semi-defined or semi-synthetic media.

1.    Simple media

Simple media include the basal media or general purpose media like peptone water, nutrient broth and nutrient agar which are very commonly used in Microbiology laboratory for cultivation of microorganisms. Peptone media is prepared using 1% peptone in 0.5% NaCl in water (pH 7.2±0.2).  Components of nutrient broth includes peptone, yeast/ beef extract, sodium chloride and distilled water (pH- 7.2 ± 0.2). When 2% agar is added, it is called nutrient agar. Semi-solid media which is prepared using same components as that of nutrient agar, but with a different concentration of agar, is also considered as a simple media. In semi-solid agar, 0.2 to 0.5% agar is used.  

2.    Complex media

Complex media contain added ingredients for special purposes. It may be for bringing out certain special characteristics of the organisms or for providing special nutrients required for the growth of fastidious organisms. Fastidious organisms are those which are more exacting in their nutritional requirements. Most of the media other than basal media, used in microbiology laboratory are coming under complex media. Eg:  chocolate agar, Blood agar, MacConkey agar, Robertson’s cooked meat (RCM) medium, Lowenstein–Jensen (LJ) medium (Cultivation and isolation of Mycobacterium), etc. Complex media is also called rich media and typically have a mixture of many different organic compounds including glucose as carbon source, a source of amino acids and nitrogen (e.g., beef and yeast extract), various salts, growth factors, vitamins etc. and water needed for bacterial growth. This is an undefined medium, because the amino acid source contains a variety of compounds of which the exact composition is not known. Meat or beef extract is usually made by hydrolysing natural products such as meat with enzymes to release monomers like peptone, tryptone etc from the macromolecules. Another amino acid source yeast extract is made by extracting small molecules from yeast cells. 

3.    Synthetic or Defined media

This media is prepared using pure chemical substances and does not contain any animal, yeast, or plant tissue or their extracts, hence called synthetic media. The exact quantity of all ingredients used in the media is known, hence also called defined media. Synthetic media are used for various special studies such as for studying the metabolic requirements of the organisms. This media consists of a defined carbon and nitrogen source, trace elements (Mn, Mo, Cu, Co, Zn etc.) and vitamins. Glucose or glycerol is often used as carbon source and ammonium salts or nitrates used as inorganic nitrogen sources. Dubos’ medium with Tween 80 used for cultivation of Mycobacterium tuberculosis is an example of this medium. Minimal media (sometimes called Mineral media) is also an example of synthetic media.  Minimal media contain just enough ingredients, generally without the presence of amino acids, to support growth of wild type microorganisms. Minimal media typically consists of  a carbon source (sugar like glucose or less energy source like succinate), various salts with essential element like Mg, N, P, S which help bacteria to synthesize protein and nucleic acids and water.

Supplementary minimal media are minimal media that also contains a single selected agent, usually an amino acid or a sugar. This supplementation allows for the culturing of specific lines of auxotrophic recombinants.

4.    Semi- synthetic or Semi- defined media

Semi-synthetic media will be containing a set of chemical substances of known quantity like the synthetic media, but differ in that at least some natural ingredients of unknown or variable composition are also present.  A synthetic medium can be made semi-synthetic by adding a substances such as yeast extract or  beef extract. Eg: Peptone water which is made up of 1% peptone with 0.5% NaCl in water

Video link: https://youtu.be/ubturhQ90Zo

 

Culture Media-Part 2-Semisolid Media- Dr Meera C R

 

Semisolid media have a consistency between Solid and liquid media. It has a soft custard or jelly like appearance.Its components are same as solid media. Only difference is in the concentration of agar used. In solid media 2% agar is used, ie: 2gm in 100 ml of the media. In semisolid media agar concentration used is 0.5% or less. They are used in demonstration of bacterial motility and to grow microaerophilic bacteria.

Bacterial motility is due to the presence of locomotory organelles like flagella. In motility test, bacteria is stabbed using a straight inoculation needle in to the semisolid media and incubated at 37⁰C overnight. After incubation tubes are observed for bacterial growth pattern. Non motile organisms will grow confined to the stab-line, having sharply defined margins and leave the surrounding medium clearly transparent. Motile organisms will move from the line of stab producing diffuse, misty growths that spread throughout the medium rendering it slightly opaque. 

Microaerophiles are bacteria that cannot tolerate atmospheric concentration of oxygen. However they need oxygen for their growth. These groups of bacteria can grow under reduced oxygen (5% to 10%) and increased carbon dioxide (8% to 10%) concentration. Higher amount of oxygen is inhibitory to their growth. Examples: Campylobacter jejuni, Helicobacter pylori, etc. Microaerophiles when inoculated to semisolid media, will move from the surface to the just lower layer where oxygen penetration is only in the required concentration.

Youtube Video: https://www.youtube.com/watch?v=214OubJf1yM&t=1s

Classification of bacteria based on Oxygen requirement- Dr C R Meera

Based on requirement of oxygen for growth, bacteria can be classified into the following groups.

