Biotechfront

Endoplasmic Reticulum (ER)

2026-01-28T14:02:00.004+05:30

Discovery and Origin

Endoplasmic reticulum (also called ergastoplasm) was discovered by Porter et al. (1945) and the term ER was given by Porter (1953). According to different views, ER originates either from invagination of the plasma membrane or from the nuclear envelope.

Occurrence

Endoplasmic reticulum is present in all eukaryotic cells except ova, embryonic cells and mature erythrocytes. It forms more than 50% of the total membrane system of the cell. In muscle cells, it is known as sarcoplasmic reticulum.

Structure

Endoplasmic reticulum is a system of membrane‑bound channels and consists of three main components:

Cisternae – flattened, parallel, interconnected sacs
Tubules – branched tubular network
Vesicles – small, round or oval sacs

Types of Endoplasmic Reticulum

1. Rough Endoplasmic Reticulum (RER)

RER bears ribosomes on its surface and therefore appears granular. Ribosomes are attached to ER by a glycoprotein called ribophorin (ribophorin I and II). RER contains minute pores through which newly synthesized polypeptides pass into the lumen for transport. RER is abundant in cells actively engaged in protein synthesis and secretion such as plasma cells, goblet cells, pancreatic cells and certain liver cells.

2. Smooth Endoplasmic Reticulum (SER)

SER lacks ribosomes and appears smooth. It plays an important role in the synthesis of glycogen, fats and sterols, and in detoxification of drugs and poisons. SER is well developed in liver cells, muscle cells, adipose tissue and steroid‑secreting cells.

Functions of Endoplasmic Reticulum

Divides the cytoplasm into compartments for separation of biochemical activities
Provides large surface area for enzymatic reactions
Acts as a site for protein synthesis (RER)
Functions as a transport and storage system
Plays an important role in lipid metabolism and glycogen synthesis

Lysosome MCQs test

2026-01-23T21:10:02.883+05:30

Lysosomes MCQ Quiz

1. Lysosomes are also known as:

A. Power house of cell
B. Suicide bags of cell
C. Brain of cell
D. Protein factory

Correct Answer: B. Suicide bags of cell

2. Lysosomes were discovered by:

A. Watson
B. Crick
C. Christian de Duve
D. Novikoff

Correct Answer: C. Christian de Duve

3. Size of lysosomes is approximately:

A. 1–2 µm
B. 0.2–0.8 µm
C. 5–10 µm
D. 10–15 µm

Correct Answer: B. 0.2–0.8 µm

4. Lysosomes are formed from:

A. Nucleus
B. Endoplasmic reticulum
C. Golgi apparatus
D. Ribosomes

Correct Answer: C. Golgi apparatus

5. Enzymes present in lysosomes are mainly:

A. Oxidative enzymes
B. Digestive enzymes
C. Acid hydrolases
D. Reductive enzymes

Correct Answer: C. Acid hydrolases

6. Lysosomes can digest all except:

A. Proteins
B. Lipids
C. Nucleic acids
D. Cellulose

Correct Answer: D. Cellulose

7. Lysosomes are most abundant in:

A. Muscle cells
B. Neurons
C. Phagocytic cells
D. RBCs

Correct Answer: C. Phagocytic cells

8. Secondary lysosomes are also called:

A. Autophagosomes
B. Ribosomes
C. Digestive vacuoles
D. Centrosomes

Correct Answer: C. Digestive vacuoles

9. Autophagy refers to:

A. Cell division
B. Digestion of foreign particles
C. Digestion of own cell parts
D. Protein synthesis

Correct Answer: C. Digestion of own cell parts

10. In plant cells, lysosomal function is performed by:

A. Mitochondria
B. Ribosomes
C. Vacuoles and sphaerosomes
D. Plastids

Correct Answer: C. Vacuoles and sphaerosomes

11. Lysosomal membrane is strengthened by:

A. Vitamin A
B. Cholesterol
C. Bile salts
D. Radiation

Correct Answer: B. Cholesterol

12. Excess of which vitamin makes lysosome fragile?

A. Vitamin C
B. Vitamin D
C. Vitamin A
D. Vitamin K

Correct Answer: C. Vitamin A

13. Hurler’s disease is caused due to:

A. Excess enzymes
B. Absence of lysosomal enzymes
C. Mitochondrial defect
D. Ribosomal defect

Correct Answer: B. Absence of lysosomal enzymes

14. Accumulation of residual bodies causes:

A. Increased metabolism
B. Cell division
C. Diseases
D. Protein synthesis

Correct Answer: C. Diseases

15. Fruit rotting can be controlled by inhibiting:

A. Protease
B. Amylase
C. Polygalacturonase
D. Lipase

Correct Answer: C. Polygalacturonase

Lysosomes: Structure, Functions and Significance

2026-01-23T21:06:00.003+05:30

Lysosome are important cellular organelles commonly known as “suicide bags” or “disposal units” of the cell. They play a crucial role in intracellular digestion and cellular defense mechanisms.

Discovery and Structure

Lysosomes were discovered by Christian de Duve in 1955 and were later named and observed under the electron microscope by Novikoff in 1956. Structurally, lysosomes are small membrane-bound vesicles, measuring about 0.2–0.8 µm in diameter. Each lysosome is surrounded by a single membrane and is formed by the Golgi apparatus.

Enzymatic Composition

Lysosomes contain nearly 40 types of hydrolytic enzymes, collectively known as acid hydrolases. These enzymes function optimally in an acidic environment and include:

Nucleases
Proteases
Phosphatases
Sulphatases

Due to the presence of these enzymes, lysosomes are capable of digesting almost all organic substances except cellulose.

Distribution

Lysosomes are found in almost all eukaryotic cells but are most abundant in phagocytic cells such as white blood cells (WBCs) and osteoclasts, where active digestion and defense functions are required.

Polymorphism of Lysosomes

Lysosomes exhibit polymorphism, meaning they exist in different forms depending on their function:
Primary lysosomes – newly formed lysosomes containing digestive enzymes.
Secondary lysosomes – formed by fusion of primary lysosomes with phagosomes; also called digestive vacuoles or heterophagosomes.
Residual lysosomes – contain undigested materials and may undergo ephagy.
Autophagic vacuoles – involved in autophagy or autolysis, where damaged or useless cell parts are digested.

Lysosomes in Plant Cells

In many plant cells, the function of lysosomes is performed by sphaerosomes and vacuoles, as typical lysosomes are less prominent in plants.

Lysosomal Membrane Stability

The lysosomal membrane is stabilized by substances such as cortisone, cortisol, antihistamines, heparin, chloroquine, and certain types of cholesterol. However, the membrane becomes fragile in:

Absence of oxygen
Excess vitamin A and vitamin E
Presence of progesterone, testosterone, bile salts
Exposure to high-energy radiations

Clinical Importance

Failure of lysosomal function, due to absence of specific hydrolytic enzymes or defective exocytosis, leads to accumulation of residual bodies in cells. This can cause diseases such as:

Hepatitis
Polynephritis

Hurler’s disease, characterized by bone deformities due to accumulation of mucopolysaccharides (glycosaminoglycans) caused by enzyme deficiency.

Role in Fruit Ripening

Lysosomal enzymes also influence fruit ripening and rotting. Fruit rotting can be slowed down by inhibiting the enzyme polygalacturonase, which is responsible for cell wall degradation.

Conclusion

Lysosomes are vital organelles responsible for digestion, recycling, defense, and maintenance of cellular health. Any disturbance in their structure or function can lead to serious metabolic disorders, highlighting their importance in normal cellular physiology.

MCQs (Questions and Answers) on Sterilization & Disinfection Methods

2026-01-18T12:19:00.004+05:30

1. Temperature and duration in Flash method of Pasteurization is

A) 62.9°C, 30 min
B) 71.6°C, 30 min
C) 71.6°C, 15 seconds
D) 62.9°C, 15 seconds

✅ Correct answer is C. 71.6°C for 15 seconds (HTST pasteurization).

2. Concentration range for ethylene oxide sterilization is

A) 15–100 mg/L
B) 800–1200 mg/L
C) 120–180 mg/L
D) 1000–1200 mg/L

✅ Correct answer is B. 800–1200 mg/L.

3. Browne’s tubes are indicators for

A) Heat sterilization
B) Radiation sterilization
C) Gaseous sterilization
D) Filtration sterilization

✅ Correct answer is A. Heat sterilization.

4. Royce sachet is indicator for

A) Formaldehyde
B) β-propiolactone
C) Isopropyl alcohol
D) Ethylene oxide

✅ Correct answer is D. Ethylene oxide.

5. Recommended time and pressure for autoclave sterilization of media

A) 15 min, 10 lbs
B) 60 min, 5 lbs
C) 15 min, 15 lbs
D) 15 min, 20 lbs

✅ Correct answer is C. 15 minutes at 15 lbs pressure (121°C).

6. Sterilization potency is highest in

A) Filtration
B) Hot air oven
C) Ultrasound waves
D) Autoclaving

✅ Correct answer is D. Autoclaving.

7. Formalin and KMnO₄ required for 1000 cu ft area

A) 280 ml formalin + 150 g KMnO₄
B) 300 ml formalin + 150 g KMnO₄
C) 180 ml formalin + 100 g KMnO₄
D) 80 ml formalin + 150 g KMnO₄

✅ Correct answer is A. 280 ml formalin + 150 g KMnO₄.

8. Suitable pore size for bacterial filtration

A) 0.22 mm
B) 0.45 nm
C) 0.22 μm
D) 30 μm

✅ Correct answer is C. 0.22 μm.

9. Time required to destroy 90% organisms is called

A) D value
B) Z value
C) F value
D) Q10 value

✅ Correct answer is A. D value.

10. Biological indicator for ionizing radiation

A) Bacillus subtilis
B) Bacillus pumilus
C) Salmonella typhi
D) Serratia marcescens

✅ Correct answer is B. Bacillus pumilus.

11. Suitable method of sterilization of powders

A) Autoclaving
B) Hot air oven
C) UV radiation
D) Gaseous

✅ Correct answer is B. Hot air oven.

12. Temperature change to produce 10-fold change in D value

A) D value
B) Z value
C) F value
D) Q10 value

✅ Correct answer is B. Z value.

13. Mechanism of action of ethylene oxide

A) Alkylation of molecules
B) Oxidation
C) Denaturing of proteins
D) Membrane disruption

✅ Correct answer is C. Denaturing of proteins.

14. Best method of sterilizing disposable syringes

A) Autoclave
B) Boiling
C) Hot air oven
D) Gamma radiation

✅ Correct answer is D. Gamma radiation.

15. Temperature and duration in Holder’s method of pasteurization

A) 62.9°C, 30 min
B) 63°C, 90 min
C) 71.6°C, 15 min
D) 62.9°C, 15 sec

✅ Correct answer is A. 62.9°C for 30 minutes.

16. Pressure of 15 psi in autoclave equals

A) 123°C
B) 72°C
C) 121°C
D) 160°C

✅ Correct answer is C. 121°C.

17. Bubble point pressure test validates

A) Autoclave
B) Hot air oven
C) Radiation
D) Filtration

✅ Correct answer is D. Filtration.

18. Suitable method for sterilization of syringes

A) Hot air oven
B) Autoclave
C) Filtration
D) Radiation

✅ Correct answer is D. Radiation.

19. Sterilization of catgut and gloves is done by

A) X-rays
B) Gamma radiation
C) IR waves
D) Microwaves

✅ Correct answer is B. Gamma radiation.

