Media Components - Water & Carbon Sources

The composition of fermentation media is dependent on a number of factors characteristic of the particular fermentation.  The major ingredients of a typical fermentation media are:

■       Water

■       Carbon source

■       Nitrogen source

■       Sources of P, S, minor and trace elements

■       Vitamins – Biotic, riboflavin etc

■       Buffers

■       Antifoam

■       Precursor

■       Inducer

■       Inhibitor

 

Water

Water is a major component of all fermentation media except solid-substrate fermentation. It also provides trace mineral elements. Water is also important for ancillary equipment and cleaning. Supply of large quantities of clean water, of consistent composition, is therefore essential. The quality of water in terms of pH, dissolved salts and effluent contamination is important.  The mineral content is important in brewing (mashing step) and has influenced the location of breweries and types of beer produced.  Before use, suspended solids, colloids and microorganisms should be removed. If the water is ‘hard’, it should be treated to remove salts such as calcium carbonate. Iron and chlorine should also be removed. For some fermentations, especially, plant and animal cell culture, the water must be highly purified. Since, water is becoming increasingly expensive, its recycle/reusage wherever possible should be encouraged. This minimizes water costs and reduces the volume requiring waste-water treatment.

Carbon Sources

A carbon source is required for all biosynthesis leading to reproduction, product formation and cell maintenance.  In most fermentations it also serves as the energy source. 

Carbon requirements may be determined from the biomass yield coefficient (Y), an index of the efficiency of conversion of a substrate into cellular material.

Y_carbon(g/g) =   Biomass produced (g)

                      carbon substrate utilized (g)

 

For commercial fermentations, the determination of yield coefficients for all nutrients is usually essential.  As most carbon substrates also serve as energy sources, the organism’s efficiency of adenosine triphosphate (ATP) generation and its utilization are key factors.  Carbohydrates are traditional carbon and energy sources for microbial fermentations, although other sources may be used, such as alcohols, alkanes and organic acids.  Animal fats and plant oils may also be incorporated into some media, often as supplements to the main carbon source.

Molasses Pure glucose and sucrose are rarely used for industrial-scale fermentations, primarily due to cost.  Molasses, a by-product of cane and beet sugar production, is a cheaper and more usual source of sucrose.  Molasses are concentrated syrups or mother liquors recovered at any one of several steps in the sugar refining process with different names depending on the step from which it is recovered.  Blackstrap molasses from sugar cane is the cheapest and most used sugar source for industrial fermentation.  This is the residue remaining after most of the sucrose has been crystallized from the plant extract.  It is a dark-coloured viscous syrup containing 50-60% (w/v) carbohydrates, primarily sucrose, with 2% nitrogenous substances, along with some vitamins and minerals.  Overall composition varies depending upon the plant source, the location of the crop, the climatic conditions under which it was grown and the factory where it was processed.  The carbohydrate concentration may be reduced during storage by contaminating microorganisms. 

Refinery blackstrap molasses is a similar product obtained from the recrystallization refining of crude sucrose.  High test or invert molasses is produced after whole cane juice is partially inverted/partially hydrolyzed to monosaccharides to prevent sugar crystallization. It contains approximately 70-75% sugar and is preferable to blackstrap molasses as it has lower levels of non-fermentable solids. Beet molasses are produced from beetroot, in a similar process as for sugarcane.  However, it may be limiting in biotin for yeast growth and a small amount of cane molasses may need to be added in these fermentations. Hydrol molasses, a by-product of maize starch processing primarily contains glucose (60%) and a relatively high salt concentration.

Malt Extract Aqueous extracts of malted barley can be concentrated to form syrups that are useful carbon sources for the cultivation of filamentous fungi, yeast and actinomycetes. The composition of malt extracts varies to some extent, but they usually contain approximately 90% carbohydrate, on a dry weight basis.  This contains 20% hexoses (glucose and small amounts of fructose), 55% disaccharides (mainly maltose and traces of sucrose), along with 10% maltotriose, a trisaccharide.  These products contain a range of branched and unbranched dextrins (15-20%), which may or may not be metabolized, depending upon the microorganism.  Malt extracts also contain some vitamins and approximately 5% nitrogenous substances, proteins, peptides and amino acids.

