Preserving Microbial Cultures

When once microorganisms are cultured in the laboratory, they are usually sub cultured on agar plates or slants at regular intervals to maintain viability.

Some of the microbes routinely used for experiments should always be available for any immediate experimentation. In order to have a ready availability of cultures, the microbial cultures should be maintained properly and in viable conditions.

Most microbiological laboratories maintain a large collection of strains, frequently referred to as ‘stock culture collection.’

Various methods are:

1.       Periodic  transfer to fresh media

2.       Preservation by overlying with mineral oil

3.       Lyophilization/freeze-drying

4.       Storage at low temperature

5.       Sordelli’s method of preservation of cultures

6.       Preservation at -40 degree centigrade in glycerin

7.       Preservation in soil

8.       Paraffin method

9.       Use of refrigerator or cold room storage

10.   Preservation of soil cultures

Periodic transfer to fresh media:

·         Strains can be maintained by periodically preparing a fresh stock culture from the previous stock cultures.
·         The culture medium i.e.; storage temperature and the time intervals at which the transfers are made vary with the species and must be ascertained beforehand.
·         The temperature and type of medium chosen should support  a slow rather than a rapid rate of growth so that the time interval between transfers can be as long as possible.
·         Many of the more common heterotrophs remain viable for several weeks or months on a medium like nutrient agar.
Disadvantages:  Changes in characteristics of a strain due to development or variants in mutants.

Preservation by overlying cultures with mineral oil:

·         Many bacteria can be successfully preserved by covering the growth on agar slant with sterile mineral oil.
·         The oil must cover the slant completely. Therefore,  to ensure this, the oil should be about ½ inch above the tip of the slanted surface.
·         Maintenance of viability under this treatment varies with species.
·         This method of maintenance has the unique advantage that one can remove some of the growth under the oil with a transfer needle, inoculate into a fresh medium and inoculate into a fresh medium and still preserve the original culture.
Disadvantages: Change in characteristics of strain.

Lyophilization or freeze-drying:

·         Many bacteria die if cultures are allowed to become dry, although spore-formers can remain viable for many years.
·         However, freeze-drying can satisfactorily preserve many kinds of bacteria that would be killed by ordinary drying.
·         In this process, a dense cell suspension is placed in small vials and frozen at -60 to -78 degree centigrade.
·         The vials are then connected to a high-vacuum line.
·         The ice present in the frozen suspension sublimes under the vacuum i.e.; evaporates without first going through a liquid water phase.
·         This results in dehydration of the bacteria with a minimum damage to delicate cell structures.
·         The vials are then sealed off under a vacuum and stored in a refrigerator.
·         Many species of bacteria preserved by this method have remained viable and unchanged in their characteristics for more than 30 years.
·         Lyophilized cultures are revived by opening the vials, adding liquid medium and transferring the rehydrated cultures to a suitable growth medium.
Advantages:
1.       Only minimal storage space is required.
2.       Hundreds of lyophilized cultures can be stored in a small area.
3.       Small vials can be sent conveniently through mail to other microbiology laboratories when packaged in special sealed mailing containers.
Storage at low temperature (liquid nitrogen):
·         In this procedure cells are prepared as a dense suspension in a medium containing a cryoprotective agent such as glycerol or dimethyl sulfoxide (DMSO), which prevents cell damage due to ice crystal formation during the subsequent steps.
·         The cell suspension is sealed into small ampoules or vials and then frozen at a controlled rate to -150 degree centigrade.
·         The ampoules or vials are then stored in a liquid nitrogen refrigerator, either by immersion in liquid nitrogen or by storage in the gas phase above the liquid nitrogen            -150 degree centigrade.
·         The liquid nitrogen method has been successful with many species that cannot be preserved by Lyophilization and most species can remain viable under these conditions for 10-30 years or more without undergoing change in their characteristics.
Disadvantages: This method is relatively expensive, since the liquid nitrogen in the refrigerator must be replenished at regular intervals to replace the loss due to evaporation.