·       Obligate Aerobes: are strict aerobes. Can grow only in the presence of oxygen (21%). Eg. Mycobacterium tuberculosis, Pseudomonas aeroginosa

·       Obligate Anaerobes: are strict anaerobes. Grow only in the complete absence of oxygen. Oxygen presence is toxic and lethal to their growth. Eg: Clostridium perfringens, Clostridium boyulinum.

·       Aerotolerant Anaerobes: Some anaerobic bacteria can tolerate low levels of oxygen and are called nonstringent or aerotolerant anaerobes.  Oxygen is actually not required for their growth but not harmful if present. Eg: Propionibacterium acnes

·       Facultative anaerobes: They can grow well in both aerobic and anaerobic conditions. They do not require oxygen for growth, but may use it for energy production if available.  e.g., Enterobacteriaceae group, Staphylococcus aureus

·       Microaerophiles: Require low levels of oxygen for growth. They can neither tolerate normal levels of oxygen in air nor can live in complete oxygen absence. These groups of bacteria can grow under reduced oxygen (5% to 10%) and increased carbon dioxide (8% to 10%) concentration.  Eg: Campylobacter jejuni, Helicobacter pylori

             Their growth when inoculated into semisolid media is as shown in the diagram.

 

Contributions of Joseph Lister to the field of Microbiology

 

“Success depends upon attention to detail”- Joseph Lister

Joseph Lister, was a British Surgeon and pioneer in Antiseptic surgery. He is known as the “Father of Modern Surgery”. Lister was born on April 5th, 1827, in England. After finishing his baccalaureate in Medicine in 1852, Lister joined the Royal College of Surgeons, England. In 1859, Lister joined as surgeon in the Glasgow Royal Infirmary, Scotland.

As a surgeon at the Glasgow, Lister observed that he was losing nearly half his amputation cases to sepsis which is due to wound infection.  At that time, people considered ‘miasma’ or ‘bad air’ as the cause of infection. He started experimenting with new ways to prevent wound infection. He started by implementing Florence Nightingale’s most elementary hygienic principles in his hospital. The floors, covered in dirt and mud, were thoroughly cleaned and the windows kept opened for fresh air to circulate. The towels and bed sheets were regularly washed.

He was attracted by Louis Pasteur’s theory that decay and fermentation are results of microbial activity. He concentrated on Pasteur’s theories and experiments and discovered that in the suppuration of wounds as well, micro-organisms play a fundamental role. Lister realised that these microbes had to be destroyed before they enter a wound. This became the basis of his antisepsis doctrine.

Pasteur had recommended filtration, pasteurization and use of antiseptics as the means to remove microorganisms. Motivated by this idea, Lister started trials to find out suitable antiseptic agents that are capable of killing these septic germs and can be safely applied on wounds. At that time, Carbolic acid (phenol) had been used to control putrid sewer stench. Lister experimented with phenol on infected frog legs, and the results were remarkable.

On August 12th, 1865, his first human trial of phenol was carried out on an eleven-year old boy, James Greenlees, brought into the Glasgow Royal Infirmary. Lister treated the wound with phenol and the wound healed without suppuration. Lister recognised the antiseptic properties of phenol and used it during operations as well as for cleaning surgical instruments. Lister started to use carbolic acid to clean hands, clothes and also in operation theatres. Wounds were cleaned and sprayed with phenol before dressing. Lister even invented a carbolic acid spray machine (donkey engine) to produce a mist of carbolic acid in the operating theatre as an attempt to reduce post-operative infections. He noticed that pure phenol is too powerful, and its dilutions up to 1:40 have germicide effect. In his “donkey engine”, he used dilutions up to 1:100 which was effective in air sanitization.

After nine months of experiments, he published his promising data in the International General Medical Journal - Lancet as “Papers on a New Method of Treating Compound Fracture, Abscess, etc., With Observations on the Condition of Suppuration”.  He visited conferences and symposia all over Europe, stating proudly that none of his patients so far have died from sepsis, wound infection or hospital gangrene.

                                                                    “Donkey Engine”

To avoid infection from wound suturing threads, he introduced suturing with catgut instead of the commonly used silver threads. He treated the thread first by prolonged “carbolization”. As an experiment, he sutured deep neck wound in a calf. To his surprise, there was no pus on the suture and also there was no trace of catgut. Catgut suture get naturally degraded by the body's own proteolytic enzymes. Catgut suture threads are derived from strands of purified collagen taken from small intestine of healthy ruminants (cattle, sheep, goats) or from beef tendon.

Honours

·  Chassaignac introduced the use of drainage tubes into surgery. Lister was fascinated by the idea and immediately applied it to the first abscess that came his way. It was not just any abscess; it was located in her Majesty Queen Victoria’s armpit. Using his antiseptic techniques, he treated the abscess so meticulously and he was honoured with “Sir “title.

·       In 1897, Queen Victoria honoured Lister with “Baron” title  

·       First  physician who received the highest noble title of “Lord”

·       In 1899, the “British Institute of Preventive Medicine” was renamed as the “Lister Institute of Preventive Medicine”.

On 10th of February, 1912, Joseph Lister passed away at the age of 85 years.