Sterilization of media containing malt extract must be carefully controlled to prevent overheating which produce Maillard reaction products. These are brown condensation products due to the reaction of amino groups of amines, amino acids and proteins with the carboxyl groups of reducing sugars, ketones and aldehydes. This occurs when reducing sugars and amino acids are heated at low pH. Such reactions cause loss of fermentable materials, colour change and some reaction products may inhibit microbial growth.

Starch and Dextrins These polysaccharides are not as readily utilized as monosaccharides and disaccharides. It can be metabolized by amylase-producing microorganisms, particularly filamentous fungi which hydrolyze the substrate to a mixture of glucose, maltose or maltotriose. The product composition is similar to that of malt extracts.  Maize starch is most widely used, but may also be obtained from other cereal or root crops. Starch is usually converted into sugar syrup, containing mostly glucose before use in fermentations.  It is first gelatinized and then hydrolyzed by dilute acids or amylolytic enzymes, often microbial glucoamylases.

Sulphite Waste Liquor Sulphite waste liquor is the product of the paper pulping industry. It is obtained after wood for paper manufacture is digested to cellulose pulp.  It can be used as a dilute fermentation medium for ethanol production by S. cerevisiae and the growth of Torula utilis for feed.   Waste liquors from coniferous trees contain 2-3% (w/v) sugar, which is a mixture of hexoses (80%) and pentoses (20%).  Hexoses include glucose, mannose and galactose, whereas the pentose sugars are mainly xylose and arabinose.   The liquors derived from deciduous trees contain mainly pentoses.  Sulphite Waste Liquor requires processing before use as it contains sulphur dioxide or calcium hydroxide or calcium carbonate which need to be stripped or removed by precipitation with lime. Supplementation with sources of nitrogen and phosphorous is also required.

Cellulose Cellulose is mainly present as lignocellulose in plant cell walls, which has cellulose, hemicellulose and lignin.  Lignocellulose is available from agricultural, forestry, industrial and domestic wastes.  Very few microorganisms can utilize it directly, as it is difficult to hydrolyze.  The cellulose component is encrusted with lignin and provides little surface area for enzyme attack.  It is mainly used in solid-substrate fermentations to produce various mushrooms. Preliminary processing by hydrolysis may be required in case of cellulose to expose fermentable sugars.  It has potential as a valuable renewable source of fermentable sugars particularly in the bioconversion to ethanol for fuel use.  

Whey Whey is an aqueous by-product of the dairy industry.  The annual worldwide production is over 80 million tonnes, containing over 1 million tonnes of lactose and 0.2 million tonnes of milk protein.  It is expensive to store and transport.  Milk proteins can be removed from whey to be used as food supplements. Whey is then evaporated and lactose concentrates are obtained which can be used for later fermentation. Lactose is generally less useful as a fermentation foodstock than sucrose, as it is metabolized by fewer organisms.  S. cerevisiae, for example, does not ferment lactose.  It was used extensively in penicillin fermentations and is still employed for producing ethanol, single cell protein, lactic acid, xanthan gum, vitamin B12 and gibberellic acid.

Alkanes and Alcohols n-Alkanes of chain length C10-20 are readily metabolized by certain microorganisms.  Mixtures, rather than a specific compound, are usually most suitable for microbial fermentations.  However, their industrial use is dependent upon the prevailing price of petroleum.  Methane is utilized as a carbon source by a few microorganisms, but its conversion product methanol is often preferred for industrial fermentations as it presents fewer technical problems.  High purity methanol is readily obtained and it is completely miscible with water.  Methanol has a high percent carbon content and is relatively cheap, although only a limited number of organisms will metabolize it.  Also unlike many other carbon sources, only low concentrations, 0.1-1% (v/v), are tolerated by microorganisms, higher levels being toxic.  During fermentations on methanol, the oxygen demand and heat of the fermentations are high, but this is even more problematic when growing on alkanes.  Several companies used methanol in microbial protein production in the 1970s and early 1980s, but these processes are currently uneconomic.