Sordelli’s method of preservation:

·         This is a method of preservation simpler than freeze drying, but as reliable as latter.
·         This method can be safely used when the samples to be preserved are small in quantity.
·         In this method, the culture should be preserved or incubated on solid medium for the required period.
·         The inoculum is emulsified in a loopful of horse serum and is deposited on the inner wall of the small tube, which is inserted into another large tube.
·         A small quantity of phosphorus pentoxide is placed at the bottom or the outer tube with the help of glass rod.
·         The inner tube must be placed in such a way that it is held over the bottom of the outer tube but not directly touching the chemical placed at the bottom.
·         The outer tube is then connected to a vacuum pump and after the air is removed, the outer tube is sealed.
·         This tube containing the culture can be stored at room temperature away from light.
 

Preservation at -40 degree centigrade in glycerol:

·         Cultures can be preserved for a number of years in glycerol at a temperature of -40 degree centigrade in a freezer.
·         In this method, about 2ml of glycerol solution is added onto the agar slant culture.
·         The culture can be emulsified by shaking the culture and emulsion is transferred to ampoules with each ampoule having 5ml of culture.
·         These ampoules are placed in a mixture of industrial methylated spirit and carbon dioxide and are frozen rapidly at -70 degree centigrade.
·         The ampoules are then removed and placed directly in a deep freeze at -40 degree centigrade.
·         For utilization of stock cultures, the ampoules are kept in a water bath at 45 degree centigrade for about a few seconds and then used.

                      Preservation in soil:

·         Soil borne bacteria and fungi can be stored in their natural habitat i.e.; soil.
·         About 5gms of soil sample is autoclaved at 15lb pressure for 30 minutes and inoculated with 1ml of aqueous suspension of cells/spores.
·         The microorganisms are allowed to grow for 10 days and the soil culture thus obtained is stored in refrigerator.
·         Microorganisms tend to undergo less variation in soil than in agar.

                      Paraffin Method:

·         Simple and cost effective method.
·         Used for preserving cultures of bacteria and fungi for several years at room temperature.
·         In this method sterile liquid paraffin is poured over the slant of microbes and stored upright at room temperature.
·         The layer of paraffin prevents dehydration of the medium and ensures anaerobic conditions.
·         As a result, the microbes remain in dormant condition.
·         It has been seen that in some instances bacteria remained viable for even up to 15-20 years.

Cold Room Storage:

·         Live cultures on a culture medium can be successfully stored in refrigerator or cold rooms. When the temperature is maintained at 4 degree centigrade.
·         At this temperature range the metabolic activities of microbes slow down greatly.
·         As a result, bacterial metabolism will be very slow and only less quantity of nutrients will be utilized.
Disadvantages: Cannot be used for long time storage because not only the nutrients get utilized but also waste products get accumulate, killing the microbes.

Preservation of Soil Cultures:

·         A sample of soil is passed through a sieve of 2mm and collected in sufficiently large test tubes.
·         1% solution of dextrose is added to the tubes and mixed thoroughly.
·         These cultures are then kept in a boiling water for about 15 min and then autoclaved for 1 hour at 13 pound pressure for 2 successive days.
·         Pure culture of soil microbes can be kept in this medium and preserved for a number of months under refrigerator.

Organs Of The Immune System

The immune system is composed of different immune organs, cells and tissues. There are two groups of immune organ systems.

• ·      Primary lymphoid organs
• ·         Secondary lymphoid organs

"Primary organs” - These are immune organs concerned with production and maturation of lymphoid cells including bone marrow and thymus gland.
“Secondary organs” - These immune organs are spots or sites in which the lymphocytes localize, identify unfamiliar antigens and triggers reaction in opposition to it. It Contains tonsils, lymph nodes, Spleen, Peyer’s patches (in the small intestines), appendix and liver.

Primary Organs

Thymus- Bilobed organ located above the heart (beneath the breast bone).It functions at its peak during adolescence producing specialized lymphocytes-T-cells and B-cells and dispatching them through lymph vessels to secondary organs. In very simple words, we can say its purpose is to initiate antibody formation.

