Pharmaceutical Microbiology Long Question And Answers

Pharmaceutical Microbiology Long Question And Answers

Question 1. Differentiate between gram-positive and gram-negative bacteria cell walls. Write the principle and procedure of the Gram staining technique.
Answer:

Cell Wall of Gram-positive and Gram-negative Bacteria- Differences

Gram-Positive Cell Wall Vs Gram-Negative Cell Wall:

These differences between the Cell Wall of gram-positive and Gram-negative Bacteria are classified based on their structure, composition of the cell, and the procedure of gram staining technique

Gram staining

Gram staining method, the most important procedure in Microbiology, was developed by Danish physician Hans Christian Gram in 1884. Gram staining is still the cornerstone of bacterial identification and taxonomic division.

This differential staining procedure separates most bacteria into two groups based on cell wall composition:

1. Gram-positive bacteria(thick layer of peptidoglycan- 90% of cell wall)- stains purple
2. Gram-negative bacteria(thin layer of peptidoglycan- 10% of cell wall and high lipid content) -stains red/ pink

Gram staining Principle:

The differences in cell wall composition of Grampositive and Gram-negative bacteria account for the Gram-staining differences. Gram-positive cell wall contains a thick layer of peptidoglycan with numerous teichoic acid cross-linking which resists the decolorization.

• In aqueous solutions, crystal violet dissociates into CV+ and Cl – ions that penetrate through the wall and membrane of both Gram-positive and Gram¬ negative cells.
• The CV+ interacts with negatively charged components of bacterial cells, staining the cells purple.
• When added, iodine(I- or I3-) interacts with CV+ to form large crystal violet-iodine (CV-I) complexes within the cytoplasm and outer layers ofthe cell.
• The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of both gram-positive and gram-negative bacteria.
• The outer membrane of the Gram-negative cell (lipopolysaccharide layer) is lost from the cell, leaving the peptidoglycan layer exposed.

Gram-negative cells have thin layers of peptidoglycan, one to three layers deep with a slightly different structure than the peptidoglycan of gram-positive cells.

• With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell.
• The highly cross-linked and multi-layered peptidoglycan ofthe gram-positive cell is dehydrated by the addition of ethanol.
• The multi-layered nature ofthe peptidoglycan along with the dehydration from the ethanol treatment traps the large CV-I complexes within the cell.
• After decolorization, the gram-positive cell remains purple, whereas the gram-negative cell loses the purple color and is only revealed when the counterstain, the positively charged dye safranin, is added.

Gram staining Procedure:

Smear Preparation:

Fix material on a slide with methanol or heat. If the slide is heat-fixed, allow it to cool to the touch before applying the stain.

Gram Staining Procedure/Protocol :

• Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent.
• Please note that the quality of the smear(too heavy or too light cell concentration) will affect the Gram Stain results.
• Wash slide in a gentle and indirect stream of tap water for 2 seconds.
• Flood slide with the mordant: Gram’s iodine. Wait 1 minute.
• Wash slide in a gentle and indirect stream of tap water for 2 seconds.
• Flood slide with a decolorizing agent (Acetonealcohol decolorizer). Wait 10-15 seconds or add drop by drop to slide until the decolorizing agent running from the slide runs clear.
• Flood slide with a counterstain, safranin. Wait 30 seconds to 1 minute.
• Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper.
• Observe the results of the staining procedure under oil immersion(100x) using a Bright field microscope.

Question 2. Explain the principle involved in sterilization by Alteration. Add a note on its merits and demerits
Answer:

Filters function by physically trapping particles that are larger than the size of the pore and retaining smaller particles by electrostatic attraction of particles to the filter.

Apart from porosity, various other aspects influence the effectiveness of filtration.

These include

• The filter is charged with electricity.
• The electric charge carried by organisms
• Nature ofthe filtering fluid

The liquids are filtered by either pushing the solution through a cellulose acetate membrane using the aid of a vacuum(i.e applying negative pressure on the membrane filter) or by pushing the solution into the filter paper by applying pressure over the liquid.

The air filtering process is carried out by using high-efficiency particulate(HEPA) filters that are designed to eliminate organisms greater than 0.3 millimeters from operating rooms and biological safe cabinets.

1. Depth Filters

It is an elongated layer or mat that is made of an array of random overlapping sheets of paper, or even the borosilicate (glass) fibrous material. The depth filter collects particles inside the web of fibers within the structure.

Depth Filters Applications:

• Sterilization of air filters in industrial processes
• Forced air cooling and heating systems that are used in homes have a simple filter that traps dust, spores, and allergens
• Utilization in biosafety-related applications, like biosafety cabinets. The cabinets for biological safety contain the HEPA, or high-efficiency particle air (HEPA) filter it is a form of depth filter.

2. Membrane filters

Membrane filters comprise the commonly used kind of filter employed for liquid sterilization in microbiology labs.

• Membrane filters are made of high-tensile strength polymers like cellulose acetate, polysulfone or cellulose nitrate.
• Membrane filters are made in the form of circular membranes that measure around 150mm in thickness.
• They contain millions of tiny pores of uniform diameters.
• The size of which is adapted according to the requirements in the process of polymerization
• The pores of membrane filters vary between 0.1mm to 10mm. The most popular membrane filter comes with pores that is between 0.22mm or 0.45mm.

The membranes are stored in special holders and usually preceded by depth filters comprised of glass fibers, which remove larger particles that could block the filter. The solution is pushed or pulled through the filter, and then stored in sterilized containers.

Membrane filters Application:

• Sterilization of fluids (pharmaceuticals Ophthalmic solutions as well as antibiotics and other substances that are sensitive to heat in labs and industries
• Enumeration and identification of microorganisms.

3. Air filtration Method

Air can also be cleaned through filtering. The air is cleared of disease by passing it through a High-Efficiency Particle Arrester(HEPA) filter.

• Laminar flow biological safety cabinets are among the most crucial air filters.
• They use HEPA filters that eliminate 99.97 percent of 0.33 mm particles of 0.33 mm in size. Certain operating theaters and rooms that are occupied by patients with bums are filtered to reduce the number of microbes that are a part ofthe air.
• HEPA filters can eliminate almost all microorganisms that are greater than 0.3mm within diameter.

Advantages of Filtration Sterilization:

• Capital intensive, but less
• It is suitable for liquids sensitive to heat (infusions and vaccines, hormones, and more).
• Large volumes of liquids can be easily filtered

Limitations of Filtration Sterilization:

• Only gases and liquids can be sterilized through this method.
• Filters can be expensive to replace, particularly nano¬ filters.
• The inherent limitations ofthe filters’ materials impact the efficiency of this method i.e breakage of glass filters breakage of membrane filters, and absorption of filtrate through a Sietz filter
• The process of clogging can occur

Question 3. Enlist various methods of evaluation of bacteriostatic and bactericidal disinfectants. Explain any one method of bacteriostatic disinfection.
Answer:

The common methods used for the evaluation of a disinfectant are as follows,

• Tube Dilution Method.
• Agar Plate Method.
• Filter Paper& Cup Plate Method.
• Ditch-Plate Method.
• Phenol Coefficient Method.
• Kelsey Sykes Method.

1. Tube Dilution Method:

Most commonly used method.

• In this method, a series of tubes is set up, each containing the same quantity of a standard growth liquid medium(Commonly Mueller-Hinton broth) and a gradually increasing concentration of the disinfectant to be tested.
• The tubes are then inoculated with the same quantity of cell suspension of the test microorganism and incubated for 2-3 days and 30-35!
• The first tube in the series where there is a complete absence of growth of the test microorganism denotes the minimal inhibitory concentration (MIC) of the disinfectant.

2. Agar Plate Method:

It is very similar to the tube dilution method.

• In this method, Petri plates containing a standard growth agar medium (usually Mueller-Hinton agar) are taken at the place of lubes containing liquid medium.
• Disinfectants of various concentrations are inoculated on the agar surface of plates already inoculated with the same quantity ofthe test microorganism.
• Plates are incubated and then examined for growth.
• The first plate in the series where there is the complete absence of growth ofthe test microorganism denotes the minimal inhibitory concentration(MIC) ofthe disinfectant.

3. Filter Paper& Cup Plate Method:

These methods are known as “Agar Diffusion Tests”.

• The agar is melted and cooled and 45!, inoculated with test microorganisms, and poured in a sterile Petri plate.
• In the case of the “Cup Plate Method” when the agar is solidified the holes of 9 mm diameter were made by using a sterile cork borer and the disinfectant was directly placed in it.
• In the case ofthe Filter Paper method the filter paper disks soaked in disinfectant solution are placed in the holes.
• The zone of inhibition is observed after incubation and 30-35!
• During incubation, the disinfectant diffuses into the agar.
• The diameter ofthe zone of inhibition indicates the activity ofthe disinfectant, larger the diameter greater is the efficiency ofthe disinfectant.

A zone of inhibition is proportional to the amount ofthe antimicrobial agent added to the filter paper disc, the solubility ofthe agent, the diffusion coefficient, and the overall effectiveness ofthe agent.

4. Ditch-Plate Method:

Agar is melted and then solidified in a Petri plate.

• A ditch is made in the Petri plate by cutting the solidified agar.
• The disinfectant solution is made to run through the ditch carefully.
• The test organisms are streaked outwards from the ditch.
• The petriplate is incubated at the desired temp. And period.
• The microorganisms that are resistant to the disinfectant grow even near the start at the ditch itself.
• The sensitive organisms show azone of inhibition near the ditch or at the center of the Petri plate.
• The width of the zone of inhibition is an indication of activity against the test organism

5. Phenol Coefficient Method:

• In this method, the efficacy ofthe disinfectant in use is rated by comparing it with the activity of Phenol taking as a standard.
• The test is carried out by adding an increasing amount of phenol and disinfectant in test tubes containing microorganisms.
• In the UK the test organism used is Salmonella typhi while the USA uses Salmonella typhi, Staphylococcus aureus, and Pseudomonas aeruginosa.