 Ethanol is less toxic than methanol and is used as a sole or co-substrate by many organisms, but it is too expensive for general use as a carbon source.  However, its biotransformation to acetic acid by acetic acid bacteria remains a major fermentation process.

Fats and Oils

Hard animal fats that are mostly composed of glycerides of palmitic and stearic acids are rarely used in fermentations.  However, plant oils (primarily from cotton seed, linseed, maize, olive, palm, rape seed and soya) and occasionally fish oil, may be used as the primary or supplementary carbon source, especially in antibiotic production.  Plant oils are mostly composed of oleic and linoleic acids, but linseed and soya oil also have a substantial amount of linolenic acid.  The oils contain more energy per unit weight than carbohydrates.  In addition, the carbohydrates occupy a greater volume because they are usually prepared as aqueous solutions of concentrations no greater than 50% (w/v).  Consequently, oils can be particularly useful in fed-batch operations as less spare capacity is needed to accommodate further additions of the carbon source.

References

1. Industrial Microbiology: An Introduction. Michael J. Waites, Neil L. Morgan, John S
2. Principles of Fermentation Technology- Peter Stanbury, Allan Whitaker, Stephen Hall

 

Media Formulation

The word “fermentation” originates from the Latin word Fervere which means to boil. The boiling appearance is due to the appearance of bubbles on fruit/malted grain extracts which indicates the CO₂ production.

Fermentation is a process for the production of a product by the mass-culture of a microorganism under optimized conditions.

Based on the products formed, there are different types of Fermentation which are:

o   Production of Microbial cells/biomass

o   Production of Microbial enzymes

o   Production of Microbial metabolites

o   Production of Recombinant products

o   Production of Modified compound than which is added at the beginning- Transformation

Major Steps in Fermentation

• Media formulation- for inoculum development and for production fermenter
• Sterilization of medium, fermenters, ancillary equipments 
• Production of an active, pure culture in sufficient quantity to inoculate production vessel (Inoculum development) (Upstream processing- USP)
• Growth of the organism in the production fermenter under optimum conditions for product formation (Fermentation)
• Extraction of the product and its processing (Downstream processing- DSP)
• Disposal of effluents produced in the process

Media Formulation

Fermentation media must satisfy all the nutritional requirements of the microorganism and promote the synthesis of the target product, which is either the cell biomass or a specific metabolite. Most fermentations require liquid media/ broth (Submerged Fermentation-SmF/surface fermentation), although some fermentations employ solid substrates (solid state fermentation-SSF).

In an industrial fermentation process, media are required in inoculum (starter culture) build up and the main production fermentation. The media formulation is different for inoculum propagation and the actual fermentation process since they have different objectives. Inoculum media should allow rapid/uniform growth of the inoculum whereas the production medium should facilitate the production of desired product in good amounts. Also, if biomass or primary metabolites are the target product, the production medium should allow optimal growth of the microorganism. For secondary metabolite production, such as antibiotics, their biosynthesis is not growth related. Hence, the media should be designed to provide an initial period of cell growth, followed by starvation conditions. Here, the supply of one or more nutrients (carbon, phosphorus or nitrogen source) may be limited and rapid growth does not occur. This induces secondary metabolite production.