20. Recommended time and temperature for hot air oven sterilization

A) 160°C, 120 min
B) 180°C, 120 min
C) 160°C, 60 min
D) 160°C, 180 min

✅ Correct answer is A. 160°C for 120 minutes.

Basic MCQs of Sterilization

2026-01-12T10:07:00.000+05:30

MCQ Quiz

Q1. Which of the following is not a physical agent of sterilization ?

A. Sunlight B. Radiation C. Dyes D. Drying

✅ Answer: C. Dyes

Q2. Bactericidal agents

A. Kill bacteria B. Prevent bacterial multiplication C. Both a and b D. None of the above

✅ Answer: A. Kill bacteria

Q3. Which of the following is not a method of dry heat sterilization?

A. Flaming B. Hot air oven C. Incineration D. Pasteurisation

✅ Answer: D. Pasteurization

Q4. Microbiological test of dry heat efficiency is confirmed by non toxigenic strain of

A. Bacillus megatherium B. E.Coli C. Clostridium tetani D. C.jejuni

✅ Answer: C. Clostridium tetani

Q5. Spores of Bacillus Stearothermocolus are used for determining efficiency of

A. Filtration B. Hot air oven C. Moist Heat sterilization D. Flaming

✅ Answer: C. Moist Heat sterilization

Q6. Which of the following is not used for filtration in sterilization?

A. Candle filter B. Membrane filter C. Asbestos filter D. Glutaraldehyde

✅ Answer: D. Glutaraldehyde

Q7. Which of the following is not a chemical agent for sterilization ?

A. Ethyl alcohol B. Methyl alcohol C. Formaldehyde D. Uranium

✅ Answer: D. Uranium

Q8. Ethylene oxide is used for sterilizing all accept

A. Heart lung machine B. Sutures C. Rooms D. Books

✅ Answer: C. Rooms

Q9. Most suitable for size for bacterial filtration in a membrane fielder is_____

A. 0.22 mm B. 0.22 µm C. 0.45 nm D. 0.32 µm

✅ Answer: B. 0.22 µm

Q10. Best method of sterilizing disposable syringes is ____

A. Autoclave B. Boiling C. Carbon dioxide D. Gamma radiation

✅ Answer: D. Gamma radiation

Biosensors Types and it's Application in Pharmaceutical industries

2024-05-29T12:58:00.001+05:30

Introduction

An analytical device used to change biological response into an electrical signal is called a biosensor. The term biosensor refers to sensor devices used for determining the concentration of substances and other biological parameters even where the biological system is not directly involved.
Biosensors use a transducer to couple a biological sensing element with a detector. The first scientifically planned and successfully commercialised biosensors were the electrochemical sensors useful for multiple analytes.

Schematic Diagram Showing Main Components of a Biosensors

The biosensor contains a biological sensing element (e.g., tissues, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, natural products, etc.), a material obtained biologically (e.g., recombinant antibodies, engineered proteins, aptamers, etc.) or agents that mimic biological system (e.g., synthetic receptors, biomimetic catalysts, combinatorial ligands, imprinted polymers, etc.) either closely associated to or integrated in a transducer.

Principle

The preferred biological material is generally a specific enzyme that is immobilised using conventional methods (e.g., physical or membrane entrapment, non- covalent or covalent binding) and brought in close contact with the transducer.
The analyte on binding to the biological material forms a bound analyte that produces a measurable electronic response. Sometimes due to the release of heat, gas (oxygen), electrons or hydrogen ions, the analyte converts into a product; and changes associated to this product is transformed by the transducer to electrical signals that are amplified and measured.

Working

The electrical signal coming from the transducer is low and superimposed by high and noisy baseline (could be due to high frequency signal component of random nature, or electrical interference generated in transducer electronic components). A baseline signal derived from a similar transducer without any biocatalyst membrane is called a reference baseline signal.
In signal processing, this reference baseline signal is subtracted from the sample signal; the signal difference obtained is amplified and the unwanted noise signals are electronically filtered (i.e. smoothened). The biosensor response is slow and eases the electrical noise filtration. The analogue signal produced directly is the output; however, it is converted to a digital signal, passed to a microprocessor for processing and manipulating the data to desired units, and then the output is displayed or stored.

Types

A biosensor is of the following different types based on the type of sensor devices and the biological materials:

Electrochemical Biosensor : It is a simple device used to measure electronic current, ionic or conductance changes carried by bio-electrodes.
Amperometric Biosensor : It determines the movement of electrons or electronic current due to a redox reaction catalysed by enzyme. Usually, a normal contact voltage moves along the electrodes to be analysed. In the enzyme-catalysed reaction, the substrate or product obtained can transfer the electrons with the surface of electrodes to be reduced; hence, an alternate current flow is measurable.
Blood Glucose Biosensor : It is employed extensively for diabetic patients. It contains a watch pen-shaped disposable electrode for single use. This electrode has glucose oxide and derivatives of a mediator (Ferrocene). The electrodes are converted using hydrophilic mesh.
Potentiometric Biosensor : It measures the changes in the concentration of ionic species with the help of ion-selective electrodes present in it. It generally employs pH electrodes, thus in the release of hydrogen ions a large amount of enzymatic reactions are involved.
Conductometric Biosensor : Many reactions occurring in the biological system bring about a change in the ionic species. This change is helpful in measuring the electronic conductivity. Urea biosensor which utilises the immobilised areas is an example of conductometric biosensor.
Thermometric Biosensor : Several biological reactions involve production of heat and form the basis of thermometric biosensors. The diagram representing a thermal biosensor consists of a heat insulated box fitted with a heat exchanger.
Optical Biosensor : It works on the principle of optical measurements, like fluorescence, absorbance, etc., and is utilised in fibre optics and optoelectronic transducers. Optical biosensor can even be safely used for non-electrical remote sensing of materials. It is involved in enzymes and antibodies in the transducer elements. This biosensor generally does not require any reference sensor, and sampling sensor is used for generating comparative signals.
Fibre Optic Lactate Biosensor : It measures the change in oxygen concentration at molecular level by identifying the effects of oxygen in fluorescent dye.
Optical Biosensor for Blood Glucose : In diabetic patients, the blood glucose level is important to be monitored. It is based on a simple technique in which paper strips saturated with reagents, like glucose oxide, Horseradish Peroxidase and a chromogen are used. The intensity of the dye colour is measured using a portable reflectance meter. The calorimetric test strips of cellulose layered with suitable enzymes and reagents are also widely used for testing blood and urine parameters.
Piezoelectric Biosensor : It is also called acoustic biosensor as its principle relies on sound vibrations. It contains piezoelectric crystals and the characteristic frequencies vibrate with the positively and negatively charged crystals. With the help of electronic devices, certain molecules on the crystal surface can be measured. The response frequencies can be changed by using these crystals with attached inhibitors. For example, the biosensor for cocaine (in the gas phase) works by attaching the cocaine antibodies on crystal surface.

Applications in Pharmaceutical Industries

Biosensors are made up of a biological element and a physiochemical detector used for detecting analytes. These devices have a wide range of applications in fields ranging from clinical to environmental to agricultural and to food industries. Given below are some of the fields in which biosensor technology is used:

General healthcare monitoring,
Screening of diseases,
Clinical analysis and diagnosis of diseases,
Veterinary and agricultural applications,
Industrial-processing and monitoring, and
Environmental pollution control.

Biosensors can be used for quantitative determination of numerous biologically important substances in body fluids, e.g., glucose, cholesterol, urea. Glucose biosensor is widely used for regular monitoring of blood glucose in diabetic patients.

Also biosensors are used for blood gas monitoring for pH, pCO₂, and pO₂ during critical care and surgical monitoring of patients. Mutagenicity of a few chemicals can be determined by using biosensors. Presence of toxic compounds produced in the body can also be detected.

Production and Purification of Monoclonal antibodies by Hybridoma Technology

2024-05-18T13:18:00.002+05:30

G. Kohler and C. Milstein in 1975 first discovered the hybridoma technology. It is used for producing hybrid cells by fusing B-lymphocyte with tumour or myeloma cells. The hybrid cells produced using hybridoma technology, are either cultured in laboratory or sub-cultured using mouse peritoneal cavity. Thus, due to the presence of B-lymphocyte genetic material the hybrid cells can produce antibodies. The tumour cells used to produce hybrid cells make them undergo indefinite division in the culture.

The B-lymphocytes involved are pre-programmed to respond to a single type of antigen or antigenic determinant, thus they produce a single type of antibody that shows specificity for a specific antigen. The reaction of an antigen with B-lymphocyte receptors triggers the rapid division of lymphocytes. As a result, a clone of B cells is produced that generates antibodies against that specific antigen.

This entire process is called clonal selection in which B-lymphocytes produce a single type of antibodies specific to a single type of antigen or antigenic determinant. However, a fully differentiated antibody producing B-lymphocytes (known as plasma cells) does not undergo division when cultured in a laboratory.

Principle

The myeloma cells used in hybridoma technology should not synthesise their own antibodies. The hybridoma cells are selected based on inhibiting the nucleotide (subsequently, the DNA) synthesising machinery. The mammalian cells can synthesise nucleotides either by De novo synthesis or salvage pathway.

Pathways for the Nucleotide Synthesis (HGPRT-Hypoxanthine Guanine Phosphoribosyl Transferase; TK-Thymidine Kinase)

In the De novo synthesis of nucleotides, tetrahydrofolate formed from dihydrofolate is required. Aminopterin (an inhibitor) isolated formed from formation of tetrahydrofolate (and therefore nucleotides)

In the salvage pathway, the purines and pyrimidines are converted into the corresponding nucleotides. The key enzyme involved in the salvage pathway of purines is the Hypoxanthine Guanine Phosphoribosyl transferase (HGPRT) which converts hypoxanthine and guanine to inosine monophosphate and guanosine monophosphate, respectively. Thymidine Kinase (TK) is involved in the salvage pathway of pyrimidines and converts thymidine to Thymidine Monophosphate (TMP). If any of these enzymes (HGPRT or TK) undergo mutation, the salvage pathway gets blocked.

When the mutated cells (i.e., the cells deficient in HGPRT) are grown in a medium containing Hypoxanthine Aminopterin and Thymidine (HAT medium). they fail to survive as the de novo synthesis of purine nucleotides is inhibited. Thus, the cells deficient in HGPRT and grown in HAT medium die.

The hybridoma cells possess the ability of myeloma cells to grow in vitro with a functional HGPRT gene obtained from the lymphocytes fused with myeloma cells. Thus, only the hybridoma cells can proliferate in the HAT medium, and this procedure is used for their selection.

Production of MAbs

The establishment of hybridomas and production of MAbs involves the following steps:
1) Immunisation,
2) Cell fusion,
3) Selection of hybridomas,
4) Screening the products,
5) Cloning and propagation, and
6) Characterisation and storage.

1] Immunisation

The first step in hybridoma technology is to immunise a mouse with a suitable antigen. The antigen and an adjuvant (like Freund's complete or incomplete adjuvant) are injected via subcutaneous route (adjuvants are non-specific potentiators of specific immune responses). The injections are repeated multiple times at many sites.

This increases the stimulation of B-lymphocytes which are responding to the antigen. Three days before the animal is slayed, a final dose of antigen is given via intravenous route. This approach gives rise to large number of immune-stimulated cells for synthesis of antibodies. The concentration of desired antibodies is assayed in the animal serum at frequent intervals during immunisation.