Immature thymocytes, also called prothymocytes, abandon bone marrow to move in to the thymus, by the way of an extraordinary maturation process at times called thymic education, T cells which are good for the body's defense mechanisms are spared, but other T cells which may stimulate a harmful autoimmune reaction are eliminated. The Release of Mature T cell into the bloodstream takes place next.

Bone Marrow - All  the cells from the immune system, before anything else, are originally produced by the bone marrow. They are formed via a process named hematopoiesis. Throughout hematopoiesis, the stem cells derived from the bone marrow separate into precursors of those cells that travel from the bone marrow to carry on their growth somewhere else or into mature cells. Bone marrow generates B cellular material, granulocytes, natural killer cells, and also immature thymocytes, as well as red blood cells.

                                                  

  Secondary Organs

Lymph nodes- Also at times termed as lymph glands, they are little spherical or bean-shaped clusters of lymphatic tissues enclosed by a sort of capsule made of connective tissue. There are about 500-700 lymph nodes spread all through our bodies. Lymph nodes separate out the lymphatic fluid the store specific cellular material that will capture most cancers cells or bacteria which are travelling throughout the human body within the lymph fluid.


Spleen - One of the most essential  immune organs, it works as an immunologic filtration system in the bloodstream. It consists of B cells, dendritic cells, T cells, red blood cells, macrophages and natural killer cells. Together along with unfamiliar materials (antigens) from the bloodstream goes to the dendritic cells, spleen, migratory macrophages and deliver antigens to the spleen through the blood stream. An immune answer is started once the dendritic cells or macrophage offer the antigen towards the proper T or B cells. This organ could be regarded as a conference centre. Within the spleen, then follows the activation of the B cells and the production of massive quantities of antibody. Also, older red blood cells, at that time, are eliminated within the spleen.


Tonsils and adenoids - They are two lumps of tissues, on either sides in the throat, inserted in a pocket beside the palate (that's the roof of the mouth). The lower edge of every single tonsil is near the tongue...way at the back of the throat. The adenoids really are a single clump made of tissue behind the nose area (nasopharynx). They're situated (in the adult) around the back, at the wall structure of the throat (pharynx)...about 1 inch over the uvula (the small teardrop shaped little bit of tissues that dangles down in the centre of the soft palate). Even though adenoids and tonsils have comparable purpose, i.e. trapping viruses and bacteria, they're entirely independent immune organs.


Peyer’s patches - They are nodules of lymphatic cells that combine to make patches or bundles and appear generally only within the lowest part (ileum) of your small intestine; they're named after 17th-century, Switzerland anatomist Hans Conrad Peyer. Saying that in a different way, they could be thought as patches of nodules, in the small intestines walls. 


Appendix - It's a thin, dead-end tube measuring about three-to-four inches in length and it hangs from the cecum (which is end in the large intestine). Even though it's typically called the "appendix," the actual term for it is "vermiform appendix."
During the past, the appendix was regarded as not having any purpose in the body or as not currently being among the immune organs. Today, it is stated, often, that this takes on a task in the body's defense mechanisms because its surfaces incorporate aggregated lymphoid tissues. Researchers say that the appendix assists in supporting the immunity process through 2 approaches. It will  tell the lymphocytes exactly where they have to head over to attack infection and it also enhances the massive intestine's defenses to a range of drugs and foods.

Selective Toxicity

Selective Toxicity, can be defined as, the capability of an antibiotic to harm one type of microorganism without harming other organism (humans) that is intimately associated with it.
This principle is used in agriculture, pharmacology and diagnostic microbiology. It is mostly important in the 'systemic chemotherapy of infectious diseases."

1. Selective action against microorganisms is based on the differences in the cell physiology of the parasite and the mammalian host.
2. Energy yielding processes do not offer possibilities for selective toxicity.
3. Chemicals that inhibit a particular step in a metabolic pathway that is important to the parasite, but that does not occur or is not accessible in the cells of the host, exhibit selective toxicity.
4. The treatment of bacterial infections is more successful than that of viral diseases. This is because viruses depend on many enzymes of the host cell for their replication.
5. A high degree of selective toxicity may be associated with a narrow antimicrobial spectrum and with the emergence of drug-resistant organisms.