The media components usually are,

■       Water

■       Carbon source

■       Nitrogen source

■       Oxygen

■       Sources of P, S, minor and trace elements

■       Vitamins – Biotin, riboflavin etc

■       Buffers

■       Antifoam

■       Precursor

■       Inducer

■       Inhibitor

Most fermentations, except those involving solid substrates, require large quantities of water in which the medium is formulated. General media requirements include a carbon source, which provides both energy and carbon units for biosynthesis, and sources of nitrogen, phosphorus and sulphur. Other minor and trace elements must also be supplied, and some microorganisms require added vitamins, such as biotin and riboflavin. Aerobic fermentations require oxygen, and even some anaerobic fermentations require initial aeration of media, e.g. beer fermentations. As the fermentation proceeds different metabolic products are formed which may change the pH of the media and it can interfere with the growth or product formation, So, to adjust the pH, media should contain buffers or the pH is controlled by addition of acid/ alkali. Antifoam agents are also required to control foam production. Some processes require compounds like precursor, inducer or inhibitor which are added certain stages of the fermentation.

The composition of a fermentation medium may be simple to complex, depending on the particular microorganism and its fermentation.  Autotrophic microorganisms require only the simplest of inorganic media (inorganic salts, water, nitrogen source, carbon source is fulfilled by CO₂ or by carbonates) and are capable of synthesizing all the complex organic compounds required to sustain life.  Fastidious microorganisms on the other hand lack the ability to synthesize many of their sustenance and growth requirements.  They require the presence of many simple to complex preformed nutrients in the medium and must have an organic carbon supply to provide for synthesis of cell substances and release of metabolic energy.  

Simple and complex media are further subdivided into two categories: synthetic and crude. In a synthetic medium, all the components are specifically defined and known compounds.  Each component is relatively pure and the exact concentrations are known.  Synthetic medium has defined components and concentrations and are expensive due to the relatively pure ingredients used. The concentration of one or several can be varied in order to determine the effect on cell growth and product yield. Individual components may be added or deleted as well. This has advantages in certain types of studies. However, yields derived from these media are relatively low.  Crude media contain crude factors or ill-defined sources of nutrients and growth.  They usually allow much higher yields.

The media adopted also depend on the scale of the fermentation. For small-scale laboratory fermentations pure chemicals are often used in well-defined media. However, this is not always possible due to cost, as media components may account for up to 60-80% of process expenditure. Industrial-scale fermentations primarily use cost-effective complex crude substrates, where many carbon and nitrogen sources are almost indefinable.  Most are derived from natural plant and animal materials, often by-products of other industries, with varied and variable composition.  The effects of such batch-to-batch variations must be determined.  Small scale trials are usually performed with each new batch of substrate, to examine the impact on product yield and product recovery.

            The main factors that affect the final choice of individual raw materials are as follows:

1 Cost and availability: ideally, materials should be inexpensive and of consistent quality and year round availability.

2 Ease of handling in solid or liquid forms, along with associated transport and storage costs, e.g., requirements for temperature control.

3 Sterilization requirements and any potential denaturation problems.

4 Formulation, mixing, complexing and viscosity characteristics that may influence agitation, aeration and foaming during fermentation and downstream processing stages.

5 The concentration of target product to be attained, its rate of formation and yield per gram of substrate utilized.

6 The levels and range of impurities and the potential for generating further undesired products during the process.

7 Overall health and safety implications.

 

References

1. Industrial Microbiology: An Introduction. Michael J. Waites, Neil L. Morgan, John S
2. Principles of Fermentation Technology- Peter Stanbury, Allan Whitaker, Stephen Hall

Leptospirosis

Leptospirosis

Leptospirosis is infection with the Spirochaete Leptospira. It is an acute zoonotic infection of worldwide significance. Leptospirosis is seen in both humans and animals.  The primary reservoir is rodents such as rats, mice, wild rodents and once infected, they shed the organisms for life. Livestock farming is the major occupational risk factor for human leptospirosis since cattle, dogs, swine etc., can also be reservoirs.