The animal is sacrificed when the concentration of antibodies in serum becomes optimal. The spleen is removed aseptically and disrupted mechanically or enzymatically to release the cells. The spleen lymphocytes are separated from the remaining cells by density gradient centrifugation.

Protocol for the Derivation of Monoclonal Antibodies from Hybrid Myelomas

2] Cell Fusion

The lymphocytes are thoroughly washed and mixed with HGPRT defective imyeloma cells. The mixture of cells is treated with Polyethylene Glycol (PEG) but for a few minutes due to its toxicity. The cells are then washed to remove PEG and kept in a fresh medium. These cells contain a mixture of hybridomas (fused cells), free myeloma cells, and free lymphocytes.

3] Selection of Hybridomas

On culturing the cells in HAT medium, the hybridoma cells grow and the remaining cells disappear slowly within 7-10 days. Selecting a single antibody producing hybrid cells is very essential, and is possible if the hybridomas are isolated and grown individually. The suspension of hybridoma cells is diluted to such intensity that the individual aliquots contain one cell each. These cells are grown in a regular culture medium to produce the desired antibody.

4] Screening the Products

The hybridomas should be screened for the secretion of the antibody of desired specificity, and the culture medium from each hybridoma culture should be tested occasionally (using ELISA and RIA techniques) for the desired antibody specificity. In both the techniques, i.e., ELISA and RIA, the antibody binds to the specific antigen (coated to plastic plates) and the unbound antibody and other components of the medium are washed off. Thus, the hybridoma cells producing the desired antibody are identified by screening. The antibody secreted by the hybrid cells is the monoclonal antibody.

5] Cloning and Propagation

The single hybrid cells producing the desired antibody are isolated and cloned by using the following two techniques:

Limiting Dilution Method: In this method, the suspension of hybridoma cells is serially diluted and aliquots of each dilution are transferred into microculture wells. The dilutions are made such that each aliquot in a well contains a single hybrid cell, thus ensuring that the antibody produced is monoclonal.
Soft Agar Method: In this method, the hybridoma cells are cultured in soft agar. Many cells can be grown simultaneously in a semisolid medium to form monoclonal colonies.

Practically, both these methods are used in combination to produce maximal MAbs.

6] Characterisation and Storage

The obtained monoclonal antibodies are subjected to biochemical and biophysical characterisation for the desired specificity. The MAbs should also be elucidated for the immunoglobulin class or sub-class, its specific epitope, and its number of binding sites.

The stability of the cell lines and the MAbs is also important. Both are characterised to check their ability to withstand freezing and thawing by freezing the desired cell lines in liquid nitrogen at several stages of cloning and culture.

Large Scale Production of MAbs

Production of MAbs in culture bottles is low (5-10µg/ml), thus to increase the yield the hybrid cells are grown as ascites in the peritoneal cavity of mice. The ascitic fluid contains 5-20mg of MAb/ml, which is much superior to the in vitro cultivation techniques. Collection of MAbs from ascitic fluid has a heavy risk of contamination by pathogenic organisms of the animal. Also many animals have to be sacrificed to produce MAbs. Therefore, workers prefer in vitro techniques over using animals.

Encapsulated Hybridoma Cells for Commercial Production of MAbs

To increase the hybridoma cell density in suspension culture, the hybridomas are encapsulated in alginate gels using a coating solution containing poly-lysine. These gels allow the nutrients to enter in and antibodies to come out of the hybridomas. In this way, the yield of MAb production can be increased (10-100µg/ml).

Purification of MAbs

The desired antibodies should be extracted from a media sample of cultured hybridomas or a sample of ascites fluid. The contaminants in the cell culture sample consists of growth factors, hormones, and transferrins. The in vivo sample however, contains host antibodies, proteases, nucleases, nucleic acids, and viruses. Other secretions by the hybridomas (like cytokines) may be present in both the cases. Endotoxins may also be present in case of bacterial contamination as they are secreted by the bacteria.

For purification, the sample is conditioned by removing cells, cell debris, lipids, and clotted materials through centrifugation and then filtration through a 0.45µm filter. Membrane fouling can be caused by the large particles in the further steps of purification. Moreover, the product concentration in the sample might not be sufficient, particularly in cases where the desired antibody is produced by a low-secreting cell line. Therefore, the sample is subjected to ultrafiltration or dialysis for condensation.

The charged impurities are mostly anions like nucleic acids and endotoxins, and are separated by ion exchange chromatography. At sufficiently low pH, cation exchange chromatography is conducted so that the desired antibody binds to the column and the anions flow through. While at sufficiently high pH, anion exchange chromatography is conducted so that the desired antibody flows through column and the anions bind to it.

Based on their Isoelectric point (pl), many proteins and anions can be separated For example, pI of albumin (4.8) is lower than the pl. of most monoclonal bodies (6.1). So, the average charge of albumin molecules at a certain pH a e negative. Size exclusion chromatography is used for removing transferrin.

Affinity purification is conducted to obtain maximum purity in a single step as the antigen used delivers antibody specificity. In this technique, the antibody generating antigen is covalently bonded to agarose support. If the antigen in a peptide, it is synthesised with a terminal cysteine that allows selective attachment a carrier protein (e.g., Keyhole Limpet Hemocyanin, KLH) during development and to promote purification. The media, containing antibody is incubated with the immobilised antigen in batch. It can also be incubated as the antibody is passed through a column, at which it binds selectively and the impurities are rinsed. The purified antibody is recovered from the support by an elution using a low pH buffer or a gentle high salt elution buffer.

Sodium or ammonium sulphate is used to precipitate out the antibodies for their further selection. Antibodies precipitate at low salt concentrations, while other proteins precipitate at higher concentrations. To obtain best separation, salt should be added in sufficient amount, and the excess salt can be removed by desalting method like dialysis.

Chromatogram is used for the analysis of final purity. Presence of any impurities can be detected by the formation of peaks and the amount of impurity is indicated by the volume under the peaks. Gel electrophoresis and capillary electrophoresis can also be performed for the analysis of final purity. Presence of any impurities can be detected by the formation of bands of varying intensity.

Type 2 Hypersensitivity Reaction and Examples

2024-05-16T15:48:00.003+05:30

An elevated activity of normal immune system that damages the body tissues is known as hypersensitivity. Hypersensitivity, also termed as hypersensitivity reaction refers to inappropriate immune responses (like damaging. discomforting, and sometimes fatal). A pre-sensitised (immune) state of the host initiates hypersensitivity reactions.

Туре 2 Hypersensitivity

The type II hypersensitivity reactions cause tissue or cell damage as a direct result of the actions of antibody and complement.

Mode of Action

The reaction during blood transfusion is an example of type II hypersensitivity reactions. In blood transfusion, reaction occurs between the host antibodies and foreign antigens present on the incompatible transfused blood cells. This reaction mediates cell destruction.

Events Following Initial and Subsequent Exposures to an Allergen Resulting in Sensitisation and Manifestation of Allergic Responses

Antibody-mediated cell destruction occurs through activation of complement system. This increases membrane porosity in foreign cell by forming Membrane Attack Complex (MAC). Cell destruction can also be mediated through Antibody Dependent Cell-Mediated Cytotoxicity (ADCC).

Haemolysis of the donor's erythrocytes occurs in the recipient's blood vessels as a result of faulty cross-matching in which the alloantigen of the donor's erythrocytes react with the serum antibodies of the recipient along with the activated complement.

Biological Effects

When the maternal IgG antibodies specific for antigens of foetal blood-group cross the placenta and destroy the erythrocytes of foetus, haemolytic disease occurs in new-born.
A haemolytic medical condition affecting the new-borns is erythroblastosis foetalis in which the Rh foetus expresses an Rh+ antigen on its blood cells that the Rh- mother does not express.
Drug-induced haemolytic anaemia occurs when some antibiotics (e.g., penicillin, cephalosporin and streptomycin) get non-specifically absorbed to proteins on erythrocyte membranes and form a complex (like hapten-carrier complex) that induces anaemia.

Examples of Type 2 Hypersensitivity

Autoimmune haemolytic anaemia
Goodpasture syndrome,
Erythroblastosis foetalis pemphigus,
Pernicious anaemia (if autoimmune),
Immune thrombocytopenia,
Transfusion reactions,
Hashimoto's thyroiditis,
Graves' disease,
Myasthenia gravis,
Rheumatic fever, and haemolytic disease of the new-born.
Hyper acute graph rejection.

Dot Blot Technique Principle, Steps & Applications

2024-03-04T07:48:00.001+05:30

In the realm of molecular biology, where precision and efficiency are paramount, the dot blot technique stands out as a simple yet powerful method for detecting and analyzing biomolecules. Originally developed in the 1970s by George Stark and colleagues, dot blotting has since become a cornerstone in various research fields, including genetics, immunology, and diagnostics.

Principle of Dot Blotting :

At its core, dot blotting involves the immobilization of target molecules, such as DNA, RNA, or proteins, onto a solid support membrane. This membrane, typically made of nitrocellulose or nylon, acts as a platform for subsequent detection and analysis steps.

The procedure begins with the application of a small volume (usually a few microliters) of the sample directly onto the membrane in the form of discrete dots. The samples can be pure substances, crude extracts, or complex mixtures, depending on the experimental objectives.

Steps Involved in Dot Blotting:

1.Sample Application: The sample is carefully spotted onto the membrane, usually using a pipette or a specialized dot blot apparatus. The arrangement of the dots can be controlled to facilitate comparison and quantification.

2.Blocking: To minimize nonspecific interactions and reduce background noise, the membrane is incubated with a blocking agent, such as bovine serum albumin (BSA) or non-fat dry milk. This step saturates any unbound binding sites on the membrane surface.

3.Primary Antibody Incubation: Next, the membrane is exposed to a specific primary antibody that recognizes the target molecule of interest. This antibody binds selectively to its target, forming an antigen-antibody complex.

4.Washing: Excess primary antibody is removed through several washes with a suitable buffer. This step helps to remove any unbound antibodies and further reduces background signals.

5.Detection: The presence of the target molecule is visualized by adding a detection reagent that interacts with the primary antibody. This can involve secondary antibodies conjugated to enzymes, fluorophores, or other reporter molecules. The detection reagent generates a signal that can be detected and quantified.

6.Analysis: The dot blot results are analyzed either qualitatively or quantitatively, depending on the research goals. Quantification can be achieved through densitometry or by comparing signal intensities relative to standards of known concentration.

Advantages of Dot Blotting:

- Speed and Simplicity: Dot blotting offers a rapid and straightforward alternative to traditional methods such as Western blotting and Southern blotting. The entire procedure can be completed in a matter of hours, making it ideal for high-throughput applications.

- Versatility: Dot blotting can be adapted to detect a wide range of biomolecules, including DNA, RNA, proteins, antibodies, and antigens. This versatility makes it a valuable tool in various research areas, from gene expression analysis to disease diagnostics.

- Sensitivity: With proper optimization, dot blotting can achieve high levels of sensitivity, allowing for the detection of low-abundance molecules in complex samples.

- Cost-Effectiveness: Dot blotting requires minimal specialized equipment and reagents, making it a cost-effective option for many laboratories.

Applications of Dot Blotting:

- Gene Expression Analysis: Dot blotting can be used to study gene expression patterns by detecting mRNA transcripts or specific DNA sequences.

- Protein Detection: Dot blotting is commonly employed to screen for the presence of specific proteins in biological samples, such as cell lysates or tissue extracts.