Selective toxicity of an antibiotic depends on the therapeutic index i.e; the ratio of toxic dose to the therapeutic dose. The larger the index, more safe is the antibiotic for human use.

Example:

• Protein synthesis inhibitors such as, streptomycin or tetracycline  act only on the protein synthesis of the microorganism because, bacterial ribosomes are different from the human or eukaryotic ribosomes.
• Penicillin antibiotics act on the cell wall of the microorganisms. They are safe for the human beings as they do not have a cell wall.

Biofilter

Biofiltration is the most cost effective, operationally simple technology with high waste-removal efficiencies; used to purify contaminated air evolved from volatile organic and inorganic compounds by using microorganisms. 

This technique has been industrially successful in Europe and Japan. It is gradually becoming popular. They were first built in U.S.A during 1960's.

The process is carried out in biofilters.These are packed with soil or compost covered by an active bio film, through which gas is blown. Microorganisms make use of the gaseous pollutants that are present in the gas blown and use these pollutants as a source of carbon and energy. This is a very beneficial technique as it does not need large amounts of energy for operation.

Microorganisms Used In The Process

Microorganisms that are present in biofilters are mainly aerobic ones(microorganisms that require oxygen for their growth). Most common organisms are Bacteria such as coryneforms and endospore formers, sometimes Protozoa, Invertebrates and few Actinomycetes are also used. most commonly used fungi are
Alternaria, Aspergillus, Fusarium etc. Microorganisms are the crucial component because they degrade the gaseous pollutants. In specific cases genetically engineered microorganisms are also used.

Biofilter Media

The packing material or the media used should have some characters which influence the efficient working of Bio-filters.

1. The filter media should allow the microorganisms to interact with oxygen and water.
2. The media should have fine pores, large surface area and a uniform pore size.A large surface area provides adsorption and support for microbial growth.
3. Should have the capacity to retain moisture to sustain bio film
layer and retain capacity of nutrient supply to microbes that form the bio film.
The most commonly used packing materials are metal oxides, glass or ceramic beads.Polyvinyl chloride is the most efficient packing material.
Natural Bio-filter media include compost, peat, soil, wood chips etc.

Mechanism Involved

Mechanism involved is a multistage process.First the contaminants are converted into liquid phase and then transported to the bacterial cell in the bio-film across the cell membrane of the organism, where the compound is degraded and used in cell metabolism to produce carbon and energy. After bio degradation the contaminants are exhausted from the biofilter.The treatment process depends on two mechanisms:
1. Directly adsorbing to biofilm and degradation.
2. Conversion into aqueous phase and degradation.

Attachment Of Microorganisms To The Media

The attachment of microorganisms to the media consists of two processes:
1. self-attachment of cells to the media by the secretion of glycocalyx or by covalent, hydrophobic and electrostatic interactions.
2. Immobilization ( artificial attachment)by carrier-binding method, cross-linking method, entrapment etc.

But the main disadvantage of the technique is that, it is not suitable for halogenated compounds.
Other drawbacks are:
1. very low aerobic degradation.
2. Size of bio degradation is inversely proportional to the degradation rate.

Despite of disadvantages, the process can be used to remove foul smelling compounds from waste water treatment plants or animal farming. This can be achieved by digging trenches, laying an air distribution system and refilling the trenches with soil, wood chips and compost.

Plants As Biofilters

Plants can be used as Bio-filters to make the ponds habitable for fish. Plants such as Sphagnum, Water lettuce and American shower weed. When the plants are grown in nutrient deprived conditions and transferred to ponds, they take up nutrients, specifically nitrates present in pond water.

This is a very old biotechnological process and research has to be done to determine the extent to which biofilters are efficient in removing the pollutants and much information has to be gathered regarding design, construction and implementing this technique.