                                   

Infected animals excrete Leptospira both in active infection and asymptomatic stage. The Leptospira survive and remain viable for several weeks in stagnant water. It is common in temperate or tropical climates - rare in North America. Transmission to humans occurs through penetration of the organism into the blood stream via cuts, skin abrasions or mucus membranes

                                                    

Leptospirosis is also known as hemorrhagic jaundice, infectious jaundice, mud fever, spirochetal jaundice, swamp fever, swineherd's disease, caver's flu, sewerman's flu, Canicola fever (canine leptospirosis-dogs) etc

First human leptospiral disease was described by Adolf Weil in 1886, as an "acute infectious disease with enlargement of spleen, jaundice and nephritis. Leptospira was first observed by Stimson in 1907 from a post mortem renal tissue slice. Stimson named it Leptospira interrogans owing to its shape resembling interrogation (question) mark.

                          

Human infection also known as Weil's disease, is caused mainly by Leptospira icterohaemorrhagiae, which was isolated in 1915 by Inada. Consequently many leptospires have been isolated.

Spirochetes are divided into two families, Spirochaetaceae and Leptospiraceae. Spirochaetaceae –include Treponemes, Serpulina and Borrelia and Leptospiraceae include Leptospira.

Leptospira is further classified into several species and subspecies, called serogroups and serovars, based on the surface (lipopolysaccharide -LPS) antigens. Genus Leptospira is divided into two species -L. interrogans includes pathogenic strains and L. biflexa includes saprophyte strains -from the environment. These two species are divided into serovars  as defined by agglutination techniques. There are approximately 60 serovars for L. biflexa, more than 200 for L. interrogans.

L. interrogans has more than 22 serogroups common examples being L. icterohaemorrhagiae, L. Canicola, L. australis, L. hebdomadis, L. andamana, L. pyrogenes etc

 

 

Cleaning of Glass Wares

                                                     

Clean glass wares are the most important requirement of lab. Even carefully done work may have the wrong results if dirty glass wares are used. After receiving and before starting a work, the glass wares must be chemically cleaned so that there are no chemical deposits on its surface. 

Dirty glass wares should be cleaned up first by removing grease with a rag soaked in benzene or chloroform, followed by soaking overnight in chromic acid. Chromic acid is widely used as a cleansing agent for glass wares. It is a mixture of sodium chromate and concentrated sulphuric acid. It has powerful oxidising and dissolving properties. If it is not effective, a mixture of concentrated nitric acid and sulphuric acid can be applied. All the traces of cleanser are then removed by repeated rinsing in the tap water followed by distilled water. 

Sometimes, new glass wares may contain some bacterial and fungal spores that come with packaging materials. The glass wares from factory also contain some amount of alkalies, hence for removal of alkali, 2-3% Hydrochloric acid is applied for 24 hours, for the process of neutralization. 

Preparation of chromic acid 

Weigh 5 gm of sodium/potassium dichromate (Na2Cr2O7/K2Cr2O7) and dissolve in 5 ml distilled water in a beaker (250 ml). Add 100 ml concentrated sulphuric acid slowly and stir it constantly. The mixture is allowed to cool to about 40°C and then stored in a dry glass stoppered bottle. The solution should be handled with care. Preferably, rubber gloves and apron should be used while using it. If clothes are spilled with the solution, wash them immediately followed by neutralization of the acid with sodium carbonate solution. This in turn is washed off with water. 

Cleaning of pipettes 

Place the used pipettes in 3% Lysol. If necessary, keep overnight in detergent or dichromate or sulphuric acid cleaning solution. Wash with tap water followed by deionized water. If required, the tip end of the pipette may be plugged with cotton wool.

Basic Rules and Regulations in Microbiology Laboratory

                            

A microbiology laboratory is a place for working with a variety of microorganisms. Most microbiological laboratory procedures require the use of living organisms and the integral part of all laboratory sessions is the use of aseptic techniques. Although the virulence of microorganisms used in the academic laboratory environment has been greatly diminished because of their long term maintenance on artificial media, all microorganisms should be treated as potential pathogens (capable of causing disease). Thus microbiology students must follow aseptic techniques (free of contaminating microorganism) in the preparation of pure cultures that are essential in the industrial and clinical applications. 