- Antibody Screening: Dot blotting can rapidly screen for the presence of antibodies in patient sera, facilitating the diagnosis of infectious diseases or autoimmune disorders.

- Quality Control: Dot blotting is often used in quality control processes to assess the purity and identity of biomolecules, such as recombinant proteins or synthetic oligonucleotides.

Application of Biotechnology in various fields

2024-02-10T14:10:00.009+05:30

Biotechnology is a multidisciplinary field that combines principles of biology, chemistry, physics, engineering, and computer science to develop technologies and products that utilize biological systems, living organisms, or derivatives thereof, for various applications. These applications can range from healthcare and agriculture to industrial processes and environmental remediation. In essence, biotechnology harnesses the inherent capabilities of living organisms or their components to solve problems, create new products, or improve existing processes. Examples include genetic engineering, biopharmaceuticals, agricultural biotechnology, biofuels, and environmental bioremediation.

Application of Biotechnology in various fields

Biotechnology, a dynamic field at the intersection of biology and technology, has revolutionized industries ranging from healthcare to agriculture. With its vast array of applications, biotechnology continues to push the boundaries of what is possible in science and innovation.

Healthcare Advancements

One of the most impactful applications of biotechnology is in healthcare. From personalized medicine to advanced diagnostics, biotechnology plays a pivotal role in improving human health. Genetic engineering techniques, such as CRISPR-Cas9, have opened doors to precise gene editing, offering hope for treating genetic disorders and diseases. Additionally, biopharmaceuticals derived from biotechnology, such as insulin and monoclonal antibodies, have transformed the treatment of various illnesses, including cancer and autoimmune diseases.

Agricultural Innovations

Biotechnology has also revolutionized agriculture, offering solutions to global challenges such as food security and environmental sustainability. Genetically modified organisms (GMOs) have been developed to enhance crop yield, improve resistance to pests and diseases, and reduce the need for chemical pesticides and fertilizers. Furthermore, biotechnology enables the development of drought-resistant and nutrient-enriched crops, addressing the challenges posed by climate change and malnutrition.

Environmental Remediation

Biotechnology presents promising solutions for environmental remediation and conservation. Bioremediation techniques utilize microorganisms to degrade pollutants in soil, water, and air, offering eco-friendly alternatives to traditional cleanup methods. Moreover, biotechnology facilitates the development of biofuels, such as biodiesel and bioethanol, derived from renewable sources like algae and agricultural waste, reducing reliance on fossil fuels and mitigating greenhouse gas emissions.

Industrial Applications

In the industrial sector, biotechnology drives innovation in various fields, including bio-based materials, bioinformatics, and bioprocessing. Bio-based materials, such as bioplastics and biofibers, offer sustainable alternatives to conventional petroleum-based products, reducing environmental impact and promoting circular economy principles. Additionally, bioprocessing techniques enable the production of bio-based chemicals, enzymes, and pharmaceuticals through microbial fermentation and enzymatic catalysis, fostering a greener and more efficient manufacturing industry.

Challenges and Ethical Considerations

Despite its tremendous potential, biotechnology also raises ethical and societal concerns, particularly regarding genetic engineering, biosecurity, and bioprospecting. The manipulation of genetic material, while offering opportunities for therapeutic interventions and agricultural improvements, raises questions about safety, equity, and unintended consequences. Furthermore, the commodification of biological resources and the potential for biopiracy highlight the importance of ethical frameworks and regulatory oversight to ensure responsible innovation and equitable access to biotechnological advancements.

Conclusion

Biotechnology continues to shape the world we live in, offering transformative solutions to global challenges while posing complex ethical dilemmas. As we navigate the evolving landscape of biotechnological innovation, it is crucial to approach its applications with a balanced perspective, weighing the potential benefits against ethical considerations and societal implications. By harnessing the power of biotechnology responsibly, we can unlock its boundless potential to improve lives, safeguard the environment, and drive sustainable development for future generations.

Koch's postulates application in Microbiology

2024-02-10T14:03:00.003+05:30

In the world of microbiology, understanding the causes of infectious diseases is crucial for effective prevention and treatment strategies. One of the fundamental principles in this field is Koch's postulates, named after the German physician and microbiologist Robert Koch, who developed them in the late 19th century. Koch's postulates provide a systematic approach to demonstrate the causative relationship between a microorganism and a disease, laying the groundwork for the field of medical microbiology.

The Four Koch's Postulates:

1. The microorganism must be present in every case of the disease but absent from healthy organisms.

This postulate emphasizes the importance of identifying a specific microorganism consistently associated with a particular disease. Koch recognized the need to isolate and characterize the microorganism responsible for causing the illness.

2. The microorganism must be isolated from the diseased organism and grown in pure culture.

Once the microorganism is identified in a diseased individual, it must be isolated and cultivated in laboratory conditions. This step ensures that the microorganism can be studied and manipulated independently of its host.

3. The cultured microorganism should cause disease when introduced into a healthy organism.

To establish a causal relationship, Koch demonstrated that inoculating a healthy organism with the isolated microorganism results in the development of the same disease observed in the original host. This step confirms that the microorganism is indeed responsible for the illness.

4. The microorganism must be re-isolated from the experimentally infected organism.

Finally, to complete the chain of evidence, Koch reisolated the same microorganism from the experimentally infected organism. This step confirms that the microorganism retrieved from the experimental host is identical to the one initially isolated from the original diseased individual.

Applications and Limitations:

Koch's postulates have been instrumental in identifying the causative agents of numerous infectious diseases, including tuberculosis, cholera, and anthrax. By rigorously applying these criteria, researchers have been able to establish causal relationships between specific pathogens and their associated diseases, paving the way for the development of vaccines, antibiotics, and other treatments.

However, it's important to note that there are limitations to Koch's postulates. For example, some microorganisms cannot be grown in pure culture or do not cause disease when introduced into a healthy host due to complex interactions with the host's immune system or other factors. Additionally, advances in molecular biology and genomics have revealed cases where multiple microorganisms or non-infectious agents contribute to disease pathogenesis, complicating the application of Koch's postulates in certain contexts.

Conclusion:

Despite these limitations, Koch's postulates remain a cornerstone of microbiology and have greatly contributed to our understanding of infectious diseases. They provide a systematic framework for establishing causation and have guided generations of researchers in their quest to unravel the mysteries of microbial pathogenesis. As technology continues to advance, researchers will undoubtedly refine and expand upon Koch's postulates, ensuring their continued relevance in the field of medical microbiology.

General Nature and Basic Structure of Enzyme

2023-12-31T09:45:00.001+05:30

A living cell is capable of performing a multitude of biochemical reactions in order to survive grow and multiply. This involves

Degradation of complex nutrients into simpler forms so that they can be absorbed by the cell.
Uptake of these simple nutrients.
Chemical transformation of these simple nutrient molecules into various precursor metabolites, so that they are available for biosynthesis.
Generation of ATP and other bio reactive molecules, which co-operate in cellular biochemical reactions.
Biosynthesis of cellular molecules and structural components of the cell etc.

All these diverse types of chemical reactions can occur with a high degree of specificity and rate, under normal growth conditions for the organism. These chemical changes are brought about by the living cell through the role and participation of a special class of molecules known as enzymes . Enzymes function in the cell by acting as a biocatalyst.

Discovery of Enzymes

The term enzyme was coined by Kuhne in 1878, suggesting in yeast (en = in, zyme = yeast). It was examined that cell free extract from yeasts is capable of causing conversion of sugar into alcohol. This lead to the understanding that the molecules present inside the cells of yeasts are responsible for these chemical transformations. Kuhne called them as enzymes.

Later on, it was established that all biochemical activities of a living cell are attributed to these magic molecules called enzymes. The first enzyme to be discovered was 'amylase'. Its presence in malt extract was detected in 1833 by two French chemists Pain and Persuses.

General Nature of Enzymes

Enzymes are regarded as organic biocatalysts which are capable of functioning both extra cellular and intra cellular. Following are the major characteristics of enzymes.

1] With exception of a few catalytic RNA molecules or ribozymes, all enzymes are protein in nature. Over 90 % enzymes are globular proteins. Many of them are conjugated proteins, having non protein component.

Enzymes may be monomeric or multimeric proteins. As they are proteins, they share all major characteristic of proteins .

They are macromolecules with high MW.
They are non dialyzable and are unable to pass through semi permeable membrane.
They are amphoteric in nature. i.e. they possess both types of lonizable groups : -NH₂ and -COOH. which on ionization . yield positively charged ammonium (NH4+) ion and negatively charged carboxyl (COO-) ion.
As they are amphoteric, they possess specific isoelectric pH. At this pH, both -NH₂ and -COOH groups get equally ionized such that their net electrical charge becomes minimum.
They show electrophoretic mobility. If the enzyme solution is at pH, above isoelectric value, they acquire negative charge and hence move towards anode and vice versa.
They are colloidal in nature.
They can be salted out by salts like ammonium sulfate
They can be precipitated out by protein denaturing solvents like acetone and alcohol.
They can absorb maximum UV light at 280 um wavelength.

2]. Enzymes are mostly thermo labile and get denatured at high temperature, usually above 60°C. However, some enzymes are found thermo stable and can withstand high temperature up to 70°C - 80°C or even more.

3]. Enzymes are highly specific in action. They can act on a specific substrate molecule to bring about specific biochemical reaction .

Catalytic RNA - Ribozyme

Apart from proteins, a few RNA have also been recognized to have catalytic activity. They are called ribozymes or catalytic RNA. They were first recognized by Thomas Cech in 1982. The ribozymes have two types of common roles :

RNA processing, where the RNA is involved in RNA splicing, RNA ligation and RNA replication.
Peptide bond formation during protein synthesis. In ribosomes, they function as a part of rRNA and participate in peptide bond formation.

Basic structure of Enzymes

In general, enzyme molecule consists of two components.

Apo enzyme and
Prosthetic group or cofactor

Apo enzyme is protein part of enzyme.
Prosthetic group or cofactors are non protein part of enzymes. They consist of vitamin or their derivatives or metal lons. (If these non protein components are bound loosely to protein, they are called cofactors) and if they are bound firmly (covalently), are called prosthetic groups. Apo enzyme and prosthetic group form complex to form active form of enzyme, known as Holoenzyme.

Classification of Enzymes

What is GMP ? and why it is important in pharmaceutical industries

2023-05-21T20:39:00.004+05:30

Good Manufacturing Practices (GMP) is the minimum standard that a pharmaceutical manufacturer must meet in their production processes. Process must:

Be of consistent high quality.
Be appropriate to their intended use.
Meet the requirements of the marketing authorization (MA) and product specification.

Pharmaceutical organizations which comply with GMP must have manufacturer license. Regulatory agencies carries out in section on these pharmaceutical organizations to check if manufacturing sites comply with GMP. Sites are inspected when applied for a manufacturer license and then periodically based on risk assessments.

CGMP Legal Principles

Quality built into product

By "taking care" in making medicine.
Quality system is based on the principle of Quality by Design instead of quality by Inspection.

Without/Inadequate cGMP

Product(s) adulterated(defects need not be shown).
Firm and its management are responsible.

Current = Dynamic

Standards evolve over time.

Good Practices

Minimal standards.
Not "best practices". (Unless "best" is, in fact, current minimal.)

Why is GMP so IMPORTANT?