Industrial Production Of Cheese

Cheese is a means to conserve many of the nutrients in milk. A well- ripened cheese is so attractive and sometimes it is hard to understand how something that has distinctly bad smell can yield wonderful flavors.

Two thousand distinct varieties of cheese are produced throughout the world. There are approximately twenty types used commonly. There are two groups of cheese:
1.Fresh cheese- which is made by coagulating milk with acid or high heat. Example is cottage cheese.
2.Ripened cheese-made through fermentation by lactic acid bacteria and then coagulated by an enzyme.

There are three categories of ripened cheese:
1.Soft cheese-ripened by enzymes from yeast and other fungi.
2.Hard cheese-ripened by lactic acid bacteria that grows throughout the cheese.
3.Semisoft cheese-ripened by proteolytic (protein degrading) and lipolytic (lipid degrading) organisms.

Microorganisms Involved In Cheese Making:

Most of the cheeses making processes employ starter organisms such as strains of Lactococcus lactis and its subspecies, thermophilic starters such as Lactobacillus helveticus, Lactobacillus casei, Lactobacillus lactis, Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus salivarius subspecies thermophilus.

The role of starter organisms is:
1.Fermentation of milk sugar lactose to lactic acid.
2.Decrease pH of cheese, to increase its shelf life. As the pH decreases, the cheese develops a good flavor.

Mechanism Involved In Fermentation Of Milk Sugar Lactose:

Different microorganisms employ different metabolic pathways for breaking down of lactose.
1.In Lactobacilli and Streptococcus salivarius, a specific permease takes up the lactose and then is broken down by beta galactosidase enzyme. The glucose that is formed is converted to galactose. Galactose is eventually converted to glucose-6-phosphate. If lactose is not available in sufficient amounts to the microorganism, galactose accumulates leading to a brown discoloration of cheese that is unwanted.
2.In most Lactococci and some Lactobacilli such as Lactobacillus casei, take up lactose by a PTS system (phosphoenolpyruvate-dependent phosphotransferase system). This system adds a phosphate to the lactose and then this lactose phosphate is broken down by phosphor-beta-galactosidase enzyme to glucose. Glucose is first converted to galactose-6-phosphate and then eventually to pyruvate.
3.Starter organisms such as Lactococcus lactis subspecies diacetylactis or Leuconostoc cremoris lead to citrate fermentation and form diacetyl. Carbon dioxide is one if the end products and it produces small eyes in Dutch cheese like Gouda.

Steps Involved In Making Cheddar Cheese:

·  Before the cow's milk is processed, it is treated with heat, so that the milk is free from antibiotics and sanitizing agents that might interfere with fermentation.

·  In the next step, milk is cooled to fermentation temperature 29-31 degrees centigrade.

·  After cooling the milk to fermentation temperature, starter culture is added. A good starter culture will multiply rapidly producing acidity.

·  To stop the multiplication of the microorganism salting is done.

·  After forty five minutes rennet is added.
Rennet is prepared from stomach of suckling calves, lambs or goats. Rennin or chymosin is an enzyme that is extracted and purified from rennet. It is added, to coagulate the protein 'casein' in the milk and also for good smell and structure of cheese. It prevents the development of bitter taste. 
Now –a-day's rennin that is extracted from microorganisms is widely used. It is extracted mostly from Fungi such as Aspergillus, Candida, Mucor, Rhizopus, Pencillium etc.

·  After thirty to forty five minutes, coagulation of milk will be completed. Then the whey is removed by cutting the curd to approximately 1cm cubes and then heating them to thirty eight to forty two degrees centigrade.
The process of expulsion of whey is called 'cheddaring' and the process is carried out in a cheddaring tower.

·  Salt at a level of 1.5-2% W/w is added. And, the salted curd is then pressed to expel trapped air and whey.

·  Finally, the cheese is ripened or matured at ten degrees centigrade for flavor development. The ripening is carried out for five months. At this stage, a combination of microbial and enzymatic reactions takes place that give the cheese characteristic flavor.

This ancient Roman food is a major industry worldwide. Brillat savarin once said "Dessert without cheese is like a pretty woman with only one eye."