Rewarding laboratory experience demands strict adherence to prescribed rules for personal and environmental safety. The former reflects concern for personal safety in terms of avoiding laboratory accidents. The latter is required maintaining a scrupulously clean laboratory setting to prevent contamination of experimental procedures by microorganisms from exogenous sources. These techniques will thus determine the success or failure of the scientific experiments conducted as well as protects the lab personnel from potentially harmful microorganisms. 

The following basic steps should be observed at all times to reduce the ever present microbial flora of the laboratory environment. 

1. Upon entering the laboratory, place coats, books and other paraphernalia in specified locations and never on working bench tops. Working space should be reserved for essential equipments and a lab book. 

2. Keep doors and windows closed during laboratory sessions to prevent contamination from air currents. 

3. A lab coat or apron must be worn at all times in the laboratory to protect clothing from contamination or accidental discoloration by staining solutions. When leaving the laboratory, remove the coat or apron and at regular intervals, get the apron washed. 

4. Wear a paper cap or tie back long hair to minimize its exposure to open flames and contamination with microbial cultures. 

5. Wear closed shoes at all times in the laboratory. 

6. Cut nails regularly and don’t put finger in eyes, ear or mouth as it can facilitate the chance of infection by microorganisms. 

7. Do not eat, drink, apply cosmetics or insert contact lenses in the laboratory. 

8. Wash the hands with liquid detergent, rinse with 70% ethyl alcohol and dry them with paper towels upon entering and prior to leaving the laboratory. 

9. Wipe bench tops with disinfectant solution, at the beginning and termination of each laboratory sessions and after every spill. 

10. Carry cultures in a test tube rack when moving around the laboratory. Also keep cultures in a test tube rack on the bench top when not in use. This helps to prevent accidents and to avoid contamination of yourself and the environment. 

 11. Open the culture tubes or plates and perform the experiments near the vicinity of the flame of the burner. The burner must be kept at a distance from organic solvents and immediately turned off after use. 

12. Always use a mechanical device for pipetting reagents or bacterial cultures. Mouth pipetting is strictly prohibited in the microbiology laboratory. 

13. Do not lick labels. Use only self stick labels for the identification of the experimental cultures. 

14. Do not place contaminated instruments such as inoculating loops, needles, pipettes and slides on bench tops. Loops and needles should be sterilized by incineration and pipettes and slides should be disposed off in designated receptacles. 

15. On completion of the laboratory sessions, place all cultures and materials in the disposal area 

16. Rapid and efficient manipulation of fungal cultures is required to prevent dissemination of their reproductive spores in the laboratory environment. 

17. Speak quietly and avoid unnecessary movements in the laboratory so as to prevent distractions which can lead to accidents. 

18. Report accidental cuts or burns or spills immediately. 

19. Spilled cultures or broken tubes must be immediately covered with paper towel and saturated with disinfectant solutions. After 15 minutes of reaction time, the towel should be removed and dispensed. 

20. Follow strict aseptic techniques for personal safety and success of experiments performed 

Indicator microorganisms

                                                 Indicator microorganisms

Indicator micro-organisms are used to suggest the presence of pathogens in water. The direct detection of pathogenic bacteria and viruses, and cysts of protozoan parasites requires costly and time-consuming procedures, and well-trained labor. These requirements led to the concept of indicator organisms of fecal contamination. 

The criteria for an ideal indicator organism are: 

1. It should be one of the intestinal microflora of warm-blooded animals.

 2. It should be present in samples when pathogens are present. 

3. It should be present in greater numbers than the pathogen. 

4. It should be at least equally resistant as the pathogen to environmental factors and to disinfection in water and wastewater treatment plants. 

5. It should not multiply in the environment. 

6. It should be detectable by means of easy, rapid, and inexpensive methods. 

7. The indicator organism should be nonpathogenic. 

8. It should be useful for all water types. 

Various microorganisms have been proposed and used for indicating the occurrence of fecal contamination, treatment efficiency in water and wastewater treatment plants, deterioration and post- contamination of drinking water in distribution systems. 