Is a legal requirement enforced and mandated through law, regulations and directives by each country government.
To protect public health of each and every individual.
Prevent contamination and mix-ups.
Prevents mislabeling and adulteration.
Consistent maintenance of Quality product supply throughout the product life cycle.
Ensure high standard quality product supplied into the market is safe and effective.
Satisfy stakeholders, customers and consumers.
To meet the requirement of the marketing authorization (MA) and product specification.
Enhance the organization image and reputation.

Responsibility of GMP

Quality and GMP compliance are independent of job title and have no boundaries.
Everyone involves in the process regulatory compliance, manufacturing, packing, Quality Control, distribution and supply of pharmaceuticals product has the responsibility to make sure it reaches to the patient with registered quality standards.
Building quality into the entire process of an operation makes sustainable compliance more achievable. However, it requires commitment by Senior management and the allocation of adequate resources (personnel, facilities, training, etc.).

Applications of Biotechnology in Various Fields

2023-05-16T12:34:00.001+05:30

Biotechnology is a branch of biology involving the use of living organisms and bioprocesses in engineering, technology, medicine, and other fields using bioproducts. The term biotechnology indicates the use of living organisms or their products for modifying the human health and environment.

Applications of Biotechnology in various fields

1] Medicine:

Modern biotechnology finds promising applications in medicine as in:

i) Drug Production :

Most of the traditional pharmaceutical drugs used for treating the symptoms of a disease are simpler molecules found through trials and errors. Small molecules are manufactured chemically, but the larger ones are created by human cells, bacterial cells, yeast cells, and animal or plant cells.
Modern biotechnology involves the use of genetically altered microorganisms (e.g., E.coli or yeast) for producing insulin or antibiotics via synthetic means. Modern biotechnology can also be used for producing plant-made pharmaceuticals. Biotechnology is also used in the development of molecular diagnostic devices used to define the target patient population for a given biopharmaceutical. For example, herceptin was the first drug to be used with a matching diagnostic test for treating breast cancer in women whose cancer cells expressed HER2 protein.

ii) Pharmacogenomics :

It is the study of how the genetic inheritance of an individual affects his/her body's response to drugs. The term pharmacogenomics was derived from the words pharmacology and genomics, thus it involves studying the relationship between pharmaceuticals and genetics.
Pharmacogenomics aims to design and produce drugs adapted to each individual's genetic makeup.

iii) Gene Therapy :

It is used for the treatment of genetic and acquired diseases like cancer and AIDS. Gene therapy utilises normal genes for supplementing or replacing the defective genes or for strengthening immunity. This therapy targets either the somatic cells (i.e., body) or the gametes (ie., egg and sperm).
In somatic gene therapy, the recipient's genome is altered; however, this alteration is not passed on to the next generation. On the other hand, in germline gene therapy, the egg and sperm cells of the parents are altered to be passed on to their offspring.

iv) Genetic Testing :

This involves direct examination of the DNA, and is used for:

Carrier screening, or identifying unaffected individuals who carry one copy of a gene for a disease that requires two copies for the disease to manifest,
Confirming the diagnosis of symptomatic individuals,
Determining sex,
Forensic/identity testing.
New-born screening.
Prenatal diagnostic screening.
Pre-symptomatic testing for determining the risk of developing adult-onset cancers, and
Pre-symptomatic testing for predicting adult-onset disorders.

2] Cloning :

In this method, the nucleus from one cell is removed and is transferred to an unfertilised egg cell whose nucleus has either been deactivated or removed. Cloning can be done in the following two ways:

i) Reproductive Cloning :

In this method, the egg cell after a few divisions is transferred to a uterus for its development into a foetus that is genetically identical to the donor of the original nucleus.

ii) Therapeutic Cloning :

In this method, the egg is placed in a petridish for its development into embryonic stem cells that are potential for treating several ailments.

3] Agriculture :

Biotechnology in the agricultural field is used for the following purposes:

i) Crop Yield :

For increasing the crop yield, one or two genes are transferred to a highly developed crop variety for imparting a new character. The current techniques of genetic engineering are best for the effects controlled by a single gene. Some genetic characteristics related to yield (e.g., enhanced growth) can be controlled by various genes, each posing a nominal effect on the yield.

ii) Reduced Vulnerability of Crops to Environmental Stresses :

Such crops which can be made resistant to biotic and abiotic stresses can be developed with the help of genes; for example, drought and salty soil are the two limiting factors in crop productivity.

iii) Increased Nutritional Qualities :

The nutritional value of proteins contained in foods can be enhanced; for example, proteins in legumes and cereals can be transformed such that they also provide amino acids required in the balanced diet of humans.

iv) Reduced Dependence on Fertilisers :

Modern biotechnology can also be used to reduce the dependence of farmers on agrochemicals; for example, Bacillus thuringiensis (Bt) is a soil bacterium that produces a protein having insecticidal properties. Conventionally, these bacteria were used to produce an insecticidal spray by a fermentation process.
In this form, the Bt toxin occurs as an inactive protoxin, which becomes effective when digested by an insect. There are several Bt toxins and each has a specificity for some target insects.

v) Production of Novel Substances in Crop Plants :

Biotechnology is also applied for novel uses apart from food; for example, oilseed is genetically modified to produce fatty acids for detergents, substitute fuels, and petrochemicals. Potatoes, tomatoes, rice tobacco, lettuce, safflowers, and other plants are genetically engineered to produce insulin and certain vaccines.

4] Biological Engineering :

It is a branch of engineering that involves biotechnologies and biological science. It includes different disciplines such as biochemical engineering, biomedical engineering, bio-process engineering, biosystem engineering, etc.

Classification of Bacteria Based on Temperature Requirement

2023-04-01T12:13:00.002+05:30

The temperature at which the growth of organisms is maximum and most rapid is called the optimum growth temperature. The range of temperature between which the organisms can grow is called the temperature range for growth. Shift in temperature on either side of optimum value retards growth. Usually the maximum temperature up to which the organisms can grow, is close to the optimum growth temperature. Whereas the minimum temperature required for growth can be much lower than the optimum value. Minimum, optimum and maximum growth temperatures are called cardinal temperatures.

Cardinal temperatures vary greatly between microorganisms. Optima normally range from 0°C to 75°C; whereas growth can take place between -20°C to 100°C. Organisms having a narrow range of growth temperature are called stenothermal while the organisms having wide range of growth temperature are called eurythermal.

Based on the temperature requirements for growth, bacteria can be divided into three groups:

Psychrophiles
Mesophiles
Thermophiles

Psychrophiles

The organisms able to grow at low temperature i.e. below 10°C, are called psychrophiles. They can grow even at 0°C or even lower, if the medium is not frozen due to high salt concentration. The optimum temperature for growth is 15°C or lower. The maximum temperature of growth is 20°C. There are two types of psychrophiles.

Obligate or true psychrophiles : These are the organisms which grow at 0°C or lower with optimum growth temperature 15°C or below. e.g. Vibrio marinus, Vibrio psychroerythreous.
Facultative psychrophiles or psychrotroph : These are the organisms which can grow at 0°C. But optimum temperature for growth is between 20°C to 30°C. e.g. Pseudoinonas flourescens.

The physiological factors for the psychrophilic nature of organisms are not much clear. But it is observed that their ribosomes and other cellular enzymes are unstable at high temperature. Hence, they cannot grow at higher temperature. Even their membrane permeability is altered at temperature above optimum value, leading to leakage of cell material, impairment of membrane permeability and hence results in loss of viability.

Mesophiles

These are the organisms that can grow at moderate temperatures of incubation, i.e. within range of 25°C to 40°C. Their optimum growth temperature falls within this range. All pathogenic bacteria for humans and warm blooded animals grow best at body temperature, i.e. at 37°C.

Thermophiles

The organisms capable of growing best at temperature above 45°C are called thermophiles. They can be grouped into two categories.

1) Facultative thermophiles

These organisms can grow even in the mesophilic range of temperature.

2) Stereothermophiles or hyperthermophiles

These bacteria cannot grow in the mesophilic range of temperature. They grow well between temperatures 45°C to 70°C. There are some organisms which can grow even at 110°C. These bacteria are also referred to as strict or obligate thermophiles. These bacteria are normally found from hot water springs, salt lakes etc.
e.g. Bacillus coaggulans, B. stereothermophilus. Thermus aquaticus can grow even at 100°C.

The basis of thermal resistance of these bacteria is the thermal stability of most of their cellular proteins.

Fermentation : Defination, Principle and Batch Fermentation Method

2023-02-15T18:03:00.003+05:30

The process of fermentation involves biochemical activity of organisms during their growth, development, reproduction, senescence, and death. Fermentation technology employs organisms to produce food, pharmaceuticals, and alcoholic beverages in industries on a large scale.

Principle of Fermentation

The principle involved in industrial fermentation technology is that organisms are grown under optimum conditions and are provided with raw materials and other necessary requirements like carbon, nitrogen, salts, trace elements, and vitamins. The end products formed due to their metabolism during their life span are released into the media. These end products are extracted by human beings as they are commercially valuable.

Some major end products of fermentation produced on a large scale industrial basis are wine, beer, cider, vinegar, ethanol, cheese, hormones, antibiotics, complete proteins, enzymes, and other beneficial products.

Batch Fermentation

In batch fermentation process, the microorganisms are inoculated in a fixed volume of batch culture medium. The organisms during their growth consume the nutrients, and the growth products (i.e., biomass and metabolites) start accumulating. Since the nutrient environment within the Fermenter is continuously changed, the rate of cell metabolism also changes, and ultimately, cell multiplication stops due to limitation of nutrients and accumulation of toxic excreted waste products.

Growth Characteristics in a Batch Culture of a Microorganism. 1) Lag Phase, 2) Transient Acceleration, 3) Exponential Phase, 4) Deceleration Phase, 5) Stationary Phase, 6) Death Phase

There is the complex nature of batch growth of microorganisms. In the initial lag phase, no apparent growth is observed; however, biochemical analyses show metabolic turnover signifying that the cells are acclimatising to the environmental conditions and will start growing. Then comes the transient acceleration phase when the inoculum begins to grow. This phase is quickly followed by the exponential phase where the organisms are growing at fastest rate as the nutrients are in excess, environmental conditions are optimum and growth inhibitors are absent.

In the batch fermentation process, the exponential growth occurs for a limited period. With the change in nutrient conditions, the growth rate decreases and begins deceleration phase followed by the stationary phase at which the growth sto completely because of nutrient exhaustion. The death phase when the grow rate has come to an end is the final phase of the cycle. Mostly t biotechnological batch processes are stopped before this stage because decreasing metabolism and cell lysis.

Advantages of Batch Fermentation

It Requires less space.
It Can be easily handled, and
There is Less chances of contamination.

Disadvantages of Batch Fermentation

It is time consuming method.
It requires more time for cleaning, sterilisation, and cooling.
Product yield is low.

Applications of rDNA Technology and Genetic Engineering in Medicine

2023-02-11T10:04:00.006+05:30

Genetic engineering has an important role to play in the production of medicines. Microorganisms and plant-based substances are used for producing various drugs, vaccines, enzymes, and hormones at low costs. Genetic engineering involves the study of inheritance pattern of diseases in man and collection of human genes that provide a complete map for inheritance of healthy individuals.

Vaccines

Recombinant DNA technology is used for producing vaccines against diseases by isolating antigen or protein present on the surface of viral particles. Vaccines contain a form of an infectious organism that does not cause disease but allow the body immune system to form antibodies against the infective organism.