The official phenol coefficient tests include:

• Rideal-Walker Test(RW Test).
• Chick-Martin Test.
• United States FDA Test for Phenol Coefficient. (FDA Test)
• The US Association of Official Agricultural Chemists Test(FDA Test)

Rideal-Walker Test:

• Introduced by Rideal and Walker the British chemists in 1903 are still in use.
• The test uses Rideal, Walker Broth, and Salmonella typhi as a test organism.
• Different dilutions ofthe phenol and test disinfectants are made and 5 ml of each is inoculated with 0.5 ml of the 24-hour broth culture of the test microorganism.
• All the test tubes are placed in a water bath at 17.5!
• Subcultures from each test tube are taken and transferred to 5 ml sterile broth after 2.5, 5, 7.5, and 10 minutes.
• The broth tubes are incubated at 37! for 2 – 3 days and are examined for the presence or absence ofthe growth and the Phenol coefficient is calculated using the formula,

Rw cofficient = Dilution of Disin Fectant Killing / Dilution of Phenol Killing In 7.5 But Not In 5 min

• The Phenol Coefficient for phenol is considered as “1” Any value for disinfectant coming below one is considered as less while above it is more

Advantages of Phenol Coefficient Test:

• Quick.
• Inexpensive.
• Reproducible results.
• Useful to eliminate useless products.
• Sets standards for the preparations.

Disadvantages of Phenol Coefficient Test:

• Most tests use only one test organism i.e. Salmonella typhi which represents inadequate information.
• Compares the activity of disinfectants at only one concentration at fixed conditions.
• The presence of organic matter at action time is not considered.
• Does not give information about the tissue toxicity ofthe disinfectant.
• Sampling errors are large.

5. Kelsey-Skyes Method:

• Developed in 1969, this test overcomes many drawbacks of the RW Test.
• The test uses several test organisms viz.
• Staphylococcus aureus, Proteus vulgaris,
• Eschericia coli and Pseudomonas aeruginosa.
• Tests can be carried out in a clean as well as dirty environment.
• In both clean and dirty cases the final bacterial concentration should be about 109/ml.
• Clean conditions are simulated by using a clean broth while dirty conditions are simulated by using a yeast suspension or inactivated horse serum.
• The samples are taken at 8, 18, and 28 minutes and are then incubated at 30 to 32! and observations of the growth of microorganisms in test tubes are recorded.
• Result interpretation: A disinfectant is considered satisfactory if
  • No growth in 2 or 5 tubes of 18 min sample or
  • No more than 5 colonies from 5 drops on the agar plate

Question 4. What are viruses? Explain the morphology and replication of the virus.
Answer:

Viruses:

Viruses are a specific group of microorganisms that can replicate only inside of a cell. These are extremely small organisms and visible only under an electron microscope. Viral infections can occur in all types of life forms including plants, animals, and other microorganisms (bacteria, archaea). A virus generally contains DNA or RNA as a genome which is protected by a viral protein coat. Some enzymes are often attached to the genome ofthe virus. A complete viral particle is known as a virion.

Morphology:

Viruses are generally categorized into four groups:

• Filamentous -Plant virus-like TMV (tobacco mosaic virus).
• Isometric/icosahedral – They are rough in shape. Like polio or herpes virus.
• Envelope – Enveloped viruses include animal virus-like
• HIV which contains an envelope protein outside their cell.
• Head and tail- Head and tail having virus infects bacteria. For example – bacteriophages have a head similar to icosahedral and a filamentous-like tail.

The morphology and shape of the virion and its outer envelope provide us a piece of information about how and what sort of infection the virus may cause which is useful in classifying the viruses.

Structure and function of viral parts:

Virus consists of major three components:

1. Nucleic acid
2. Capsid, a protein coat
3. And sometimes an envelope

1. Nucleic acid:

• Nucleic acid are either DNA or RNA and viruses use it as genetic material.
• Depending upon taxonomy, genomes can be either double-stranded or single-stranded and circular or linear.
• DNA-containing viruses like chicken pox, Hepatitis B, and herpes virus direct the replication of the host’s cell protein to synthesize new similar copies of DNA with further transcription and translation processes to obtain viral proteins.
• RNA-containing viruses like Hepatitis C, measles and rabies virus encode RNA polymerase enzyme that converts RNA intoDNA which is carried out by the virus cell.
• There often occurs a misinterpretation during transcription which is the reason why RNA viruses have more mutations than DNA viruses which causes them to rapidly adapt according to several changes.

2. Capsid:

• Capsid functions as a proteinous shell to protect the viral genetic material i.e, nucleic acid from the digestive enzymes and also helps in attachment to the specific receptor of the host cell during replication and injection of infectious nuclei
• Capsomere is a composition of protein capsid subunits.
• The capsid must be assembled to build a three-dimensional structure.
• Two basic patterns that the virus capsid follows are:
• Helix symmetry
• Icosahedral symmetry
• Larger viruses have a complex structure consisting of both these symmetries.
• The T4 bacteriophage has a complex structure that infects E. coli bacterium

Lipid Envelope:

• Virus envelope consists of phospholipids and protein structure which is acquired from the part of the membrane of host cells.
• Some of the viruses contain an envelope as the additional protective covering consisting of a glycosylated transmembrane protein on the surface. This protein has a main role in identification, attachment, and causing viral infection to the host cell.
• It also determines the range of host and composition of antigenicity.
• Adenovirus causes respiratory illness in humans which is a non-enveloped animal virus.
• Poliovirus, Papillomavirus, and Hepatitis A are also non-enveloped viruses.
• Enveloped viruses like HIV, chicken pox, influenza virus, and mumps virus are more fragile towards temperature, pH, disinfectants, and some antiviral drugs. Therefore, non-enveloped viruses are more resistant to these changes.

Replication of virus:

Virus mode of replication involves major steps:

1. Attachment: Protein produced on the surface ofthe virus capsid or envelope binds to the specific receptor ofthe host cell surface. This binding process acts as a lock and key method where each key fits only with a specific lock.
2. Penetration: HIV and influenza virus with an envelope enters the cell with a fusion of host cell membrane whereas non-enveloped virus enters by translocation or through receptor-mediated endocytosis. While penetrating, viral capsid protein goes into several conformational changes
3. Uncoating/disassembly: The viral nucleic acid is released by the viral enzyme or host enzyme that degrades the viral capsid uncoats the genetic material and makes it available for transcription and translation.
4. Genome replication: Transcription and translation are initiated after the uncoating of viral genetic material where DNA and RNA viruses replicate in terms of different taxonomic grouping.

DNA viruses use protein cells and enzymes ofthe host to transcribe into mRNA and translate to protein synthesis. While RNA viruses use the RNA template and enzymes to get transcribed into mRNA. These replication processes lead up the de novo synthesis.

• Assembly: After replication, the newly replicated genomic material is assembled and packed inside the new nucleocapsid ofthe virus. This process is also the maturation stage before release into the host cell.
• Release: New viruses get ready to be released in the host organism by lysis or by budding. Enveloped viruses are released by budding and usually do not kill the infected cell. So, they are termed cytopathic viruses.

Whereas, lysis is termed as cytolytic which results in the death ofthe host cell. After viruses are released by various processes, some remain in the host cell which proceeds in making the circulating antibodies, and the remaining viral proteins are processed and presented to the MHC class I molecules which are recognized by memory cells
.
Question 5. Enumerate the various methods involved in the identification of bacteria.
Answer:

Identification of bacteria is done by various methods:

1. Identification by morphology:

This method is to determine the individual bacterial physical appearance.

Based on size:

Here bacteria are identified basedon their physical size. Like small size or big one expressed in microns. For this bacteria are viewed under a microscope to view their size. One can apply some stain to the culture and use methods like glass slit or hanging drop method

• In the glass slit method, a layer of bacterial culture is applied, stained, and viewed by a microscope above 40x magnification.
• In the hanging drop method, a drop of culture is made to hang between the glass slide and slit and viewed under a microscope.
• The glass slide has a concave aperture on one side at the center where the glass slit with culture drop is placed over such that drop suspends in the concave aperture.
• The advantage ofthe hanging drop method is we can identify motile bacteria.

2. Based on the shape:

The structure of bacteria varies spherical (cocus), bar or stick-like (bacilli), chain-like (strep), and comma-shaped like vibrio-cholera bacteria. Even the shape of the bacteria colony grown on the nutrition medium also differs for a different strain of bacteria. Hence under the microscope bacterial colony radius, the shape can be viewed and the strain of bacteria be identified.

3. By extra-cellular characters

• Here some extra-cellular characters are used in the identification of bacteria. Some bacteria have flagella for motion.
• So they can be identified based on the number of flagella and the arrangement of flagella on the bacterial surface.
• For example: Atrichous bacteria have no flagella, Monotrichous have one flagellum, and Polytrichous have many flagella.
• Even some bacteria have two flagella one on each side ofthe cell.

4. By Cultural characteristics:

Here bacteria are identified as group or culture as a whole and not individual bacteria. Since most bacteria grow in colonies and also divide fast, they can be easily grown into a culture in suitable nutrition media.

Based on the characteristics of the culture they can be identified as

• The shape of culture: Circular, irregular, rhizoid, etc.
• Size of culture or colonies in millimeters.
• Type of elevation of culture like effuse, convex, concave, etc.
• Margins ofthe colony like dentate, rough, smooth, etc.
• The surface of the colony: Smooth, wavy, papillate, etc.
• Color of culture.