The main indicator groups are: 

 Coliforms 

 Fecal Streptococci 

 Sulphite Reducing Clostridia 

Faecal indicator: A group of organisms that indicates the presence of faecal contamination, such as the thermo tolerant coliforms (E. coli) or fecal streptococci. Their presence denotes that pathogens may be present. 

1. Coliforms

 The total coliform group belongs to the family Enterobacteriaceae. These are aerobic and facultative anaerobic, gram-negative, nonspore-forming, oxidase-negative, rod-shaped bacteria that ferment lactose (β-galactosidase positive) with gas and acid production within 24-48 hours at 35°C. They are not necessarily specific indicators of faecal pollution.

 eg. Escherichia coli, Enterobacter, Klebsiella, and Citrobacter. 

Coliforms are discharged in high numbers in human and animal feces, but not all of them are of fecal origin. These indicators are useful for determining the quality of potable water. They are less sensitive than viruses or protozoan cysts to environmental factors and to disinfection. Some members (e.g., Klebsiella) of this group may sometimes grow under environmental conditions in industrial and agricultural wastes. 

Fecal Coliforms 

Fecal coliforms or thermo tolerant coliforms include all coliforms that can ferment lactose with gas and acid production at 44.5°C within 24-48 hours, in addition to 35°C. The fecal coliform group comprises bacteria such as Escherichia coli. 

Thermotolerant coliforms produce indole from tryptophan, cannot utilize citrate as sole source of Carbon and produce βglucuronidase. They are the specific indicators of recent faecal pollution from warm-blooded animals. 

The presence of fecal coliforms indicates the presence of fecal material from warm-blooded animals. However, human and animal sources of contamination cannot be differentiated. They are much less resistant to disinfection than viruses or protozoan cysts. 

2. Fecal Streptococci 

Gram-positive, catalase-negative cocci. This group comprises Streptococcus faecalis, S. bovis, S. equinus, and S. avium. Streptococcus faecalis, S. faecium, S.durans are commonly isolated from humans, while S. bovis, S. equinus, and S. avium are isolated from animal sources, cattle, horse and birds, respectively. Because they commonly inhabit the intestinal tract of humans and warm-blooded animals, they are used to detect recent fecal contamination in water.

 All fecal streptococci that grow at pH 9.6, 10° and 45°C and in 6.5% NaCl are designated as Enterococci. They show resistance to 60°C for 30 min and ability to reduce 0.1% methylene blue. Faecal streptococci can also grow in presence of 40% bile, sodium azide and potassium tellurite concentrations which normally inhibit other organisms including coliforms. 

The fecal coliform to fecal streptococci ratio (FC/FS ratio) is an indicator of the origin of pollution of surface waters. A ratio of >4 indicates a contamination of human origin, whereas a ratio of 0.7 is indicative of animal pollution. This ratio is only valid, however, for recent (24 hours) fecal pollution and is unreliable for chlorinated effluents. 

3. Sulphite Reducing Clostridia - Clostridium perfringens. 

Clostridia are mostly opportunistic pathogens, but are also implicated in human diseases such as gas gangrene (C. perfringens). Clostridium perfringens are gram-positive, strictly anaerobic rods which are endospore-forming, non-motile and can reduce sulphite to H2S. 

Clostridium perfringens can ferment lactose, sucrose and inositol with the production of gas, produce a stormy clot fermentation with milk, reduce nitrate, hydrolyse gelatin and produce lecithinase and acid phosphatase. These sulfite-reducing bacterium found in the colon form approximately 0.5 percent of the fecal microflora. The spores are quite resistant to environmental stresses, and to disinfection by oxidizing agents and UV than bacterial and phage indicators. It is commonly found in human and animal feces and in wastewater-contaminated aquatic environments. 