When an individual receives vaccination against any viral disease, the antigens produce antibodies to act against and inactivate the viral profeins. The scientists with the help of recombinant DNA technology have transferred the genes for some viral sheath proteins to vecinia virus which was used against small pox. Vaccines produced by gene cloning are non-contaminated and safe as they contain only coat proteins against which antibodies are produced. Vaccines against viral hepatitis influenza, herpes simplex virus, virus-induced foot and mouth disease in animals are being produced by gene cloning.

Hormones

Insulin was commercially produced in 1982 through biogenetic or recombinant DNA technology. Its medicinal use was approved by Food and Drug Administration (FDA) of the USA in the same year. The human insulin gene has been cloned in large quantities in E. coli bacterium that can be used for synthesising insulin. Humilin is the commercially available genetically engineered insulin.

Lymphokines

Lymphokines are proteins regulating the immune system in the human body. u. Interferon is an example of lymphokines that are used to fight viral diseases (such as hepatitis, herpes, and common cold) and cancer. Such drugs can be manufactured in large quantities in bacterial cells. Lymphokines are also helpful in AIDS. Interleukin-II is a commercially available genetically engineered substance that stimulates the multiplication of lymphocytes.

Somatostatin

Somatostatin is used in some of the growth related abnormalities. This drug appears to be species specific and the polypeptide obtained from other mammals has no effect on humans, hence it is extracted from the hypothalamus of cadavers. Genetic engineering has helped in the chemical synthesis of gene which is joined to the PBR 322 plasmid DNA and cloned into a bacterium. This transformed bacterium is converted into a somatostatin synthesising factory.

Production of Blood Clotting Factors

Blockage of coronary arteries by cholesterol or blood clots causes heart attack. Plasminogen is a substance found in blood clots. Genetically engineered tissue Plasminogen Activator (tPA) enzyme is used to dissolve these clots in individuals who have suffered heart attacks.

Cancer

Antibodies cloned from a single source and targeted for a specific antigen (monoclonal antibodies) have proved useful in the treatment of cancer. Monoclonal antibodies have been targeted with radioactive elements of cytotoxins (e.g., Ricin from castor seed) to make them more deadly. Such antibodies seek cancer cells and kill them with their radioactivity or toxin.

Bergey's Manual of Systemic Bacteriology

2022-10-22T18:41:00.004+05:30

Bergey's manual is an accepted reference on the identification of bacteria. It has undergone gradual transformations and expansion since the time of its first publication.

The American Society of Microbiology published first edition of Bergey's Manual of Determinative Bacteriology in 1923. Professor David H. Bergey (Chair Person) and other four colleagues acted as the members of the editorial board. Afterwards, there was a sequel of eight editions, an abridged version and several supplements. At present, ninth edition (published in 1994) is available. It is used to classify bacteria based on their structural and functional attributes by arranging them into specific familial orders. However, this process has become more empirical in recent years.

Bergey's Manual of Systematic Bacteriology Manual of Systematic Bacteriology

It is the main resource for determining the identity of prokaryotic organisms, emphasizing bacterial species, using every characterizing aspect. First edition of this manual consists of four volumes. It's first volume was published in 1984, the second in 1986 and final two volumes in 1989. This manual has much broader scope. It includes all information of earlier manuals. In addition it includes the information on taxonomy, ecology, cultivation, maintenance and the preservation of organisms. It includes many kinds of information such as...

Descriptions and photographs of species,
Test of distinguish to distinguish among genera and species,
DNA relatedness among organisms and
Various taxonomic studies.

There are four divisions of kingdom Procaryotae according to Bergey's Manual of Systematic Bacteriology.

Division I : Gracilicutes

Includes prokaryotes with thin cell walls e.g. Gram negative bacteria.

Division II : Firmicutes

Includes prokaryotes with thick cell wall e.g. Gram positive bacteria.

Division III : Tenericutes

Includes the prokaryotes that lack cell wall.

Division IV : Mendosicutes

Includes the prokaryotes lacking peptidoglycan in their cell wall.

Second edition of Bergey's Manual of systematic Bacteriology consists of five volumes. Volume I was published in 2001, Volume II in 2005 and the other three volumes in 2007. In comparison to the first edition, second edition has certain changes. e.g .,

Volume - I includes The Archaea and The Deeply Branching And Phototrophic bacteria.
Volume II includes the Gram-negative Proteobacteria. It includes medically important genera are Escherichia, Neisseria, Pseudomonas, Rhizobium, Rickettsiae, Salmonella and Vibrio.
Volume - III includes the Gram- positive bacteria with low G + C content in their DNA. They are the members of phylum Firmicutes . It includes rods and cocci and also pleomorphic Mycoplasma. They may form endospores. Its classes are Clostridia, Mollicutes and Bacilli.
Volume - IV includes the Gram - positive bacteria with high G + C content in their DNA. They have more than 50- 50 % G + C content.
Volume - V includes ten phyla. They are located here for convenience. Includes morphologically diverse gram - negative organisms. They may not be related. Organisms included are Plantomycetes, Chlamydia, Spirochaetes, Bacteroilds, Fusobacteria, Chlamydiae, Acidobacteria, Verrucomicrobia and Pictyoglomus.

Effect of pH on Microbial growth

2022-06-27T14:05:00.005+05:30

The self-ionization of water is a continuous process when it is in pure form. In this process, when there is collision of two H₂O molecules they dissociate into H⁺ and OH^- ions. There is no free hydrogen ions in the water. Because the free hydrogen ions have tendency to attach to the H₂O molecule to form hydronium ion. This means, you will always find H₃O⁺ or hydronium ions in water, instead of free H+ ions.

What is pH ?

pH stands for potential of hydrogen, or power of hydrogen. pH can be defined as measurement of hydrogen ion concentration in a solution, or mathematically it can be defined as negative logarithm of hydrogen ion concentration.

Formula of pH : -log [H+]

pH is applied only to aqueous solutions. That means where there is water, there is pH. pH was first described by Sorensen in 1909. pH reveals the acidity or basicity of water.
The pH scale ranges from 0 to 14 where the value 7 indicates the neutral pH. pH less than 7 indicate acidic conditions, and greater than seven indicate basic or alkaline. In other word pH is about calculating the free hydronium ions and free hydroxyl ions in a given solution.

A solution with more H+ ions is acidic and gives a pH value less than 7 Similarly, a solution with more OH- ions, is basic and gives a value greater than seven.

Importance of pH

Certain reactions in our body require certain value of pH. Anything higher or lower value may cause damage. For example, if too much of hydrochloric acid is produced in the stomach, the patient will be advised to take antacids like magnesium hydroxide in order to neutralize the excess pH.
In the same way plants cannot grow if there is continuous rising and falling of pH of soil. Animals in the river cannot survive when acid rains fall on the river.
Similarly, pH plays an important role on microbial growth.

There were cases where microbes did not show growth in the absence of right pH conditions, even they were supplied with all the required nutrients.
The pH value where a microbe can grow its best, is called the optimum pH.
Most bacteria grow best at a pH value near to 7, which means, most bacteria are neutrophils.
Some bacteria can grow at a pH range between 3 and 4. These are called acedophiles.
Alkaliphiles are the bacteria that can tolerate pH between 8 and 11.

Impact of pH on Microbial growth

The bacterial cell consists of several protein molecules, lipids, and nucleic acids. And the cell hosts several biochemical reactions. All these activities are regulated when there is optimum pH.
In lower pH conditions, the increased hydrogen ions break the weak hydrogen bonds of protein side chains and finally change the shape of the protein.
When a protein is not in its original shape, it cannot perform its routine function, and ultimately the bacteria cannot survive.

There are two methods to measure pH in a microbiology lab. The first one is using pH paper. The pH paper changes its color when it is dipped into a solution. This color change is based on acidity and basicity of the sample solution.
Later, the color of the paper will be compared with the color chart provided along with the paper. These papers are coated with a pigment called Flavin, which is extracted from red cabbage. Flavin has ability to change color when it comes in contact with an acid or base. This method will not provide the exact pH value. However, this will provide a value which is closer to the actual pH value.
The other method to measure pH that gives an accurate value is using a pH meter.

INTRODUCTION TO BIOGECHEMICAL TRANSFORMATIONS IN SOIL : MINERALIZATION AND IMMOBILIZATION OF ELEMENTS

2022-06-19T17:35:00.000+05:30

Erth is a closed system, where the over all quantity of matter remains constant. Microorganisms need electron, energy and nutrients to grow. They are responsible for cyclic transformation of compounds, and therefore they are called biogeochemical agents. They carryout transformation of carbon, nitrogen, sulphur, phosphorus, iron etc. This cycling of elements is called biogeochemical cycling. Both biological and chemical process are involved in biogeochemical cycling.

The oxidation reduction reactions are mainly responsible for biogeochemical cycling of compounds. This changes the chemical and physical characteristics of different compounds. These cyclic turnover of elements are brought about by different types of microorganisms resulting into continuous change in chemical states of matter.

The life of earth depends on cyclic conversion of chemicals from inorganic state to organic (complex state) to the elemental state. The break in the cycle at any point would dramatically affect all life forms. Various processes carrying out these transformation include :

Mineralization :

It is a process of conversion of complex organic compounds to simple inorganic forms. Many heteroprophic microbes play role in mineralization.
The resultant simple compounds are made available to plants and microbes. Energy is released in the process. The process of mineralization is very important as it increases soil fertility.

Carbon mineralization :

Organic carbon is mineralized to inorganic state.
Under aerobic condition the main products of carbon mineralization are CO₂ and water.
In absence of O₂ organic carbon is incompletey metabolized to produce organic acids, alcohols and gases.

Assimilation :

It is the process of conversion of substrate elements to protoplasmic elements.
Microbes take up the simple materials from the environment (soil) and convert them into cellular materials. It is known as assimiliation or biosynthesis.
In this process synthesis of energy and cellular material takes place. Through assimilation microbes store the excess of simple inorganic chemicals, and prevent their loss due to erosion.

Immobilization :

It is a process in which the quantity of plant available nutrients are reduced in soil by microorganisms. The nutrient assimilation is an important method of immobilization.
The uptake of various elements like carbon, nitrogen, phosphorus, sulphur, etc. cause immobilization.

Intermediary substances accumulate abundant quantities of CH4 and smaller amount of H₂ is evolved. The factors affecting mineralization are level of organic matter, temperature, moisture, pH, depth and aeration.
All these factors affect the growth and metabolism of microbes and the process of mineralization.

In nitrogen mineralization ammonium, nitrite and nitrates are accumulated from organic nitrogenous compounds like proteins, nucleic acids, etc.
In phosphorus mineralization organic phosphorus present in nucleic acid, phytin, lecithin, etc. are converted to inorganic phosphorus.
Sulfur mineralization involves aerobic breakdown of sulfur containing amino acids : cystine, cysteine, methionine, and vitamins, thiamine, biotin, thioctic acid releasing sulfates. Whereas in absence of oxygen , H₂S and odoriferous mercaptans accumulate in soil.

Lytic cycle: Multiplication of T4 Bacteriophage

2022-04-07T12:36:00.003+05:30

T4 coliphage is a phage that infect coliform bacteria especially E.coli. T4 is a double stranded DNA phage, it has a contractile sheath and an unique base 5-hydroxyl methyl cytosine (5-HMC). It is the best known member of large virulent phages.