5. Based on resistance:

Antibiotics are specific to certain bacteria. Hence bacteria develop resistance to certain types of antibiotics. By adding the antibiotics to the culture and measuring the resistance of the microbe to the said antibiotic, the type of bacteria can be identified.

Example: TB bacteria i.e. Mycobacterium tuberculosis is not affected by penicillin but killed by ciprofloxacin. So this differentiation can be used to identify a few bacteria.

Based on metabolism features:

• Few bacteria are so specific for metabolic requirements. Based on this specificity, they can be identified.
• Example: Aerobic bacteria require oxygen for survival. Anaerobic bacteria get killed when exposed to oxygen. Similarly, some require carbon dioxide, blood or other pigments for metabolism.
• Identification of bacteria by biochemical tests.

This is one of the most widely used methods and more important:

• Sugar fermentation test: Bacteria is grown in a sugar media.
• Litmus milk test: When bacteria are grown in this medium, there may be the production of acids or alkalis or even no change in pH.
• Indole production test: Bacteria are grown in the peptone water culture. After 48 to 96hrs, of incubation at 37°C, it is checked for the presence of red color. The red color indicates the production of indole from the amino acid tryptophan. The reagent used is called Kovac’s reagent.
• Methyl Red test: Bacteria is grown in glucose phosphate medium at 30°C for five days. Then few drops of 0.04% of methyl red are added and mixed to observe for color change. The red color indicates a positive while the yellow color indicates a negative test for glucose-fermenting bacteria.
• Citrate utilization test: This test helps identify mostly intestinal gram-negative bacteria. The citrate medium also called Koser’s medium is used here. Since the only carbon source is citrate here, its utilization is seen in terms of turbidity. If positive indicates the above-said microbes. See in detail on citrate test.
• Voges Proskauer Test: This is a test used to detect bacteria like Enterobacter, and Klebsiella. See more details on the VP test.

6. By differential staining:

The identification depends on the staining of bacteria. Most bacteria can be stained by specific stains

Example: Gram +ve bacteria are stained by Gram satin while Gram -ve bacteria don’t take up Gram stain. Tuberculous bacteria can be stained by acid-fast stain specifically and other strains don’t take up this stain

7. Serological methods:

Here the identification of bacteria is done by the use of antibodies and antigens which are specific against the suspected bacteria. Antigens and antibodies are very specific and bind to a single type of bacteria.

8. By protein and nucleotide analysis:

The cellular constituents like protein content, and nucleotide sequence in DNA are used for the identification of bacteria. This requires methods like Polymerase chain reaction(PCR), gel electrophoresis, radio Immunoassay, etc.

Identification of bacteria is necessary because:

• Identify the disease: It helps to know the type of infection and the disease caused by the individual. Ex. Tuberculosis can be identified by an acid-fast stain test for mycobacterium. So bacterial identification is important in health care.
• Select a suitable drug: Not all drugs (antibiotics) are active against all the bacteria. So identification helps to target that specific bacteria with a suitable choice of drugs. For example, rifamycin is best suited for tuberculosis and cannot be controlled by other antibiotics.
•  Evaluation of treatment progress: Identification of bacteria is also necessary to know how far the drug is effective and if the patient is freed from the said bacteria by the treatment.
• During the treatment: The patient’s body samples are repeatedly checked during the course to see if there is a recovery in the patient. This is ascertained by performing an identification test for the causative bacteria. An indication ofa negative result in the test indicates he is free from infection.
• For industrial purposes: It also helps isolate a specific strain of bacteria needed for research or other industrial productions like fermentation etc. Many vitamins(B12), hormones(insulin), and supplements are produced by rDNA technology by using the principles of biotechnology.
• For storage: Some bacteria are specifically stored with the intention of future requirements. Identification is also necessary to store the bacteria in pure form without foreign contamination. These stored bacteria are supplied when required for research or industrial manufacture.

Question 6. Write the principle and procedure to carry out the sterility testing of aqueous solutions and suspensions.
Answer:

1. Method A: Membrane Filtration

The method is to be preferred where the substances examined is

1. An oil
2. An ointment that can be put into a solution
3. A non-bacteriostatic solid not readily soluble in the culture medium, and
4. a soluble powder or liquid that possesses inherent bacteriostatic and fungistatic properties.

For liquid products where the volume in the container is 100 ml or more, only Method A should be employed.

This method needs good skill and special knowledge, and it also calls for the routine use of positive and negative controls. A positive control is a small number (not more than 100 CFU) of microorganisms specified in separate portions of each medium.

Apparatus:

• The sterility test apparatus. consists of a closed reservoir and a container to collect the filtrate, between which a properly supported membrane of appropriate porosity is placed.
• A membrane generally suitable for sterility testing has a nominal porosity of 0.45 pm, a diameter of about 50 mm, and a flow rate of 55-75 ml of water/ minute at a pressure of 70 mm of mercury.
• Cellulose nitrate filters are used for aqueous, oily, and weakly alcoholic solutions, and cellulose acetate filters, for strongly alcoholic solutions.
• The complete unit should be free from microorganisms including the membrane, and operation should be carried out aseptically. Preferably assemble and sterilize the entire with the membrane in place before using

Dilution fluids: Generally two types of dilution fluids are used:

1. Fluid A: Dissolve 1 gm of peptic digest of animal tissue(such as bacteriological peptone) in one liter of water. Filter and adjust the pH to 7.1 ± 0.2. Dispense fluid into the flasks(100ml) and sterilize at 121 °C for 20′ minutes.
2. Fluid B: If the test sample contains lecithin or oil, use fluid A, to each liter of which has been added 1 ml of polysorbate 80. Adjust pH to 7.1 ± 0.2 and sterilize at 121 °C for 20 minutes using an autoclave.

Method of test:

1. For aqueous solutions:

• Transfer aseptically the quantities of the preparation being examined prescribed in the two media onto one membrane.
• Draw the liquid rapidly through the filter with the aid of a vacuum.
• The membrane is removed aseptically and cut into two, one part is then immersed in 100 ml of soybean casein digest medium and incubated in 100 ml of liquid thioglycollate medium and incubated at 30 to 35°C for not less than 14 days.

For liquids immiscible with aqueous vehicles and suspension: Carry out the test described under, for aqueous solutions but add a sufficient quantity of fluid ‘A’ to the pooled sample to achieve rapid filtration.

2. Method B: Direct Inoculation

Quantities of sample to be used:

The quantity of the substance or preparation being examined which is to be used for inoculation in the culture media varies according to the quality in each container.

Method of test:

For aqueous solution and suspensions

• Remove the liquid from the test container with a sterile pipette or syringe.
• Transfer the quality ofthe preparation under examination directly into the culture medium so that the volume ofthe preparation under examination is not more than 10% ofthe volume of the medium unless otherwise prescribed.
• When the quantity in a single container is insufficient to carry out the tests, the combined contents of two or more containers are to be used to inoculate the media.
• The inoculated medium is incubated for 14 days at 30 to 35°C in the case of fluid thioglycollate medium and at 20 to 25°C in the case of soybean-casein digest medium.
• If the preparation under examination has antimicrobial activity, carry out the test after neutralizing this with a suitable neutralizing substance or by dilution in a sufficient quantity of culture medium.
• Medium is in contact with the entire lumen or full lumen with medium and then immerses the intact unit

Interpretation of Results

Observation and interpretation of sterility testing results.

The test may be considered invalid if only one or more ofthe following conditions are fulfilled:

• Microbial growth is found in the controls.
• Data on microbial monitoring ofthe sterility testing facility show a fault.
• A review ofthe testing procedure used for the test in question reveals a fault.
• After identifying the microorganisms from the containers showing microbial growth, the growth may be ascribed without any doubt to faults concerning the materials or technique used in conducting the test procedure

Question 7. With the help of a labeled diagram. Explain the principle, construction, working, and applications of Hot air ovens.
Or
Explain the principle, procedure, and applications of anyone thermal method of sterilization.
Answer:

A hot air oven is a laboratory instrument that uses dry heat to sterilize laboratory equipment and other materials.

Hot air oven Principle:

Sterilization by dry heat is performed by conduction. The temperature is consumed by the surface of the objects and then moves towards the core of the object, coating by coating. The whole object will ultimately attain the temperature needed for sterilization to take place.

Dry heat causes most ofthe injury by oxidizing particles. The primary cell components are damaged and the organism dies. The temperature is kept for about an hour to eliminate the most ambitious ofthe resistant spores.

Hot air oven Construction:

A hot air oven consists of various parts that can be broadly classified under two heads viz. exterior components and interior components.

Exterior Components

• Main switch: It is used to switch on or switch off the instrument. A green indicator lights up when the device is switched on.
• Temperature regulator: It is used to regulate the temperature as per one’s requirement. The temperature increases when the knob is rotated to the right side and decreases when turned to the left.
• Thermostat: It is present at the top, whose one end is inside the oven. Its function is to check and maintain the desired temperature inside the hot air oven.
• Asbestos door jacket: This is the outer double-walled jacket, made of glass-wool fibre. Its primary function is to confine the heat generated within the chamber, thus conserving energy. It provides maximum thermal efficiency and also safeguards the instrument surfaces against heat damage.