The hardy spores make this bacterium too resistant to be useful as an indicator organism. It is an indicator of past pollution. 

4. Bacteriophages 

To evaluate the virological quality of water, the use of bacteriophages as indicators has been proposed. Three groups of phages have been suggested: somatic coliphages (infect mostly E. coli), F-specific RNA bacteriophages and phages infecting Bacteroides fragilis. 

The occurrence of specific pathogens is seasonal. Also, viruses and other pathogens not part of the normal faecal microbiota and are excreted by infected individuals. 

So, the idea of indicators of microbial water quality help in alerting and managing waterborne microbial risks, where the pathogens may escape chances of detection.

ABO Blood Grouping

 

Blood Grouping

Aim

To understand the ABO blood group system and to determine the blood group.

Principle

The ABO blood grouping system was discovered in the year 1901 by Karl Landsteiner. ABO Blood Group System is based on the presence or absence of two specific antigens and antibodies - A and B: The presence or absence of these two similar carbohydrate antigens located on the surface of red blood cells is determined using specific antisera. 

Haemagglutination occurs when anti A antiserum is mixed with type A red blood cells. People with blood type AB possess both A and B antigens on their red blood cells and those with O blood type lack A and B antigens.  Agglutination reactions occur between high molecular weight particulate antigen and antibody. Because many antigens are there on cells, agglutination reaction leads to clumping or agglutination of cells. When the clumping involves red blood cells, it is called haemagglutination. Haemagglutination reactions are used in the typing of blood.

Many other antigens exist on human red blood cells. Another surface antigen on red blood cells is designated as Rh factor or D antigen. Individuals are Rh positive when D antigen is present. The presence of Rh factor is determined by a haemagglutination reaction between antigen D antiserum and red blood cells with antigen D on their surface.

A person possess antibodies to the alternate antigen. Thus people of blood type A have antibody to the B antigen in their sera. Rh negative individuals do not naturally have anti D antibody in their sera. Anti D antibodies are produced when red blood cells with D antigen are introduced into Rh negative individuals.

Type O (O-ve) blood group is called the universal donor, because type O blood group have neither A or B antigens. It can be donated to recipients of any blood type. Type AB (AB-ve) blood group is called the universal recipient. They lack anti-A nor anti-B antibodies in their plasma and can receive blood from the donors of any blood type. AB-negative is considered to be the rarest blood type. 

ABO and Rh blood types are important as they are essential during the blood transfusions and to avoid further complications.  An incompatible transfusion results when the antigens of the donor red blood cells react with the antibodies in the recipient’s serum or induce the formation of antibodies. Small samples of the recipient's and donor's blood are mixed to check compatibility in a process known as cross-matching before transfusion.

Materials Required

• Sterile cotton balls
• Sterile lancet
• Clean glass slide
• Anti-A, B, and D antiserum
• 70% alcohol
• Marking pen

Procedure

1. With the marking pen three circles are drawn in a clean glass slide and labelled A, B and D.
2. The ring finger disinfected with the cotton ball dipped in 70% alcohol
3. The ring fingertip pricked with the lancet and added a drop of the blood each to the circles labelled A, B and D
4. The bleeding stopped using a sterile cotton ball and a bandage applied to the wound
5. To the circle labelled A, added one drop of anti A antiserum, to the circle labelled B, added Anti-B and to the third circle labelled D, added Anti-D with the help of a dropper.
6. Mixed each suspension gently with the help of a toothpick and observed the result.

Observation

The test sample showed no agglutination in the circles “A” (with anti A) and “B” (with anti B) and agglutination in circle D (with anti D)

Result

The tested sample is O+

 

ABO blood grouping (left hand side)

Blood Group	Antigen	Antibody
A	A	B
B	B	A
AB	A and B	neither A nor B antibodies
O	No A or B antigens	both A and B antibodies
Rh+	D antigen	No D antibody
Rh-	No D antigen	No D antibody in usual cases, till an exposure to D antigen occurs