The lytic cycle of T4 phage involve following steps: ADSORPTION
PENETRATION
BIOSYNTHESIS
ASSEMBLY
RELEASE

1]. Adsorption :

The lytic cycle begins when a bacteriophage comes in contact with a susceptible host cell by random collision.
Phage possess an adsorption organs or anti-receptors and host cells possess receptors.
Host cell surface components -Flagella, Pilli , Teichoic acids, Proteins, Carbohydrates , LPs and Lipopolysaccharides serves as receptors.
Phage components such as tail fibers, tail proteins and spikes serves as adsorption organs or anti-receptors.
Each phage has its specific receptor to which it adsorbs.
Adsorption takes place only when the anti-receptor is chemically complementary to the receptor.
T4 phage possess tail fibers that serves as an adsorption organ or anti-receptor.
Normally, tail fibers are present in folded form around the tail.
Whiskers hold the tail fibers in folded form.
When phage come in contact with host, tail fibers unfold.
Unfolding of fibers requires tryptophan & co-factors - Mg++ & Ca++.
Thus, the phage and host binding is favoured by ionic environment.
T4 host E.coli possess outer membrane protein C (OmpC) – lipopolysaccharide complex as receptor.
Initial attachment occurs when tail fibers attach to the OmpC- lipopolysaccharide complex.
Initial adsorption is weak and reversible.
It becomes irreversible when tail pins attach to lipopolysaccharide.

2]. Penetration :

Once attached, the bacteriophage injects DNA into the bacterium.
Bacteria possess rigid cell wall and therefore the phages directly cannot penetrate into the bacterial cells.
They inject only their nucleic acids inside the host cell.
In the T-even phage, irreversible binding of the phage to host results in the contraction of the sheath and the hollow tail tube is inserted through host cell wall.
Some phages have enzymes like lysozyme that digest the cell wall components of the bacterial cell.
The penetration of T4 phage DNA occurs when -

There is irreversible attachment of phage to host cell,
Contraction of sheath, pushing tail tube through cell envelope
Injection of DNA into cell like injection of vaccine/drug by a syringe

3]. Biosynthesis :

Biosynthesis divided into three steps:

Formation of immediate early and delayed early protein
Replication of phage genome
Formation of late proteins

i) Formation of immediate early and delayed early protein :

Part of phage DNA is immediately transcribed by host RNA polymerase to form immediate early m-RNAS.
These early m-RNA translate to following enzyme proteins -
a) Nucleases - Breaks down host DNA & make nucleotides available for its own synthesis.
b) α-subunit modifying enzyme - modifies α-subunit of host RNA polymerase.
Modified host RNA polymerase transcribes part of viral genome to delayed early m-RNAS.

Delayed early mRNAs are translated to following enzymes-

a) Phage enzymes that produce 5-hydroxyl methyl cytosine (5-HMC), a unique base in phage DNA
b) Polymerases and ligases - that play role in phage DNA replication and recombination.
c) Glucosylation enzyme-adds glucose to HMC & protects phage DNA from host restriction endonuclease
d) σ-subunit modifying enzyme - modifies σ-factor of RNA polymerase so that is transcribes late mRNAs.

ii) Replication of Phage Genome :

Two modes have been proposed for the replication of T4 phage DNA.
By bi-directional mode - at early stage
By rolling circle mode - at later stage

Initial replication is bi-directional and semi-discontinuous.
Leading strand is synthesized continuously and lagging strand is synthesized discontinuously leading to the formation of eye structure.
Bi-directional replication is initiated at several origins along the DNA and is catalyzed by phage coded enzymes.

In the rolling circle mode of replication, a cut is made in one of the DNA strands by a specific endonuclease and 3'end is made free.
DNA polymerase extends the free 3'OH end by adding complementary bases.
Intact strand serves as template for addition of complementary bases.
Due to extension of 3'OH end, the 5'end is displaced.
Displaced strand is synthesized discontinuously by adding Okazaki fragments.
This mechanism produces multi-genome length molecules.
Such molecules are referred to as concatemers.
The concatemers are later cleaved to head sized molecules by headful cutting mechanism.

III] Formation of late proteins

Soon after the replication of phage DNA, transcription of late m-RNAs occur.

These late m-RNAs translate to different proteins.
These proteins include the structural proteins.
They are proteins involved in phage assembly and an enzyme lysozyme that degrades the peptidoglycan layer of bacterial cell wall.
For example - head (capsid) proteins, tail tube protein, sheath proteins, collar, whiskers, base plate, tail fiber, tail pins, lysozyme etc.

4. Assembly of Phages :

Assembly of new phage particles begins after accumulation of structural proteins and nucleic acid molecules in the cell.
Process of assembling phage particles is known as known as maturation.
There are four different pathways that lead to the formation of phage particles.
These include base plate, tail tube & tail sheath, tail fibers and head.
About 50 genes take part in the morphogenesis of T4 phage.
Subunits of base plate assemble to form a base plate.
Then tail tube and sheath subunits polymerize on base plate to form mature tail.
The subunits of head assemble together to form prohead and then DNA is inserted in the prohead to form complete head.

5. Release :

The release of newly synthesized phages occurs by sudden explosion or bursting (lysis) of bacterial cell.
Lysis begins after about 22 minutes.
One of the gene products involved in the process include lysozyme.
It cleaves glycosidic bonds in the peptidoglycan making the cell wall susceptible to the rupture.
There is another protein termed as holin that make holes in the cell membrane and makes the way for lysozyme action.

Eijkman Test Principle and Procedure

2022-03-05T12:36:00.010+05:30

The IMViC test has two drawbacks. The first, It has many controversial procedures and second is test results do not give satisfactory differentiation between fecal and non-fecal coliforms. In 1904 Eijkman proposed another test to differentiate fecal and non-fecal coliform.

Principle of Eijkman Test

Only fecal coliforms of warm blooded animals grow at 46°C and ferment lactose with Acid & Gas production
Most strains of fecal E.coli can ferment lactose in a special buffered broth when incubated at 45°C, where as very few or less frequently the Enterobacter aerogenes do so.
The test is called as Eijkman test or elevated temperature test.

Procedure of Eijkman Test

A buffered tryptose lactose broth in tubes with inverted Durham's tube is inoculated with a culture of coliforms.
It is then incubated in water jacketed incubator at 45°C for 48 hours.
Gas production after incubation constitutes a positive test for fecal coliforms.
Another method, instead of tryptone lactose broth, buffered boric acid lactose broth (BALB) medium is used.
The advantage is that, the medium used is selective for fecal Escherichia.
It selectively inhibits growth and gas production by Enterobacter and other intermediate members of coliforms.
Sterile medium is first warmed to 37°C and then inoculated with culture & incubated at 45°C for 48 hrs.
Gas production indicates positive test.
Eijkman test gives better result than IMViC tests.
Therefore, it is generally preferred in water examination.

IMViC Biochemical Tests: Principles Procedures and Results

2022-03-05T12:21:00.011+05:30

IMViC tests are a group of individual tests used in microbiology lab testing to identify an organism in the coliform group. In this the first test is Indole test, the second one is Methyl Red, the third one is Voges Proskauer and the fourth one is Citrate Utilization test.

In the quantitative test for coliforms if completed test is positive, further testing is essential to differentiate fecal and nonfecal coliforms.
Escherichia coli and Enterobacter aerogens are the important contaminants of water respectively.
Escherichia coli is a fecal coliform as it is mainly found in human feces while Enterobacter aerogenes is considered as non fecal as it also occurs in soil & plant material.
They closely resemble each other in their morphological and cultural characteristics.
Therefore, the biochemical tests are performed to differentiate them.
Tests are collectively designated as the IMViC tests.
The name was coined by Parr from the first letters of the four tests namely - I for Indole, M for Methyl Red, V for Voges Proskauer and C for Citrate Utilization test.
The letter i between V and C is added solely for euphony.

Indole Test

Indole Test is used to detect indole production from amino acid tryptophan.
E. coli has the ability to breakdown the tryptophan by enzyme tryptophanase with release of indole, pyruvic acid and ammonia.
Enterobacter doesn't produce enzyme tryptophanase. Therefore, they are not producing indol from an amino acid tryptophan.
Test is performed by inoculating the test organism in 1 % tryptone water or 2 % peptone water, incubation at 37°C for 24 hrs.
Indole production can be detected by adding few drops of xylene and Kovac's or Ehrlich's reagent which contains p-dimethyl aminobenzaldehyde.
This p-dimethyl aminobenzaldehyde reacts with the indole and produce a cherry red(pink) coloured compound. This is a reduction type of reaction.
Xylene extracts the indole in upper layer of the medium.

Methyl Red Test

Methyl Red Test is carried out to detect acid production ability of test organism from glucose.
It is performed by inoculating test organism in glucose phosphate broth tube and incubating at 37°C for 24 hrs
Methyl red indicator is then added to detect acid production which gives red colour in positive reaction and yellow in negative.
Escherichia coli rapidly ferments glucose with production of acids and reduce the pH to about 5.0
This pH / acidity prevents further growth of E.coli in glucose phosphate broth tube.
Enterobacter aerogens initially produce acids but later on it is converted to non acid products such as ethanol, acetyl-methyl-carbinol (acetoin) and 2, 3- butane-di-ol ( reduction product of acetoin).
Due to this, E.aerogens continues to grow without producing its limiting pH.
Thus, Escherichia coli gives positive methyl red test while Enterobacter aerogeys gives negative test.
Methyl red is a pH indicator which is red at pH 4.4 while yellow at pH 6.2

Voges Proskauer

The test used for the detection of acetyl methyl carbinol (acetoin) production from glucose, by the test organisms.
It is also performed by inoculating the test organisms in glucose phosphate broth medium and incubating at 37°C for 24 hrs.
This is followed by addition of 40 % potassium hydroxide and 5 % a-naphthol solution with the shaking of the tube.
For the Detection of acetoin requires its further oxidation to diacetyl, In presence of catalyst α-naphthol, alkali(KOH) and air, acetoin is further oxidised to diacetyl.
Diacetyl in presence of peptone, gives a red colour.
The constituent of peptone responsible for red colour is guanidine nucleus of the amino acid arginine.
Thus, a positive test is indicated by development of red colour.
Enterobacter aerogenes produces acetoin from pyruvic acid(Positive test) while Escherichia coli doesn't produce it(Negative test).

IMViC tests Results

Citrate Utilization Test

Citrate Utilization test is carried out to detect the ability of organism to utilize citrate as a sole source of carbon and energy.
The utilization of citrate depends upon the enzyme citrate permease that facilitates citrate transport into the cell.
E. aerogenes produce citrate permease and are able to utilize citrate as the sole source of carbon while E.coli do not produce
The test is performed by inoculating the test organisms in Koser's citrate medium which sodium citrate as the sole source of carbon; and incubating at 37°C for 24 hrs.
Ability to use citrate is indicated by the development of turbidity in medium.
Citrate Utilization test can also be performed by inoculating the test organisms on the Simmon's citrate agar slant and incubating at 37°C for 24 hrs.
The Simmon's citrate agar is the modified processed agar media which contains bromothymol blue as a pH indicator
Enterobacter converts citrate to oxaloacetate and acetic acid by enzyme citrase.
These products are further converted to pyruvic acid and CO2.
The CO2 reacts with sodium and water to form sodium carbonate
Sodium carbonate is an alkaline product and it raises the pH of medium Bromothymol blue (pH indicator) is blue in alkaline and green in acidic condition.
The change in colour of slant from green to blue indicates positive test.