Interior Components

• Inner chamber: It is made of stainless steel which supports long-lasting operation and also provides corrosion-resistant usage. On both sides, tray slots are found onto which the tray shelves can be adjusted or removed accordingly, considering the height and volume ofthe articles to be sterilized. The tray holder is made of stainless steel wire mesh cable.
• Temperature sensor: It senses the rise and fall in temperature inside the oven.
• Air vents: It is located towards the top ofthe chamber, which releases hot gases and excessive heat outside the oven.
• Aluminum trays: The articles like glassware, metals, etc. are kept on the aluminum trays of the hot air oven. The trays are generally perforated to facilitate the equal distribution of heat to the articles that are kept inside.
• Circulating fans: It performs a central role in the uniform flow of heat inside the hot air oven along with the improved temperature distribution.
• Heating element: Electrical coils are placed at the bottom of the instrument, which allows the upward movement of heat.

Hot air oven Procedure:

• Wrap the articles or enclose them in a container of cardboard, aluminum, or paper. Mouths of flasks, test tubes and both ends of pipettes must be plugged with cotton wool.
• Articles to be sterilized such as Petri plates and pipettes may be arranged inside metal canisters and then placed. Place the articles at sufficient distances to allow free circulation of air between them and to ensure uninterrupted airflow.
• Shut the door and switch on the hot air oven. When the thermometer shows that the oven air has reached sterilizing temperature, heating is continued for the required period oftime (for example: 160°C for an hour).
• Allow the temperature to fall up to 40°C (approximately 2 hours), before the removal of sterilized materials; which prevents breakage of glassware.

Hot air oven Applications:

• It is used to dry glassware and sterilize N95 masks, general instruments, and packaging items in life science, and microbiology laboratories.
• It is also used in the chemical and pharmaceutical industries, food and beverage industries, and textile industries.
• It helps in the elimination ofmoisturefromthe material thus it is used in curing, drying, baking, and annealing.
• It is also used for the Measurement of mixed liquor-suspended solids(MLSS).
• In certain laboratories and hospitals, it is used to store materials at a constant temperature.

Question 8. Draw a neat labeled diagram of a virus cell and explain the stages of replication.
Answer: 

Stages of virus replication:

Although the specific details of virus replication vary from one virus to another, general replication is the same for most virus. t,

The stages include:

Attachment of virus to the outer surface of suitable host cell; a process called Adsorption

• Penetration of the virus into the host cell
• Release of viral genome from capsid; a process called un-coating that sometimes occurs simultaneously with penetration)
• Synthesis of viral proteins
• Synthesis of viral genome
• Assembly of viral progeny(virion)
• Release of progeny virus from the host cell.

Attachment (Adsorption):

• This is the first step in virus infection in which interaction of virion with a specific receptor site on the surface of the host cell occurs.
• The receptor sites are normal cell surface components of the host cells such as proteins, polysaccharides, or lipoprotein-polysaccharide complexes to which the virus attaches.
• For example: HIV binds to the CD4 cell receptor of T lymphocytes
• Rhinovirus binds to ICAM-1
• Epstein Barr virus binds to the C3 complement receptor.
• Each host cell contains up to 100,000 receptor sites for a given virus.
• In general viral receptors carry normal functions in cell
• For example: In some bacteriophages, receptors are pilli and flagella, and in other virus receptor sites may be transported binding protein, etc
• The receptor of the influenza virus is a glycoprotein found in RBC and on another cell of mucus membranes of susceptible host.

 2. Penetration:

After the binding of the virus, the virus is taken up inside the cell which is referred to as penetration or engulfment.

• The entry of the virus into the host cell may involve;
• Transfer of only genome across cytoplasmic membrane
• Transport of the entire virus across the cytoplasmic membrane by endocytosis
• Fusion of viral envelope with cytoplasmic membrane of host cell.

3. Uncoating;

• Shortly after penetration, the uncoating of viruses takes place.
• Uncoating is defined as the release of the viral genome from the capsid and is accessible to enzymes required to translate, transcribe, and replicate it.
• The uncoating process varies from virus to virus.
• Transcription of viral genome is usually the next step in all viruses except in that virus whose genome acts directly as mRNA
• For example: Picoma virus).
• RNA viruses that carry minus(-) stranded RNA first transcribe their DNA to plus (+) stranded RNA that functions as mRNA.
• The transcription is catalyzed by viral RNA polymerase released during uncoating.

4.  Biosynthesis:

The biosynthesis process of virus replication can be divided into early events and late events.

• Early event: In most viruses, only part of nucleic acid is initially transcribed into mRNA. The early mRNA codes for early proteins (enzymes) required for nucleic acid replication After nucleic acid replication, many copies of progeny nucleic acids formed
• Late event: Late mRNA is transcribed from the progeny genome. Late mRNA codes for structural proteins by the process of translation. The translation process always occurs in the cytoplasm of the host cell, even if the mRNA is synthesized in the nucleus, it enters the cytoplasm for translation

5. Assembly:

• When criticalnumberofvarious viral components have been synthesized, they are assembled into mature viruses.
• The assembly occurs in the nucleus or cytoplasm of the host cell depending upon the type of virus.
• DNA viruses are assembled in the nucleus and RNA viruses are assembled in the cytoplasm except
• Influenza virus and Reo virus.

6. Release:

• The release of a mature virus from the host cell is the final event in virus replication. The mechanism of virus release varies with the type of virus.
• The naked viruses are generally released by cell lysis.
• The enveloped viruses are released by budding through a special area of the host cell membrane; during which the virion acquires a portion of the host cell membrane.
• In some animal and plant viruses, host cells are not killed, the virus is released through special channels.

Question 9. What is pure culture? Write in detail about the isolation of pure
Answer:

A pure culture is a culture that contains the same types of cells. Pure cultures arise from a single cell or a single organism. Pure cultures are used for preparing antimicrobial assays.

The methods for isolating pure cultures are as follows:

1. Streak plate method
2. Pour plate method
3. Spread plate method
4. Serial dilution method
5. Single-cell isolation method
6. Enrichment culture method.

1. Streak Plate Method:

•  This method is used most commonly to isolate pure cultures of bacteria. A small amount of mixed culture is placed on the tip of an inoculation loop/ needle and is streaked across the surface of the agar medium.
• The successive streaks “thin out” the inoculum sufficiently and the micro-organisms are separated from each other.
• It is usually advisable to streak out a second plate by the same loop/needle without reinoculation. These plates are incubated to allow the growth of colonies.
• The key principle of this method is that, by streaking, a dilution gradient is established across the face of the Petri plate as bacterial cells are deposited on the agar surface.
• Because of this dilution gradient, confluent growth does not take place on that part of the medium where a few bacterial cells are deposited. Presumably, each colony is the progeny of a single microbial cell thus representing a clone of pure culture.
• Such isolated colonies are picked up separately using a sterile inoculating loop/needle and re-streaked onto fresh media to ensure purity

2. Pour Plate Method:

• This method involves the plating of diluted samples mixed with melted agar medium.
• The main principle is to dilute the inoculum in successive tubes containing liquefied agar medium to permit a thorough distribution of bacterial cells within the medium
• Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar medium maintained in the liquid state at a temperature of 42-45°C (agar solidifies below 42°C).
• The bacteria and the melted medium are mixed well.
• The contents of each tube are poured into separate Petri plates, allowed to solidify, and then incubated. When bacterial colonies develop, one finds that isolated colonies develop both within the agar medium (subsurface colonies) and on the medium (surface colonies).
• These isolated colonies are then picked up by inoculation loop and streaked onto another Petri plate to insure purity

Pour Plate Method Disadvantages:

• The picking up of subsurface colonies needs digging them out of the agar medium thus interfering with other colonies, and
• The microbes being isolated must be able to withstand temporary exposure to the 42-45° temperature ofthe liquid agar medium; therefore this technique proves unsuitable for the isolation of psychrophilic microorganisms.
• However, the pour plate method, in addition to its use in isolating pure cultures, is also used for determining the number of viable bacterial cells present in a culture

3. Spread Plate Method:

• In this method, the mixed culture or microorganisms are not diluted in the melted agar medium (unlike the pour plate method); it is rather diluted in a series of tubes containing sterile liquid, usually, water or physiological saline.
• A drop of so diluted liquid from each tube is placed on the center of an agar plate and spread evenly over the surface using a sterilized bent-glass rod. The medium is now incubated.
• When the colonies develop on the agar medium plates, it is found that there are some plates in which well-isolated colonies grow. This happens as a result of the separation of individual microorganisms by spreading over the drop of diluted liquid on the medium ofthe plate.
• The isolated colonies are picked up and transferred onto a fresh medium to ensure purity. In contrast to the pour plate method, only surface colonies develop in this method and the microorganisms are not required to withstand the temperature of the melted agar medium.

4. Serial Dilution Method:

• This method is commonly used to obtain pure cultures of those microorganisms that have not yet been successfully cultivated on solid media and grow only in liquid media.
• A microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions. The inoculum is subjected to serial dilution in a sterile liquid medium, and a large number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution.
• This dilution aims to inoculate a series of tubes with a microbial suspension so dilutes that some tubes show the growth of only one individual microbe. For convenience, suppose we have a culture containing 10 ml of liquid medium, containing 1,000 microorganisms i.e., 100 microorganisms/ml ofthe liquid medium.
• If we take out 1 ml of this medium and mix it with 9 ml of a sterile liquid medium, we would then have 100 microorganisms in 10 ml or 10 microorganisms/ml. If we add 1 ml of this suspension to another 9 ml. of fresh sterile liquid medium, each ml would now contain a single microorganism.

If this tube shows any microbial growth, there is a very high probability that this growth has resulted in from the introduction of a single microorganism in the medium and represents the pure culture of that microorganism

5. Single Cell Isolation Methods:

An individual cell ofthe required kind is picked out by this method from the mixed culture and is permitted to grow.

The following two methods are in use:

Capillary pipette method:

• Several small drops of suitably diluted culture medium are put on a sterile glass coverslip by a sterile pipette drawn to a capillary.
• One then examines each drop under the microscope until one finds a drop, which contains only one microorganism.
• This drop is removed with a sterile capillary pipette to fresh medium.
• The individual microorganism present in the drop starts multiplying to yield a pure culture.

Micromanipulator method:

• Micromanipulators have been built, which permit one to pick out a single cell from a mixed culture.
• This instrument is used in conjunction with a microscope to pick a single cell (particularly a bacterial cell) from a hanging drop preparation.
• The micro-manipulator has micrometer adjustments using which its micropipette can be moved right and left, forward, and backward, and up and down.
• A series of hanging drops of diluted culture are placed on a special sterile coverslip by a micropipette. Now a hanging drop is searched, which contains only a single microorganism cell.
• This cell is drawn into the micropipette by gentle suction and then transferred to a large drop of sterile medium on another sterile coverslip.
• When the number of cells increases in that drop as a result of multiplication, the drop is transferred to a culture tube having a suitable medium. This yields a pure culture of the required microorganism.
• The advantages of this method are that one can be reasonably sure that the cultures come from a single cell and one can obtain strains within the species.
• The disadvantages are that the equipment is expensive, its manipulation is very tedious, and it requires a skilled operator. This is the reason why this method is reserved for use in highly specialized studies.

6. Enrichment Culture Method:

• It is used to isolate those microorganisms, which are present in relatively small numbers or that have slow growth rates compared to the other species present in the mixed culture.
• The enrichment culture strategy provides a specially designed cultural environment by incorporating a specific nutrient in the medium and by modifying the physical conditions of the incubation.
• The medium of known composition and, the specific condition of incubation favors the growth of desired microorganisms but, is unsuitable for the growth of other types of microorganisms 

Question 10. Describe the industrially important techniques for preserving bacteria.
Answer:

Techniques for preserving bacteria:

1. Repeated Sub-Culturing:

• This is the most common, simplest, and routine method of preservation of microorganisms.
• Selected microorganisms are initially grown on agar slants. After sufficient growth has taken place, they are transferred to a fresh medium before they lose their viability.
• The appropriate period for such transfer ranges from a week to a few months (generally four to eight months).
• Though an organism may be kept viable by this method there is a probability of occurrence of mutations in the organism, which may lead to strain degeneration and subsequent uselessness of the organism for commercial usage.
• That is why it is less frequently used for preserving microorganisms.

Repeated Sub-Culturing Advantages:

• This method is cheap,
• Needs no special equipment,
• Recommended for small collection centers, and
• Retrieval easy

Repeated Sub-Culturing Disadvantages:

• Change in physiological and genital characteristics, and
• Time consuming.

2. Storage under Liquid Nitrogen:

• This method is also called a cryogenic storage method because a cryoprotective agent in the form of 10% glycerol is used. Industrially useful microorganisms are stored under very low
• In this method ranging, low temperatures are created by employing liquid nitrogen. Metabolic activities of microorganisms are reduced
• This method is generally employed for the preservation of fungi, bacteriophages, viruses, algae, yeasts, animal and plant cells, and tissue cultures.
• This technique involves growing the desired microorganism in sufficient quantity either in the form of cells spores or fragments of fungal mycelium. The grown-up culture is suspended in 10% glycerol.
• The suspension is then introduced into small ampoules at the rate of approximately 0.5 ml each. The ampoules are usually made up of borosilicate glass.

The ampoules containing culture suspension are frozen and sealed hermetically.

• Freezing is done either by directly dipping the ampoules into the liquid medium or by hanging the ampoules initially over the column of liquid nitrogen for some time and finally dipping into the liquid.
• The frozen ampoules are then dipped one above the other on small aluminum containers at the rate of six ampoules per can.
• The cans are then packed in aluminum boxes, 20 per each box.
• The perforations allow the free flow of liquid nitrogen.
• There may be a loss of viability in a few cells during the freezing process but there is virtually no loss of viability during the storage phase.

Storage under Liquid Nitrogen Advantages:

• Viable cultures may be preserved for many years by this method, especially those cultures that do not withstand preservation by freeze-drying.
• Though the equipment is costly, the process is economical.
• The cultures remain viable under these conditions for 10-30 years without undergoing any change in their characteristics.

Storage under Liquid Nitrogen Disadvantages:

• Evaporation of liquid nitrogen and replacement of lost liquid nitrogen regularly and periodically.
• If this is not done the apparatus will fail due to which there will be a loss of valuable cultures, and
• The method is relatively expensive.

3. Employment of Dried Cultures:

This technique has been used extensively for the storage of fungi and actinomycetes particularly for sporulating mycelial organisms.

• Moist soil is generally used as a preservating medium.
• Moist soil is first sterilized and then inoculated with a desired culture and incubated for several days to allow some growth to occur.
• The soil with growing organisms is dried at room temperature for two weeks.
• The dried soil is then stored in a dry atmosphere or in a refrigerator. Silica gel and porcelain beads may be used alternatively for soil.
• It is possible to preserve a culture for more than 20 years.
• Pridham and his associates reported that out of 1800 actinomycetes preserved by this method about 50% were viable, after 20 years of storage.

4. Lyophilization:

It is one of the best methods for long-term preservation of micro-organisms.

• It is generally used for the preservation of fungi, viruses, bacteria, enzymes, toxins, sera, and other microorganisms.
• It is a convenient method for the preservation of a large number of cultures.
• Lyophilization, which is also called freeze drying involves the freezing of a culture followed by its drying under a vacuum which results in the temporary inhibition of metabolic activities of microorganisms

The technique consists of the following stages:

1. The organism is allowed to grow to the maximum stationary phase on a suitable sterilized medium.
2. The cells are suspended in a protective medium like milk, serum, or sodium glutamate
3. A few drops of suspension are transferred to a glass ampoule.
4. The ampoules are then frozen by immersing into a freezing mixture of dry ice and alcohol at 78°C and are subjected to a high vacuum until evaporation takes place completely.
5. The ampoules are then sealed and stored in a refrigerator.

Lyophilization Advantages:

• Culture once dried needs no further attention,
• It needs very cheap storage equipment like a refrigerator, and
• It is easy to transport freeze-dried ampoules to far-off places in large numbers in relatively small boxes.

Lyophilization Disadvantages:

This is expensive and needs expertise.

5. Silica Gel Storage:

Mc Cortney bottle is filled partly with medium grain non-indicating silica gel and sterilized by dry heat. The bottles are kept in a tray of water to a depth above the level of the gel.

• The water is frozen by placing the tray in a deep freeze with a temperature 17°C to 102°C.
• Spore suspensions are prepared in sterilized and cooled 5% skimmed milk and added to silica gel crystals in the tray of frozen water using a
• Pasteur pipette and wetted three quarters to avoid over saturation. The gel bottles are left in the ice bath for about 20 minutes until the ice around them have melted a little.
• The crystals are agitated to ensure thorough dispersion ofthe suspension.
• Bottles are dried with the caps loose for 10-14 days at 25°C until the silica gel crystals separate. Bottles are reversed down tightly and stored over indicator silica gel in airtight container at 4°C.
• The indicator gel requires replacement once or twice a year.

Silica Gel Storage Advantages:

• This method is simple and mites-free.
• Suitable for oomycetous fungi.
• Stability in some cultures like Neurospora and
• Aspergillus is more.

Silica Gel Storage Disadvantages:

• Repeated retrieval can result from contamination, and
• Suitable only for fungi

Question 11. Explain the principle, procedure, and applications of sterilization using a hot air oven
Answer:

Hot air oven Principle:

Sterilizing by dry heat is accomplished by conduction.

• The heat is absorbed by the outside surface of the item, then passes toward the center ofthe item, layer by layer. The entire item will eventually reach the temperature required for sterilization to take place.
• Dry heat does most of the damage by oxidizing molecules. The essential cell constituents are destroyed and the organism dies. The temperature is maintained for almost an hour to kill the most difficult of the resistant spores.

The most common time-temperature relationships for sterilization with hot air sterilizers are

• 170°C (340°F) for 30 minutes,
• 160°C (320°F) for 60 minutes, and
• 150°C (300°F) for 150 minutes or longer depending on the volume.

Hot air oven Procedure: Aim: Procedure:

• Connect the power supply.
• Switch “ON” the main power supply and instrument mains. Temperature setting
• Press the SET POINT (x/w) key to set the required temperature, press (↑) to increase the temperature and (↓) to reduce the temperature
• The temp. The sensor will maintain the set temp which is indicated by the blinking of the set temp on the display screen.
• The duration of time can also be adjusted using the time adjustment knob
• After use, SWITCH OFF the power supply.

Hot air oven Applications:

• Items that are sterilized in a hot air oven include:
• Glassware (Petri dishes, flasks, pipettes, and test tubes).
• Powders (starch, zinc oxide, and sulfadiazine).
• Materials that contain oil Metal equipment (scalpels, scissors, and blades)
• Glass test tubes can be sterilized using a hot-air oven
• Glass test tubes can be sterilized using a hot-air oven

Question 12. Explain different factors affecting disinfection.
Answer:

• Disinfection is a process of removal or destruction of microorganisms and reducing their number to a nonharmful level
• Disinfection usually kills the vegetative form of microorganisms and doesn’t affect the endospores.
• The chemical that is used for disinfection of the nonliving objects (Inanimate objects) is called a “Disinfectant”
• The chemical which is used for the disinfection of living objects is called an “Antiseptic”.
• Mostly the disinfectants are “Bactericidal” while some may be “Bacteriostatic.

Factors affecting the action of Disinfectants:

The rate and extent of antibacterial action of the disinfectant depends on many factors like,

1. Concentration of the disinfectant.
2. Chemical Structure of the disinfectant.
3. Formulation ofthe disinfectant.
4. Interfering substances in the environment.
5. pH ofthe surroundings.
6. Potentiation and antagonism ofthe disinfectants.
7. Surface Tension.
8. Temperature.
9. Time of Contact.
10. Type and no. of microbes present.

1. Concentration of the disinfectant:

• The rate of killing the microbes varies directly with the concentration ofthe disinfectant.
• The rate of killing is related exponentially with the concentration ofthe disinfectant, not linearly.
• There is an optimum concentration of the disinfectant at which it shows maximum efficacy, below and beyond this concentration the efficacy 5 > decreases.
• The Dilution Coefficient is an important characteristic of a disinfectant that determines how much dilution is to be made for maximum efficiency.

The dilution coefficient is calculated using the following formula:

n = Log t₁ – Log t₂ / Log c₁ – logc₂

Where,

n = Dilution Coefficient ofthe Disinfectant.

t₁ = Death time and Concentration c₁.

t₂ = Death time and Concentration c₂.

2. Chemical Structure of the disinfectant:

• Chemical activity is largely dependent on the chemical structure of the disinfectant
• The introduction of an alkyl chain at the para position of Phenol increases the activity, however, when the chain increases more than 6 carbon atoms it decreases the solubility and disinfectant action.
• Halogenation increases the antimicrobial action ofthe phenol while nitration reduces it.

3. Formulation of the disinfectant:

• A good formulation increases the effectiveness of the disinfectant.
• Iodine is virtually insoluble in water and hence is made to dissolve by using alcohol and potassium iodide solution.
• The addition of a surfactant in iodine solution decreases its odor, and staining problem and increases the stability of the preparation.
• Chlorhexidine and Quaternary ammonium compounds show increased efficiency in a 70% alcohol solution than in the aqueous solution.

4. Interfering substances in the environment:

• The organic materials like pus, blood, etc present at the site of the action of disinfectant greatly reduce the activity of disinfectant.
• The presence of fats and oils at the site of action of phenol greatly reduces its activity.

5. pH of the surrounding:

• Most bacteria show optimum growth at 6-8 pH.
• Acidic disinfectant shows maximum activity at an acidic pH as they remain ionized.
• Basic dyes like Acridine and Quaternary ammonium compounds show maximum activity at basic pH as they remain ionized at that pH.
• Amphoteric surfactants
  • For example: Tego compounds show good activity at a variety of pH.

6. Potentiation and antagonism of the disinfectants:

Some disinfectants potentiate the activity of other disinfectants while some antagonize the actions of each other.

7. Surface Tension:

• Surface tension is the tendency of liquid surfaces to shrink into the minimum surface area possible.
• The lowered surface tension in an aqueous solution ofthe disinfectant increases its adsorption on the microbial cell and increases the wetting properties and solubility ofthe solution.
• A combination of Phenol with soap shows increased disinfectant action as soap by its property lowers surface tension.

8. Temperature:

• The action of disinfectant normally increases with temperature at a certain point above which it decreases.
• The effect of temperature on disinfectant action is expressed by using the “Temperature coefficient” which is denoted by “p”, whereas per 10°C is denoted by Q10.

Q10  = Time Require edtokillat T / Time Require edtokillat (T/10)

9. Time of Contact:

• Sufficient time of contact must be allowed for the disinfectant to show its action.
• The lesser time of contact results in decreased activity ofthe disinfectant.

10. Type and no. of microbes present:

• Disinfectants are mainly active against vegetative forms of microbes and not their spore form.
• Bacterial spores are very difficult to destroy however, aldehydes like formaldehyde are known as sporicidal.
• Acid-fast bacilli due to the presence of fats in their cell membrane are virtually immune to aqueous solutions of disinfectants but can be killed using phenols, aldehydes, and halogen derivatives

Question 13. Outline the working of TEM and SEM.
Answer:

TEM (Transmission Electron Microscope):

TEM Principle:

• The working principle of the Transmission Electron
• Microscope (TEM) is similar to the light microscope.
• The major difference is that light microscopes use light rays to focus and produce an image while the
• TEM uses a beam of electrons to focus on the specimen, to produce an image.
• Electrons have a shorter wavelength in comparison to light which has a long wavelength.
• The mechanism of a light microscope is that an increase in resolution power decreases the wavelength of the light, but in the TEM, when the electron illuminates the specimen, the resolution power increases increasing the wavelength of the electron transmission.
• The wavelength of the electrons is about 0.005nm which is 100,000X shorter than that of light, hence TEM has better resolution than that of the light microscope, of about 1000 times.
• This can accurately be stated that the TEM can be used to detail the internal structures ofthe smallest particles like a virion particle

TEM Parts:

• Electron gun
• Image producing system
• Image recording system

TEM Working:

• A heated tungsten filament in the electron gun produces electrons that get focused on the specimen by the condenser lenses.
• Magnetic lenses are used to focus the beam of electrons of the specimen.
• By the assistance offered by the column tube of the condenser lens into the vacuum creating a clear image, the vacuum allows electrons to produce a clear image without collision with any air molecules which may deflect them
• On reaching the specimen, the specimen scatters the electrons focusing them on the magnetic lenses forming a large clear image, and if it passes through a fluorescent screen it forms a polychromatic image.
• The denser the specimen, the more the electrons are scattered forming a darker image because fewer electron reaches the screen for visualization while thinner, more transparent specimens appear brighter.

NOTE: If the screen is moved aside, a photographic image can be captured in pixels forming a permanent image.

SEM (Scanning Electron Microscope):

SEM Principle:

• Unlike the Transmission Electron Microscope which uses transmitted electrons, the scanning electron Microscope uses emitted electrons.
• The Scanning electron microscope works on the principle ofapplying kinetic energy to produce signals on the interaction of the electrons.
• These electrons are secondary electrons, backscattered electrons and diffracted backscattered electrons which are used to view crystallized elements and photons. Secondary and backscattered electrons are used to produce an image.
• The secondary electrons emitted from the specimen play the primary role of detecting the morphology and topography of the specimen while the backscattered electrons show a contrast in the composition ofthe elements of the specimen.

SEM Working

The source of the electrons and the electromagnetic lenses are from tungsten filament lamps that are placed at the top of the column and it is similar to those of the transmission electron Microscope. The electrons are emitted after thermal energy is applied to the electron source and allowed to move in a fast motion to the anode, which has a positive charge. The beam of electrons activates the emission of primary scattered (Primary) electrons at high-energy levels and secondary electrons at low-energy levels from the specimen surface.

The beam of electrons interacts with the specimen to produce signals that give information about the surface topography and composition ofthe specimen.

• The specimen does not need special treatment for visualization under the SEM, even air-dried samples can be examined directly.
• However, microbial specimens need fixation, dehydration, and drying to maintain the structural features of the cells and to prevent collapsing of the cells when exposed to the high vacuum ofthe microscope.
• The samples are mounted and coated with a thin layer of heavy metal elements to allow spatial scattering of electric charges on the surface of the specimen allowing better image production, with high clarity.
• Scanning by this microscope is attained by tapering a beam of electrons back and forth over a thin section of the microscope.
• When the electrons reach the specimen, the surface releases a tiny staw ofelectrons known as secondary electrons which are then trapped by a special detector apparatus.

When the secondary electrons reach and enter the detector, they strike a scintillator (a luminescence material that fluoresces when struck by a charged particle or high-energy photon).

• This emits flashes of light which get converted into an electric current by a photomultiplier, sending a signal to the cathode ray tube.
• This produces an image that looks like a television picture that can be viewed and photographed.
• The quantity of secondary electrons that enter the detector is highly defined by the nature ofthe specimen i.e raised surfaces receive high quantities ofelectrons, entering the detector while depressed surfaces have fewer electrons reaching the surface and hence fewer electrons entering the detector.
• Therefore raised surfaces will appear brighter on the screen while depressed surfaces appear darker.

Question 14. Define and classify cultural media with examples.
Answer:

A culture media is a special medium used in microbiological laboratories to grow different kinds of microorganisms. A growth or a culture medium is composed of different nutrients that are essential for microbial growth. Since there are many types of microorganisms, each has unique properties.

Types of cultural media:

Classification based on physical state (or solidity):

• Solid medium
• Semi-solid medium
• Liquid medium

Classification based on chemical contents :

• Synthetic media: contains organic or inorganic compounds that are chemically defined (i.e. known molecular formula).
• Non-synthetic (or complex) media: contains ingredients that are not chemically defined or pure (i.e. animal extract)

Classification based on function (or the purpose of using):

• General purpose media
• Enriched (complex) media
• Selective media
• Differential media
• Transport media
• Anaerobic media
• Assay media

Question 15. Explain the principle involved in autoclaving
Answer:

An autoclave is a machine that provides a physical method of sterilization by killing bacteria, viruses, and even spores present in the material put inside ofthe vessel using steam under pressure

Autoclaving Principle:

The autoclave works on the principle of moist heat sterilization where steam under pressure is used to sterilize the material present inside the chamber.

• The high pressure increases the boiling point of water and thus helps achieve a higher temperature for sterilization.
• Water usually boils at 100 °C under normal atmospheric pressure (760 mm of Hg); however, the boiling point of water increases if the pressure is to be increased.
• Similarly, the high pressure also facilitates the rapid penetration of heat into deeper parts of the material, and moisture present in the steam causes the coagulation of proteins causing an irreversible loss of function and activity of microbes.
• This principle is employed in an autoclave where the water boils at 121°C at a pressure of 15 psi or 775 mm of Hg.
• When this steam comes in contact with the surface, it kills the microbes by giving off latent heat.
• The condensed liquid ensures the moist killing ofthe microbes.
• Once the sterilization phase is completed (which depends on the level of contamination of the material inside), the pressure is released from the inside of the chamber through the whistle.
• The pressure inside the chamber is then restored to the ambient pressure while the components inside remain hot for some time.

Autoclaving Procedure:

In general, an autoclave is run at a temperature of 1210 C for at least 30 minutes by using saturated steam under at least 15 psi of pressure. The following are the steps to be followed while running an autoclave: 1.

• Before beginning to use the autoclave, it should be checked for any items left from the previous cycle.
• A sufficient amount of water is then put inside the chamber.
• Now, the materials to be sterilized are placed inside the chamber.
• The lid is then closed, and the screws are tightened to ensure an airtight condition, and the electric heater is switched on.
• The safety valves are adjusted to maintain the required pressure in the chamber.
• Once the water inside the chamber boils, the air-water mixture is allowed to escape through the discharge tube to let all the air inside to be displaced.
• The complete displacement can be ensured once the water bubbles cease to come out from the pipe.
• The drainage pipe is then closed, and the steam inside is allowed to reach the desired levels (15 lbs in most cases)
• Once the pressure is reached, the whistle blows to remove excess pressure from the chamber.
• After the whistle, the autoclave is run for a holding period, which is 15 minutes in most cases. 10.
• Now, the electric heater is switched off, and the autoclave is allowed to cool until the pressure gauge indicates the pressure inside has lowered down to that of the atmospheric pressure.
• The discharge pipe is then opened to allow the entry of air from the outside into the autoclave.
• Finally, the lid is opened, and the sterilized materials are taken out of the chamber.

Question 16. Write a note on the cultivation of viruses.
Answer:

Virus lacks an independent metabolism and they can only replicate inside the host cell, so viruses cannot be cultured in non-living mediums such as bacteria and fungi.

The virus can only be cultured in embryonated egg, cell line culture and animal inoculation.

Techniques of virus cultivation:

• Animal inoculation
• Eryonated egg culture
• Cell culture

1. Animal inoculation:

•  Animal inoculation is one ofthe primary methods for the isolation of certain viruses and for the study of the pathogenesis of certain viral diseases
• Lab mice (white mice) particularly suckling ones are the animal of choice for virus cultivation. Suckling mice of age less than 48 hrs are used for culture of Toga virus and Coxsackie virus.
• Other animals such as hamsters, guinea pigs, chimpanzees, etc are sometimes used as alternatives for virus culture.
• After inoculation of the virus sample, the animals are observed for symptoms of disease till death. And finally, the virus is isolated from the tissue of animals.

2. Embryonated egg culture:

• For virus cultivation, an egg embryo of 7-12 days is used.
• At first egg is kept in an incubator for embryo development up to 7-12 days and then the virus sample is inoculated into the egg.
• Opening in egg should be shielded with paraffin and it is incubated for sufficient time.
• Viruses can be cultured in different parts of the embryonated egg, such as the chorioallantoic membrane, amniotic sac, allantoic cavity, or yolk sac depending upon the type of virus.

 Chorioallontoic membrane (CAM):

• Pox viruses are cultured in the chorioallantoic membrane.
• The growing virus produces grey-white lesions called Pocks.
• Each Pock is developed by a single virus.
• The number of stocks indicates the number of viruses present in the inoculated sample.

Allantoic cavity:

• Viruses such as Influenza virus, Mumps virus,
• Yellow fever virus and Rabies virus are cultivated in the allantoic cavity.
• Allantoic cavity culture of the virus is mainly done for vaccine preparation, to obtain a large amount of virus load.

Amniotic cavity:

Influenza viruses are cultured in the amniotic cavity for isolation of viruses from clinical samples.

Yolk sac:

• Herpes virus is cultured by inoculating in the yolk sac.
• This is also used for the cultivation of some bacteria such as Chlamydia and Rickettsia

3. Cell culture (tissue culture) technique:

• This technique is the most commonly used technique for the cultivation of viruses.
• There are three types of cell culture techniques.

Organ culture:

• Small bits of organs from human or animal is maintained in tissue culture media.
• This technique is used in specific purposes only. For eg, to culture Coronavirus tracheal ring culture is done.

Explant culture:

• In this small fragment, the issue is extracted from human or animal and used for virus culture.
• This technique is very rarely used.
• Cell line culture:
• This is the most commonly used technique.
• Cell line culture is routinely used in the lab for virus culture, isolation, and identification.
• In cell line culture, at first growth media is prepared by maintaining balanced salt concentration, all essential amino acids, glucose, buffering agents, some antibiotics, serum etc.
• Some tissue fragment is obtained, it is trypsinized to dissociate cells.
• The dissociated cells are washed and suspended in culture media in a tube or Petri plates and incubated for a sufficient time.
• On incubation, the cell divides and spreads out on the glass surface to form a confluent monolayer of cells, which is now used for virus culture.

Based on origin, chromosomal characteristics, and number of generations through which cell culture can be maintained, cell line culture are of three types.

1. Primary cell line
2. Semicontinuous cell line
3. Continuous cell line

1. Primary cell line:

• These are normal cells, obtained from fresh organs of animals or humans and cultured.
• Once the cells are attached to the surface of the culture vessel, they divide by mitosis until a confluent monolayer of cells covers the surface.
• These cells are capable of limited growth for limited generations. They cannot be maintained in the serial subculture.
• This primary cell line culture is used for the isolation of viruses and for the preparation of vaccines.

Examples: Monkey kidney cell line, Human amnion cell line, etc

Semi-continuous cell line (Diploid cell):

• These cells are fibroblastic cells.
• They are diploid cells containing the same number of chromosomes as the parent cell.
• Fibroblastic cells are obtained from embryo tissue.
• This diploid cell can be sub-cultured for a limited generation.
• There is a rapid cell division and after 50 serial sub cultures, they undergo senescence.
• The diploid cell are susceptible for a wide range of
• Human virus culture and also used for vaccine production.

Examples: Rhesus embryo cell, human embryonic lung strain, etc

3.  Continuous cell line:

• These are cells single type capable of infinite growth in vitro.
• These are usually cancer cells derived from cancerous tissue. These cells grow faster and they are haploid cells.
• They are termed continuous cell lines as they can be serially subcultures for infinite generations without going senescence.
• Examples: HeLa cell is obtained from cervical cancer, HEP-2 (Humman Epithelioma of larynx cell line), Vero (Vervet monkey) kidney cell lines, and BHK-21 (Baby Hamster Kidney cell line).
• Continuous cell line is maintained by serial subculture or by deep freezing at -70C so that these cells be reused when necessary.
• The continuous cell line is used for virus culture but it is not used for vaccine preparation because vaccines prepared by continuous cell culture are not considered safe for Human use

Question 17. Explain the principle involved in the microbiological assay of vitamin B₁₂.
Answer:

Method: Agar cup method

Culture maintenance medium: Nutrient agar.

• Dipotassium hydrogen phosphate: 0.7gm
• Potassium dihydrogen phosphate: 0.3gm
• Sodium citrate: 0.05gm
• Magnesium sulphate: O.Olgm
• Ammonium sulphate: 0. lgm
• Sodium chloride: 0.005gm
• Distilled water: 100ml

Dissolve all the ingredients separately to avoid precipitation and shake and mix the solution, if necessary adjust the pH ofthe solution to 7. Add 1.5gm of agar powder and sterilize at 151bs pressure for 20 minutes.

Dextrose solution:

Prepare a 40%w/v solution of dextrose using distilled water. Sterilize at 151 bs pressure for 15 minutes. Cool and store in the refrigerator

Inoculum medium:

• Nutrient broth: 1.3gm
• Distill water: 100ml

Adjust the pH ofthe solution to 7. Dispense in screw-capped test tubes and autoclave at 1 51bs pressure for 15 minutes. Cool and store at 4degree Celsius.

Preparation of seeding inoculums:

• Using a bacteriological loop inoculate a test tube of inoculum medium with growth from a fresh agar slope and incubate at 37° C for 16-18 hrs.
• Store at 4°C. centrifuge the required amount of inoculum medium for 20 minutes at 2200rpm and decant the supernatant. Repeat the process. Finally, suspend the cells in 1ml of sterile saline mix well, and use it as inoculums.

Preparation of assay plates:

• To 100ml of sterile and cooled at 450C. Vitamin B 12 assay medium, add 1ml of 40% sterile dextrose solution and shake well. Add 1ml of inoculums ofE.coli of required thickness. Mix gently but thoroughly.
• Distribute 25-30ml ofthe inoculated medium in sterile petri plates. Allow to set the medium and store in refrigerator until use. Plates should be used on the same day within one day.
• Preparation of agar cups:

A standard 8mm diameter borer is taken. Dip the borer in isopropyl alcohol and bum the remaining isopropyl alcohol from the borer on flame, cool the borer properly, and bore cups in preseeded agar plates. Bore four cups per plate.

Preparation of standard stock solution:

Weigh accurately lOmg of cyanocobalamine powder. Transfer it to 10ml volumetric flask and make up the volume to 10ml. take 1ml and dilute it to 100ml with water. Take the reading of this solution at 361nm and calculate the cone taking E 1 % as 0.207.

Calculation:

O.D at 361 / 0.207 × 100

Preparation of standard solutions:

Take 5ml of standard stock solution and make up the volume to 50ml, treat it as standard high. Take 5ml of std high dilution and make up the colume to 50ml. Treat it as standard low.

Preparation of test dilution:

Weigh and transfer a sample quantity eq to 5mcg to 50ml of volumetric flask. Add distilled water, shake vigorously, and dilute to 50ml with distilled water. Further, dilute 5ml of distilled water to 50ml.

Application of standard and test dilutions:

With the help of sterile micropipette tips apply lOOmicrolitre of different dilutions to different cups. Mark every cup with proper dilution and keep the plates at a low temperature for 10-20 minutes for diffusion. Incubate for overnight.

Measurement of diameter:

Calculations:

Sum up the reading of all three sides.

Formula: Assay% = antilog (2±a log 1)

Question 18. Write briefly the general procedure of cell culture.
Answer:

The following three stages are performed while isolating a primary culture,

• Isolation of the tissue.
• Disaggregation of the tissue
• Seeding of culture into the culture vessel.
• All these stages are performed under a Laminar
• Air Flow hood in order to avoid any chance of contamination.

1. Isolation of the tissue:

• A piece from a surgically removed portion ofthe body of an animal is used for the culture of animal cells in a suitable nutrient medium
• The major explant tissues for animal cell culture are collected from lab animals like rabbits, mice, guinea pigs, etc.
• The human cells like muscle cells, erythrocytes, and leukocytes are also collected and cultured in suitable media.
• Organs from which cells to be collected are surface sterilized with 70% alcohol and then aseptically removed.
• The collected tissues are immediately transferred to a sterile nutrient medium or balanced salt solution (BSS) containing antibiotics.
• The tissue is usually used immediately or stored in the freezer.

1. Disaggregation of the Tissue:

• A tissue collected is disaggregated (Separated) from surrounding tissues by mechanical, enzymatic means or using chelating agents.
• a. Mechanical disaggregation:
• Also called “Physical Disaggregation”.
• The mechanical approach involves slicing or harvesting tissue and subsequent harvesting of spill-out cells.
• This can be achieved by sieving, syringing, and pipetting.
• This procedure is inexpensive, rapid, and simple, however, all these approaches involve the risk of cell damage, thus mechanical disaggregation is only used when the viability of the cells in the final yield is not very important.

2. Enzymatic Disaggregation:

• This approach involves efficient disaggregation of cells with high yield by using enzymes such as trypsin, collagenase, and others.
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• Enzyme-based disaggregation allows hydrolysis of fibrous connective tissue and the extracellular matrix.
• Currently, the enzymatic method is extensively used as it offers high recovery of cells without affecting the viability of cells.

1. Trypsin-based disaggregation or trypsinization:

The process of enzymatic disaggregation of tissues using trypsin is called trypsinization.

Trypsinization is of two types,

1. Warm trypsinization.
2. Cold trypsinization.

This allows disaggregation of tissue using trypsin, usually crude trypsin because this trypsin contains other proteases.

In addition, cells can tolerate crude trypsin well and the ultimate effect of crude trypsin can easily be neutralized by serum or trypsin inhibitor (supplementation of trypsin inhibitor is required in the case of serum-free media).

1. Warm trypsinization:
   • This approach is extensively utilized for the disaggregation of cells.
   • During the initial step, sliced tissue is washed with dissection basal salt solution and is subsequently transferred to a container of warm trypsin (37°C).
   • At regular intervals of 30 min the contents are stirred properly.
   • Then, the supernatant dissociated, the cells are separated to disperse in a suitable medium.
   • Efficient dispersion of cells can be achieved by placing the container over ice.
2. Cold trypsinization:
   • This method is also called trypsinization with cold pre-exposure.
   • In this process the chance of cellular damage due to constant exposure to trypsin is reduced, which results in a high yield of viable cells with an improved survival rate for the cells (after 24 h of incubation).
   • Since this method does not involve frequent stirring or centrifugation, it can be conveniently adopted in the research laboratory.
   • During this process, after washing and chopping, tissue pieces are kept over ice in a vial and then subjected to treatment with cold trypsin for 6-24 h.
   • Then, after the cold trypsin treatment, the trypsin is removed and discarded
   • However, the tissue fragments still contain residual trypsin. These fragments are incubated at 37 °C (for 20-30 min) followed by repeated pipetting.
   • This will encourage the dispersion of cells. The fully dispersed cells can be counted using a cell counter properly diluted, and then further utilized.

Drawbacks of trypsin disaggregation:

1. Trypsinization of cells can damage some cells, such as epithelial cells, and sometimes it is not effective for certain tissues, such as fibrous connective tissue, thus other enzymes are also recommended for dissociation ofcells.

2. Collagenase-based disaggregation:

• Collagenase is an enzyme that is responsible for the cleavage of peptide bonds in collagen.
• Collagen is a structural protein that is abundantly found in higher animals, mainly in the extracellular matrix of connective tissue and muscle.
• Collagenase, mainly crude collagenase, can be successfully used for the disaggregation ofseveral tissues that may or may not be sensitive to trypsin.
• This process involves an initial transfer of the desired tissue into a basal salt solution which contains antibiotics.
• This is followed by washing with settling and then transfer into a medium containing collagenase.
• The solution is incubated for 1-5 days, followed by repeated pipetting for uniform dispersal of cells.

Separation of these dispersed cells is encouraged by keeping the solution in a stationary phase to further encourage the settling of cells.

Chelating Agents:

• These are mainly used for preparation of cell suspensions.
• These agents form chalets along with ions like Calcium, Magnesium etc. which are essential for maintaining integrity ofepithelial cells.

3. Seeding of Culture:

• The primary culture cell grows well when seeded into culture plates at high density.
• Most Of the cells require the support of substrate for growth, these cells are called “Anchorage
• Dependent Cells” e.g. liver, kidney, etc.
• Some cells don’t require support, these cells are called “Anchorage-independent Cells”.
• The support materials used for Anchorage Dependent Cell are glass, and plastic metals like stainless steel, titanium etc.

The Anchorage-independent cells are cultivated in a liquid medium.

Question 19. Write the contributions of Edward Jenner.
Answer:

Edward Jenner was a scientist sometimes known as the Father of Immunology. Jenner’s biggest contribution to the world of immunology was his vaccine against smallpox.

• In the late 1700 Jenner noticed that milkmaids did not contract smallpox, a deadly disease that killed one out ofevery three people and also left survivors maimed. Jenner was not only a scientist; he was a physician who after training to become a doctor spent time as an army surgeon.
• He then went on to spend his time as a country doctor in England. His research into smallpox came from his case studies and clinical observations that he made. Jenner’s research ended up preventing him from running his regular medical practice, but he received monetary support from colleagues and Parliament in order to continue his research.
• Jenner observed that pus from blisters in milkmaids who developed the less deadly cowpox was somehow protecting these women from the more virulent smallpox. In 1796, Jenner tested his theory by injecting pus from cowpox into an eight-year-old boy’s two arms.

The boy was inoculated again and later tested but showed no signs of disease. Jenner himself coined the word vaccination after his work with cowpox was so successful against smallpox

Question 20. Differentiate between flagella and fimbriae
Answer:

Differentiate between flagella and fimbriae:

Question 21. Write bio-indicators of thermal sterilization.
Answer:

Bioindicators or biological indicators is a microbiological test system consisting standardized viable population of special micro-organisms inoculated on a carrier contained within its primary back ready for use providing a defined resistance to a specified sterilization process. They provide means to directly assess the microbial lethality of a sterilization process.

When used in conjugation with physical or chemical process monitors, biological indicators provide an indication of the effectiveness ofa given sterilization process.

Question 22.  Mention pharmaceutical uses offungi.
Answer:

• Medicinal production of antibiotics, vaccines, enzymes, vitamins, hormones, etc.
• As foodstuff
• Environmental cleaning
• In agriculture as biofertilizers and also as biopesticides
• In rDNA technology
• Fermentation and other industrial applications

Question 23. Define disinfection.
Answer:

Disinfection is defined as a procedure, the result of which is transient and that eliminates or kills microorganisms and/or deactivates undesirable viruses that are carried by inert contaminated environments.

Example: Propylene glycol

Question 24. Write the significance of positive control in sterility testing.
Answer:

It involves the repetition of the test using working treatment. It helps the analyst confirms the correctness of the results of a particular test.

Question 25. Write the principle of membrane Alteration method ofsterility testing.
Answer:

Membrane filters have a known uniform porosity of predetermined size (generally 0.45 pm ) sufficiently small to trap microorganisms. Using the membrane filter

• Technique, the sample is passed through the membrane using a filter funnel and vacuum system.
• Any organisms in the sample are concentrated on the surface ofthe membrane. The membrane, with its trapped bacteria, is then placed in a special plate containing a pad saturated with the appropriate medium.
• The passage of nutrients through the filter during incubation facilitates the growth of organisms in the form of colonies, on the upper surface of the membrane.
• Discrete colonies thus formed can be easily transferred to confirmation media.

The membrane filter technique is an effective, accepted technique for testing fluid samples for microbiological contamination.

• It involves less preparation than many traditional methods and is one of a few methods that will allow the isolation and enumeration of microorganisms.
• Membrane filters are used extensively in the laboratory and in the industry to sterilize fluid materials.

Question 26.  What is grade 100?
Answer:

• It is defined as the area where there should not be more than 100 particles of 0.5 microns per cubic foot of air.
• List out the different types of spoilage in pharmaceuticals.
• Infection induced due to contaminated pharmaceutical products.
• Physic-chemical spoilage: Viable growth, color change ofthe formulation, gas production.
• Physical spoilage: Cracking of emulsion and odor changes.
• Biological spoilage: Microbial toxins, microbial metabolites.
• Chemical spoilage: Due to hydrolysis, depolymerization, degradation.

Question 27. What is primary cell culture?
Answer:

This is the cell culture obtained straight from the cells of a host tissue. The cells dissociated from the parental tissue are grown on a suitable container and the culture thus obtained is called primary cell culture. Such culture comprises mostly heterogeneous cells and most of the cells divide only for a limited time. However, these cells are very similar to their parents.