Results :

The IMViC test has two drawbacks as-

It has many controversial procedures
Test results do not give satisfactory differentiation between fecal and non-fecal coliforms.

Microbial Metabolism

2022-02-12T20:20:00.074+05:30

Every living organism has the fundamental capability to grow and synthesize new cell material. This requires processing of the nutrient molecules taken up by the cell and involves a series of biochemical transformations. The set of biochemical reactions occurring in cell includes degradation, synthesls as well as modification of the molecules.

These chemical reactions, which operate by the living cells are collectively referred to as metabolic reactions and the phenomenon is called metabolism. Thus, metabolism is defined as the sum total of all biochemical reactions carried out by a living cell.

The metabolic reactions are further categorized as

Catabolism
Anabolism
Primary metabolism
Secondary metabolism
Intermediary metabolism

Catabolism

Catabolism includes the set of biochemical reactions which involve degradation of the molecules taken up by the cell and generation of substances essential for biosynthesis of cell constituents. The products of catabolism are catabolites.

They, generally, involve formation of various precursor metabolites, energy rich compounds and reducing power. Hence, these metabolic reactions are often called as fuelling reactions, which provide essential fuels required for cellular synthesis. The precursor metabolites provide basic carbon skeleton for the synthesis of building blocks of the called cellular macromolecules.

The energy rich compounds are ATP, GTP, CTP, TTP, UTP and acetyl CoA, which provide necessary form of biochemical energy required to drive various energy requiring blochemical reactions. On the hydrolysis of high energy bond of these compounds, necessary free energy is available for the purpose.

Reducing power, generated during the catabolism. is in form of reduced pyridine compounds. NADH and NADPH. They provide essential reducing conditions required for several blosynthetic as well as assimilatory reactions of the cell.

Anabolism

Anabolism includes the set of blochemical reactions which involve synthesis cellular molecules.
These include blosynthesis of

Building blocks of cellular macromolecules e.g. amino acids, nucleotides, fatty acids, sugars etc.
Vitamins and coenzymes, which are essential for driving various enzyme catalyzed reactions.
Cellular macromolecules such as proteins, lipids, nucleic acids, polysaccharides as well as synthesis of cell structural compounds.

These anabolic reactions are fuelled by the products of catabolism.

Primary metabolism

The part of cellular metabolism which is very much essential for cell growth is termed as the primary metabolism. The products of primary metabolism are called primary metabolites.

The primary metabolism includes the metabolisms associated with generation of energy rich compounds, reducing power, precursor metabolites as well as the synthesis of bullding blocks of cellular macromolecules.

The products of primary metabolism include

Energy rich compounds such as ATP and others.
Organic acids such as lactic acid, citric acid, acetic acid etc.
Organic alcohols and solvents such as ethanol, glycerol. acetone, butanol etc.
Amino acids, vitamins coenzymes, nucleotides etc.

The primary metabolism, in general, is found operative lin the cell during log phase of the growth.

Secondary metabolism

The part of cellular metabolism, which is not essential for cellular growth is called secondary metabolism. Products of secondary metabolism are called secondary metabolites.

It becomes active during late log phase and stationary phase of the growth. It involves utilization of the excess remains of carbon precursors and energy for synthesis of new molecules which may have secondary role in growth of a cell.
e.g. Antibiotic synthesis, which is not essential for growth. but does help the organisms to survive in the environment in the presence of various antagonistic organisms by destroying them.

Intermediary metabolism

The part of cellular metabolism which occurs after the entry of nutrients into the cell, leading to the synthesis of bullding blocks of the cellular macro molecules is generally referred to as the intermediary metabolism.

Central metabolic pathways

The central metabolic pathways are basically catabolic in nature. These pathways Include Glycolysis, pentose phosphate pathway and TCA Cycle.They participate in generation of energy, reducing power and precursor metabolites required for cellular synthesis.

Role of reducing power in metabolism

All elements, except phosphorus, present in cell are in reduced form. Carbon exists in organic form. Nitrogen is present as amino group. sulfur as úSH group etc. Therefore, all these elements must be reduced at the cellular level of reduction, before they are assimilated in to cell during biosynthetic reactions.

Most of these elements exist in oxidized state in nature. Therefore, before they are assimilated in the cell, they must be reduced. This requires avallability of sultable reducing power. NADPH serves as the principle reducing power in all such assimilatory and biosynthetic reactions of anabolism. In addition to NADPH, flavin coenzyme, FADH2 can also serve as immediate reducing agent in biochemical reactions.

Generation of reducing power :

Reducing power is generated on oxidation of a suitable electron donor during catabolic reactions of metabolism. These electron donors can be either organic or inorganic or both depending on the type of organism. NADPH acts as the reducing power in all anabolic reactions. In addition to NADPH, flavin coenzyme, FADH2 can also serve as reducing power in certain biochemical reactions.

NADPH is generated during oxidative pentose phosphate pathway, where glucose 6 PO4 is oxidized to pentose phosphate in all most all living organisms. However in arche bacteria, where pentose phosphate pathway does not function, alternate glycolytic pathway function to generate NADPH.

NADPH can also be generated in cell by transhydrogenase reaction, where reduced NAD will participate in reduction of NADP. This reaction also helps in maintaining adequate cellular levels of NADH / NADPH ratio. NADH mainly participates In ATP formation, by transferring electron through electron transport chain.

Role of precursor metabolites

Precursor metabolites are the intermediate molecules in the metabolic pathways. They are produced during operation of catabolic pathways. The precursor metabolites can

Provide basic carbon skeleton for the synthesis of all the building blocks required to synthesize macromolecules.
Undergo oxidation via catabolic pathways to provide ATP and other energy rich compounds that fuel anabolic pathways.

About 150 different low molecular. weight compounds are required for cellular synthesis. They include
1. Building blocks for synthesis of cellular macromolecules. They include

Amino acids for synthesis of proteins.
Fatty acids for synthesis of lipids.
Monosccharides for synthesis of polysaccharides.
Purines and pyrimidines for synthesis of Nucleic acids

2. Soluble molecules required for cellular metabolic activities. They include vitamins, co-enzymes and polyamines.

There are only 12 compounds, which act as precursor metabolites. They are virtually the same in all living organisms. They include

Acetyl CoA
Pyruvate
Phospho enol pyruvate (PEP)
3 phospho glyceraldehydes (3PGAL)
Dihydroxy acetone phosphate (DHAP)
Glucose 6 Phosphate
Fructose 6 Phosphate
Erythrose 4 Phosphate
Ribose 5 Phosphate
Xylulose 5 Phosphate
Aplha Keto glutaric Acid (Alpha KG)
Succinate

The precursor metabolites are the intermediates of three Indispensible pathways of catabolism

TCA cycle
Glycolytic or gluconeogenic pathways
Pentose phosphate pathway

Role of energy rich compounds

To perform all cellular activities, a suitable form of blochemical energy is required by the cell. This biochemical energy is obtalned as the energy rich compounds, which possess high energy rich chemical bonds.
The necessary energy required to drive a blochemical reaction is released on hydrolysis of this energy rich bond. In living cells, a variety of energy rich compounds are formed, which are utilized for general purpose or to drive a specific biochemical set of reactions.

Energy rich compounds of cell

There are mainly two classes of energy rich compounds formed in the cell, which satisfy need of energy requiring reactions. They are

Compounds having high energy anhydrous phosphoester bond.
Compounds having high energy thiolester bond.

Compounds having high energy anhydrous phosphoester bond
Most energy rich compounds of the cell belong to this category. They are obtained as nucleoside triphosphate derivatives. These include

ATP Adenosine triphosphate
GTP Guanosine triphosphate
CTP Cytidine triphosphate
TTP Thymidine triphosphate
UTP Uridine triphosphate

ATP and its role

ATP is considered as one of the most commonly used energy currency of the cell. It possesses two energy rich anhydrous phosphoester bonds.

Hydrolysis of each of this energy rich bond releases 7.3 Keal energy.

ATP + H₂O ➞ ADP + Pi + 7.3 Kcal.
ADP + H₂O ➞ AMP + Pi + 7.3 Kcal.
AMP + H₂O ➞ Adenosine + Pi+ 4 Kcal.
ATP is most commonly required for

Uptake of nutrients
Activation of most substrate molecules so that they are able to enter the cell metabolism
Biosynthesis of most cellular molecules, nucleic acids, chromosome replication and cell division.

Other energy rich compounds and their role

Apart from ATP, various other kinds of energy rich compounds are formed in the cell. They have specialized role in cellular metabolism. Their specialized utilization in specific metabolic reactions may be considered useful for adequate supply of energy for the concerned biosynthetic reactions, so that they can operate at optimal level in the cell. These energy rich compounds and their role are summarised in below table.

Energy rich compounds and their role in metabolism.

Mechanism and Specificity of Enzyme Action

2022-01-19T15:04:00.004+05:30

Enzymatic reactions include the formation or the destruction of chemical bonds. When two or more substrate molecules are Joined, chemical bonds are formed. When a complex molecule is split into simpler compounds chemical bonds are destroyed.

Both the formation and destruction of bonds generally requires the prior stretching or bending of existing bonds, creating a transition (Intermediate) state. The energy required to acquire transition state is called activation energy. Enzymes acts as catalyst and lowers the requirements for this activation energy. Therefore, the reaction can occur even at normal temperature and pressure.

Enzyme catalyzed reaction requires a lower activation energy as compared to uncatalyzed reaction.

Enzymes act by reacting with substrate through its active site. They are specific in action. Only a specific substrate can bind at the active site of the enzyme to form ES complex, which finally yields product. This can be expressed as under.
E+S ➞ ES ➞ ES* ➞ EP ➞ E + P.

First the substrate binds to the active site of enzyme to form ES complex.
Now the ES complex gets structurally Induced in such a manner that it is able to convert into product and forms EP complex. This involves a chemical change. The reaction energy required for this chemical conversion is lowered dowm by the enzyme.
Now, the product dissociates from the complex and the enzyme molecule is made free. So that It is able to react with another substrate molecule again.

Specificity of enzyme action :

Enzymes are highly specific in their action. They are specific for the substrate on which they attack and the reaction they catalyze. The basis of this specificity is their active site. The enzyme specificity, therefore, can be divided into two types as under:

1. Substrate specificity
2. Reaction specificity

Substrates specificity

Enzymes are specifie for the substrate on which they attack. This substrate specificity may further be categorized in to different types.
1. Absolute specificity
2. Group specificity
3. Stereo specificity

Absolute specificity

When the enzyme possesses specificity for the entire substrate molecule, the specificity is called absolute specificity.
e.g. Urease acts on urea to degrade It into CO2 and NH3.

Group specificity

Enzymes may be specifie for a particular group or chemical bond within the substrate and attack on them. This kind of specificity is called group specificity.
e.g. Transaminase acts only on (NH2) group of substrate and cause Its transfer.

Stereo specificity

Almost all enzymes are able to recognize orientation of groups within the structure of molecule. Therefore, they are able to distinguish structural as well as optical Isomers and attack them specifically. Such specificity is called stereo specificity.
e.g. Alanine recemase. It causes isomerization of alanine and is able to convert L-alanine to D-alanine.

Reaction specificity

Enzymes are also specific for the reaction they catalyze. One particular enzyme will catalyze one particular type of blochemical reaction only. Such specificity is called reaction specificity. This kind of specificity is given prime importance for enzyme classification by IUB. Accordingly, they are divided Into six classes:

Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases