MEDIA FORMULATION and SCALE-UP

BY: RAHUL ANDHARIA (MSIWM001)

Growth medium is used for support of micro-organisms, cells and small plants. Microbial growth medium can be solid or liquid depending on the type of Micro-organisms to be grown and cultured. It is necessary to select an appropriate culture medium for in-vitro cultivation.

Components and media Formulation:

The requirement of media components depends on the type of cell lines used. Every component used has a specific role to perform. Basic components of media are glucose, amino acids, salts, vitamins, other nutrients, energy source, nitrogen source, water and carbon source.

Carbon and Energy Sources:

  • Carbon metabolism has a role to play in product synthesis. Formation of products directly depends on the rate at which carbon is metabolized.
  • Carbohydrates, oils and fats and hydrocarbons are some of the common carbon sources.
  1. Carbohydrates:
  2. Most commonly used source of carbon in fermentation processes.
  3. Maize, cereals and potatoes contains starch, which is an essential carbohydrate. Primarily used in fermentation of alcohol.
  4. Barely grains contains are rich in  amount of carbohydrates like sucrose, cellulose, starch and other sugars.
  5. One major source of sucrose is sugarcane and Molasses. Molasses(obtained after refining of sugarcane or sugar beets) are one of the major source of carbohydrates.

B.    Oils and Fats:

  • Oils provide more energy compared to sugars. Vegetable oil is a common source of carbon.
  • Oils and fats are generally used more as additives rather than as sole carbon source. It also has anti-foaming properties.
  • Examples include: vegetable oil, olive oil, linseed oil, soya been oil.

C.    Hydrocarbons:

  • Hydrocarbons used as carbon sources are C12- C18 alkanes.
  • They have more carbon and energy content per weight when compared to sugars. They are relatively cheap.
  • Can be used in antibiotics, organic acids, amino acids and protein fermentation.

Nitrogen Sources:

  • Most commonly used nitrogen sources includes, Ammonia, ammonium salts and urea.
  • For pH control, ammonia is used.
  • Soya meal, corn-steep liquor, peanut meal, cotton seed meal, amino acids and proteins.

Essential Metals and Minerals:

  • Na, K, Ca, P, S, Cl, Mg and Fe are known as macronutrients and are required in larger quantities.
  • Zn, Mn, Br, B, Cu, Co, No, V, and Sr are the micronutrients required in relatively less amounts.
  • Concentration of these elements used depends on the type of Micro-organisms to be cultured.

Inorganic Salts:

  • helps in maintaining osmo-regulatory balance.
  • Helps to Increase membrane potential by providing calcium, phosphate and sodium ions.

Growth Factors: common growth factors includes Vitamins, Minerals and fatty acids.

  1. Amino acids:
  2. Amino acids being building blocks of proteins, becomes an essential component of any type of culture media.
  3. Microbial cells cannot synthesise Essential amino acids, and hence it must be supplied for cell proliferation.
  4. Nitrogen for NAD and NADPH, and nucleotides is provided by L- Glutamine.
  5. As L-glutamine is an unstable amino acid, it gets converted to other form with time and hence it must be added to medium just before use.
  6. Non-essential amino acids can also be provided for those that are deprived of growth.

B.   Vitamins:

  • Cells cannot synthesise vitamins in sufficient quantities and must be supplied. Vitamins serves as important growth factor for cell proliferation.
  • B group vitamins like Thiamine, niacin Pantothenic acids are commonly added.
  • Serum is the major source of vitamin in media.

C.    Fatty acids: mostly important in serum free media, as fatty acids are commonly present in serum. Example- Linoleic acid.

Chelating Agents:

  • Insoluble metal precipitation can be prevented by using chelating Agents.
  • Chelating Agents forms complexes with metal ions present in media, and than this can be utilised by micro-organisms.
  • Example- EDTA( calcium and magnesium complex), citric acid, pyrophosphates.

Buffers: role of buffer is to regulate the pH of medium. Micro-organisms growth is affected by changes in pH and hence role of buffers is vital in any type of media Formulation.

  1. Natural Buffer systemBalance of CO2 along with co3/hco3 content of culture medium is termed as natural Buffer system. Air atmosphere has to be maintained in natural buffering system.(5-10% CO2).
  2. HEPES: can be used as buffering system. It increases the sensitivity of the media towards phototoxic effects.
  3. Phenol Red: it is used as an indicator in most of the commercially available media. pH of medium changes due to release of metabolites, as pH changes colour of the solution also changes. Phenol Red turns medium yellow at lower pH, while at higher pH it turns the medium purple.

Anti-foaming Agents:

  • Large amounts of foam is produced during microbial processes. Because of excess foaming cells gets removed from the media and leads to Autolysis.
  • Hence Anti-foaming agents are required to stop excess foaming and prevent cells from Autolysis.
  • Examples- Stearyl alcohol, cotton seed oil, linseed oil, silicones and sulphonates.

Selective agents:

  • Mostly  antimicrobials are used. This agents makes them selective for certain Micro-organisms.
  • They are added in a fixed concentration which is specific and prevents growth of unwanted Micro-organisms.
  • Examples- Selenite, bile salts, dye stuffs.

Gelling Agents:

  • Most commonly used gelling agent is Agar. It is mostly obtained from sea weeds like Gracilaria and gellidium.
  • Gelatine, polyacrylamide, carrageenan scan also be used as gelling Agents.

Serum:

  • Most important component of cell culture media. It is a complex mixture of Albumins, growth factors and growth inhibitors.
  • For cell cloning and fastidious growth of cell, fetal serum is used.
  • Due to its lower growth promoting ability, calf serum, is used for contact inhibition studies.
  • 2-10% of serum is present in normal media.
  • Serum provides multiple components like proteins(fibronectin), Albumins, amino acids, provides protease inhibitors(protects cells from proteolysis) and it can also act as buffer.

Scale Up of industrial Microbial process:

  • According to its name, Scale up simply means increasing something (process) in terms of size, production and it’s amount.
  • Scale up of industrial Microbial processes is essential to meet the customer needs and to mass produce a particular product or process for larger profit gains and for greater supply needs.
  • For scale up, any process developed in laboratory has to be converted into full manufacturing scale process. For example- 20000- 2000,000L fermenters are used for scale up process.
  • Scale-up industrial process is done in 2phases generally:

Pilot Scale- 100-10,000 L fermenters and downstream equipments. The main purpose of pilot Scale- is to convert lab based process to a smaller version of manufacturing process with medium production and scale up.

Demo Scale- 10,000-100,000 L fermenters, with downstream equipments. It minimises the larger investments risk by continuous supply chain, process validation and fulfilling market demand.

Roadmap for proper and successful scale-up process (For large scale plant)

  • For an extended period, fully integrated process, including recycle streams can be operated with full industrial materials and equipments.
  • Different design models of equipments such as fermenters and suppliers can be evaluated.
  • More number of people can be trained to operate large scale plant. Proper operation methods, if followed can lead to faster production.
  • Operating-know how and pilot plant data is used to create solutions and planning for preparing large scale plant.
  • Large quantities of product can be produced at end application, to build healthy customer relationship and increasing demand for higher commercial plant output.

Scale-up process is essential in terms of increasing production and fulfilling the demand based on increasing needs and supply chain of products.

CHROMATOGRAPHY

BY: RAHUL ANDHARIA (MSIWM001)

Enzyme Purification: The process in which enzymes are purified to obtain pure biological catalysts to study their nature and further using it in industries and in applied sciences to develop various                by-products.

Chromatography: It is a technique in which components are identified, separated and purified from a mixture for qualitative and quantitative analysis. Based on Polarity, net charge and hydrophobic interactions, enzymes are separated by using chromatography. In the year 1903, Chromatography technique was first used for colourful separation of plant pigments through a calcium carbonate column by Mikhail Tswett. (Coinedthe term chromatography)

General Chromatography Principle: The basic principle involved is, mixture of molecules on the surface of solid or liquid gets separated in the stationary phase(which is also called stable phase) from each other and moves with the help of mobile phase. Factors leading to separation generally includes adsorption (liquid-solid), partition(liquid-solid) and affinity, that is molecules separate based on their molecular weights. S- phase is solid or liquid while M- phase can be liquid or gas.

Various chromatography methods involved in enzyme purification:

  1. Ion exchange chromatography
  2. Affinity chromatography
  3. Size exclusion chromatography/gel permeation chromatography
  4. Immunoaffinity chromatography
  5. Hydrophobic interaction chromatography
  1. Ion exchange chromatography:
  2. In this method polar molecules or ions gets separated depending on their affinity to ion exchangers.
  3. Cationic exchangers: They are basically negatively charged and attracts positively charged cations. Due to ionisation of acidic group, they are charged negatively and hence also known as acid ion exchange materials.
  4. Anionic exchangers: also called basic ion exchange materials. They are charged positively and attracts negatively charged anions.

  Working Principle of ion exchange chromatography:

  • The technique is based on attractions between oppositely charged stationary phase, which is basically an ion and an analyte.
  • Ion exchangers are charged groups, linked covalently to surface matrix.(can be positive or negative).
  • Charged groups, when suspended in aqueous solution will be surrounded by oppositely charged ions.
  • This forms an ‘ion cloud’ . In this ion cloud exchange of ions occurs without altering the property and nature of the matrix.

 Instrumentation:

  • Pump- IC pump is present which helps in continuous supply and flow of eluent(carrier portion- portion carrying the molecule). In this the eluent used is liquid.
  • Injector: The instrument is equipped with an injector valve that injects or allows the liquid to pass through. Solid substances are first dissolved in solvent and than they are injected through the valve.(injecting range of liquid ranges from 0.1-100ml of volume).
  • Columns: material of the column depends on the application of use. It can be various types like, glass, steel, titanium and inert plastic such as PEEK. Column diameter ranges from 2mm-5cm. Guard column is placed inside separating column for ensuring the safety of the column and its usage for longer durations.
  • Suppressor: To reduce background conductivity of chemicals used for sample elution, suppressors are used. IC suppressors are employed to convert ionic eluent water.(enhances the sensitivity).
  • Detector: commonly used detector is electrical conductivity detector.

Data system: connected to a data system to obtain high throughput data.

Procedure of ion exchange chromatography:

  • Columns are used for packaging and packed with ion exchangers.
  • Commercially available ion exchangers are made up of Styrene and Di-vinyl chloride.
  • Based on charge of particle to be separated, ion exchangers are selected.
  • The column contains sample, ion exchanger and buffer.( Tris and acetate buffer are used widely).
  • Particles gets separated based on its affinity towards ion exchangers. Particles with higher affinity for ion exchangers, settles down at the bottom of the column along with buffer.
  • Spectroscopy methods are used for sample analysis.

Merit of the method: used for separating charged particles. In-organic ions can also be separated by using this method.

Demerit: Major drawback is, only charged molecules can be separated.

  • Affinity Chromatography:
  • The technique is based on affinity phenomenon, in which atoms are held intact in combination in a mixture by exerting an attracting force between the atoms.
  • Example can be enzyme and inhibitors.
  • Affinity based chromatography was first used and demonstrated Meir Wilcheck and Pedro Cuatrecasas.

Principle:

  • Substrate(ligand) molecules binds covalently on the support medium in stationary phase. The reactive molecules required for binding to the target are exposed.
  • Using chromatography column, the mixture is allowed to pass through. Substances binding to Immobilized substrate binding sites, will also bind to stationary phase, while the leftover mixture is eluted in the volume void of the column.
  • Target molecules which remain attached to the target can be eluted by altering pH, polarity or ionic strength of the solution.

Components of affinity chromatography:

  • Matrix: coupling of ligand to the target molecule takes place in the matrix. It is very important to have a proper matrix for affinity chromatography. The matrix should be chemically and physically inert, it should have a larger surface area for more adsorption of molecules, matrix should be insoluble in buffers and solvents. Polyacrylamide and agarose are the common materials used in matrix preparation.
  • Spacer Arm: Target molecule binds with the ligand with the help of spacer arm. Spacer arm facilitates this binding by avoiding steric hindrance.(rate of reaction is slow due to bulking of large molecules and atoms).

Ligand: molecule binding reversibly to the target molecule is ligand. Based on the nature of macromolecules isolated, ligand can be selected. For purification of enzymes, cofactor, substrate analogue or inhibitor is used as a ligand.

Procedure:

  • Column preparation: materials like cellulose, cephalose or agarose are used for column packaging. Ligand selection is based on the sample opted for affinity chromatography.
  • Sample loading: The loaded mixture of substances is poured into an elution column. Elution column allows the sample to run at controlled rate.
  • Elution: Target substance recovery is done using elution by changing pH, ionic strength and polarity conditions.
  • Used in most of the enzymatic assays for identifying binding sites of enzymes.

Merits: Specificity is very high in this method, target molecules in pure form can be obtained, the matrix used can be reused and gives higher yields.

Demerits: number of solvents required are more, if pH is not adjusted properly, proteins gets denatured, and one major drawback is eliminating non-specific adsorption.

  • Size Exclusion chromatography:
  • This technique can be used in particular for high molecular mass specie.
  • Porous material is S-phase and mobile phase is liquid.
  • Diameter of the pores of porous material used generally ranges from 50 to 3000 A.(A- angstrom).
  • Molecules which are smaller in size penetrates the membrane faster than larger molecules.
  • Size of solute molecule is used as a separation measure in this chromatography method.

Principle:

  • In this method, molecules are selected based on their molecular weights and size.
  • For molecules to be separated, porous glass granules and the selected liquid solvent are in equilibrium.

As smaller molecules move faster than larger molecules, larger molecules which are excluded from the column will pass through the interstitial spaces, where generally smaller molecules are distributed between inside and outside of the solvent sieve, which will than move at much slower rates.

Theory:

  • Volume of column in total is given as:

(Vt= Vg +Vi+ Vo), where Vg, is volume occupied by solid matrix, Vi is solvent volume and Vo is free volume, volume outside particles.

Components and procedure:

  • Dextran, agarose, polyacrylamide are the commonly used gels.
  • Column packaging: done by using silica glass granules or by  cross-linked organic gels such as dextran.
  • Detectors: detectors to be used are decided based on UV fluorescence and refractive index.
  • Size Exclusion chromatography can be implied in purification of enzymes and is extensively used in enzymatic assays.

Merits: larger components can be separated from smaller ones, shorter analysis time, no loss of the sample, gives narrow bands with good sensitivity.

Demerits: filtering in mobile phase is mandatory to avoid columns interfering with detectors and one major disadvantage is that it takes shorter time, the mount of peaks resolved are less, selectivity is also poor compared to other techniques.

  • Immunoaffinity Chromatography:
  • This method is used for separating antigen or antibody from heterogeneous mixture.
  • This method is used as combined method with LC(liquid chromatography) for binding of specific antibody or antigens.

Principle:

  • S-phase is antibody or antibody related agent.
  • The method is column based in which solution is allowed to flow through the column followed by elution.
  • Antigen or antibody is pre-functionalised in the column before the start of experiment.
  • Resin bound capture protein absorbs the target protein, while the leftover solution is eluted.
  • The fraction with target protein is also eluted and purified.

Immunoaffinity Chromatography Considerations:

  • Good column material is essential for the efficiency of the technique. The column should have:  Higher efficiency, mechanical stability, lower nonspecific binding.
  • Optimal size of pores. Smaller the pores, higher the surface area, but it won’t be accessible to larger proteins.
  • Larger pores have lower immobilization.

Antibody attachment:

  • Antibodies attach to column via covalent bonding.
  • Orientation of antibody and it’s attachment point are of important consideration in this type of method.
  • At FAB(fragment antigen binding) region antibody binding is not possible.(this site’s have to be unbound, for antigen to bind).
  • Antibody can be attached via carbohydrate residues, or direct attachment via amines or carboxyl’s.

Components and working:

  • Traditional columns in this method are made up of cellulose or agarose.
  • In high performance Immunoaffinity methods, columns are made up of silica and Azalactone beads.
  • Hydrophobic interactions chromatography: (HIC)
  • Molecules are separated based on hydrophobicity, that is lack of affinity towards water molecules.
  • Molecules elute by decreasing polarity of buffer.
  • If the molecule is more hydrophobic, the binding will be more stronger.

Principle:

  • Samples are loaded with high salt buffers containing hydrobhic and hydrophilic regions.
  • Solvation of sample solutes is reduced by salt buffer.
  • Due to decrease in solvation, hydrophobic regions gets adsorbed by the media.
  • Less salt is required for binding of the molecule is more hydrophobic.
  • During elution, decreasing salt gradient is used.
  • Sample elution can be also done by detergents or modifiers added in the elution buffer.

General considerations in HIC:

  • Ligand: Behaviour of enzymes or proteins can be determined by Immobilized ligand used. For example- straight chain alkyl ligands exhibits hydrophobic nature.
  • Degree of substitution: degree of ligand substitution is directly proportional to binding capacity of enzyme or protein.
  • Temperature: correlation between hydrophobic interactions and temperature is affects solubility and structure of enzyme or protein.
  • pH- mobile phase used in HIC, usually have pH in the range of 5-7. Effect of pH differs from protein-protein.
  • Salt concentrations: salt concentrations shows higher ligand-protein binding but higher concentrations can lead to protein precipitation.

Procedure of HIC:

  • Media is made of alkyl or aryl ligands.
  • Space between matrix is filled by moderate salt buffers.
  • Non-bound proteins can be eliminated by washing the column.
  • Lower the salt concentration, during elution.
  • Ethanol(70%) can be used to remove unbound proteins.

Merits: high ionic strength samples can be used. Large volume of samples can be loaded easily.

Demerits: major drawback is requirement of non-volatile mobile phase.

Thus, these chromatography techniques are essential for purification of enzymes.

ISOLATION OF MICROBES FROM SOIL

Content:

  • Theory
  • Requirements
  • Procedure
  • Observation
  • Result

Theory:

  • Soil contains diversity of microbes include bacteria, fungi, algae and protozoa.
  • Bacteria is the most abundant and important microorganism.
  • They are unicellular, non-chlorophyll containing, very small and primitive microorganism.
  • There are various methods for isolation and enumeration of microorganism from any material but serial dilution is the most simple and commonly used method.
  • Principle of this method is that when any sample containing microbes spread on agar plate, each single microorganism will develop into colony.
  • Since the no of microorganism in soil is high, so spreading the sample in an undiluted manner leads to the dense growth of microorganism with overlapped colonies makes harder to isolate and study.
  • So it is necessary to dilute the sample before spreading and this is done by the serial dilution method.
  • Serial dilution made the concentration low to 10, 100, 1000 and more folds.  The extent of dilution is depends on the type of sample or material.

Requirements:

  • Sterile culture tubes
  • Nutrient agar petriplates
  • Micropipettes and tips
  • Autoclaved water
  • Cotton plugs
  • Glass spreader

Procedure:

  • First prepare stock solution by adding 1 g of soil to 10 ml autoclaved water in a test tube.
  • Take 5 test tubes and mark 10-2, 10-3, 10-4, 10-5 … with a glass maker and add 9 ml autoclaved water to each test tube.
  • From the stock solution pipette 1 ml solution with the help of micropipette and transfer it to the second test tube containing 9 ml of autoclaved water marking 10-2.
  • From second test tube transfer 1 ml solution to the next tube, repeat this to desirable dilution.
  • Now from each test tube pipette out  80-100μl solution and spread on the solidified agar media plates with a spreader.
  • Incubate the plates for 18-24 hours at 37OC.

Observation:

  • After incubation observe the number of colonies and also observe the different growing patterns of the colony as well as morphology.

Results:

  • By using the following formula calculate the number of microorganism per gram of the soil.
  • Viable cells/ gram of soil =  X dilution factor

Isolation And Culturing Of Microbes From Food

Theory:

  • Microorganisms are abundant and found in food stuff as well.
  • The microbes found on food can be pathogenic in nature and may cause various diseases.
  • The identification of such microbes is necessary to study and research on infectious agent.
  • Primary culture from food stuff is a mix culture of various microbes which has to be isolate form each other and identify for research.
  • The procedure below is used to isolate and cultivate pure culture form food stuffs.

Requirements:

  • Nutrient agar plate
  • Food sample
  • Spreader
  • Micropipette
  • Test tube

Procedure:

  • Take nutrient agar plate and well label them.
  • In one test tube take 4.5 ml autoclaved water and add small quantity of food sample in it
  • Mix thoroughly, after that with the help of micropipette take 100 microliter of the mixture and spread onto the petri plate with a spreader.
  • Finally put the petri plates in the incubator for 18-24 hours at 37o C.
  • After the incubation period mark the different colonies with marker.
  • Now pick each single colony with an inoculating loop and streak on nutrient agar plate.
  • Put the petri plates inside the incubator for 18- 24 hours at 37o C.

Observations:

  • After 18-24 hours examine the plates for bacterial growth.

Result:

  • Record the result of isolated colonies in tabular form.

ACID-FAST STAINING

Acid-Fast Staining

Content:

  • About
  • Principle
  • Requirements
  • Procedure
  • Observation and result

About:

  • It was developed by Paul Ehrlich in 1882.
  • Acid fast staining is a kind of differential staining.
  • This method is used for the identification of mycobacterium and other bacteria which retain carbol fuchsin (primary stain) after the treatment of strong acid and methylene blue.

Principle:

  • Mycobacteraium contain a waxy substance composed of mycolic acid in its cell wall.
  • These mycolic acid are carboxylic acid with up to 90 carbon atoms chain.
  • Mycolic acid in addition with other lipids serves as barrier and prevents the entry of dye inside the bacterial cell.
  • The dye used in this method is carbol fuchsin (lipophilic dye) which binds with the acid and lipid in the cell wall and gives red colour.
  • Binding property of the dye is related to the carbon chain length of the mycolic acid.

Requirements and reagents:

  • Bacterial culture (fresh)
  • Carbol fuchsin
  • Acid alcohol
  • Methylene blue
  • Water bath
  • Glass slide
  • Inoculating loop
  • Blotting paper
  • Microscope

Steps:

  • On a clean slide prepare a smear of Mycobacterium smegmatis and Staphylococcus aureus on another slide.
  • Air dry and heat fix.
  • Pour some drops of carbol fuchsin on both the smears.
  • Place the slides in steam for 3-5 min, to avoid smear from drying add more stain time to time.
  • Cool the slide for some time and wash with distilled water.
  • Pour acid alcohol for 20-30 second to decolorize the smear or until the smear gives pink colour.
  • Wash slide with distilled water.
  • Add few drops of counter stain i.e. methylene blue to the smears for 1-2 minutes.
  • Wash and blot dry with a blotting paper.

Observation:

  • Observe under the microscope and record the colour test
  • Classify the bacteria i.e. acid-fast and non-acid fast.
  • Also describe their morphology and arrangement of cells.

Results:

  • M. smegmatis cells appear red coloured indicate acid fast reaction and S. aureus appear blue colour and show non-acid reaction.

Example:

TypesExample
Acid-fastMycobacterium smegmatis, Mycobacterium tuberculosis
Non-Mycobacterial bacteriaNocardia

ENDOSPORE STAINING

Endospore Staining

Content:

  • About
  • Principle
  • Requirements
  • Procedure
  • Observation and result

About:

  • During unfavourable condition some bacteria goes in its dormant structure that is metabolically inactive and unable to reproduce and grow.
  • They develop structure called endospore which protects the cell from lethal agents like heat, radiation and chemicals.
  • In clinical procedure, pathogen identification include the describing the shape and position of endospores present in the bacterial cell.

Principle:

  • This method of staining is a differential staining which is used to detect, identify and differentiate endospore from vegetative cell.
  • The role of the method is to detect presence or absence of endospore some modification is done by increasing concentration of dye, increasing heat fixing duration and application of ultraviolet radiation.

Requirements and Reagents:

  • Fresh culture of B. cereus or B. subtilis and Staphylococcus aureus
  • Malachite green (5% aqueous)
  • Safarnin
  • Staining tray
  • Glass slide
  • Blotting paper
  • Microscope

Procedure:

  • Take separate clean slides and make smear of B.subtilis and S. aureus.
  • Air dry and heat fix.
  • Add few drops of malachite green on the smear.
  • Heat the slide under steam and add stain to avoid dryness.
  • Wash the slide with water running slowly on the slide.
  • Add few drops of counter stain i.e. safranin for 30 seconds.
  • Wash out the smear with slowly running distilled water.
  • Dry the slide with blotting paper.

Observation:

  • Observe the slide under oil-immersion microscope. From the microscopic field represent the size and position of the endospore. Write the colour of the spore and vegetative cell.

Result:

  • In B.cereus the endospore stain green and in vegetative cell stain red. Vegetative cell are rod shaped and S. aureus are spherical in shape.

Example:

TypeExample
PositiveBacillus anthracis, C. botulinum, Clostridium perfringens
NegativeSalmonella spp, E. coli

GRAM STAINING

Gram staining

Content:

• About
• Principle
• Materials required
• Procedure
• Observation
• Results
• Limitations.

About:

  • Gram staining is one of the most useful techniques used for the identification of bacterial population.
  • Developed by Hans Christian Gram a Danish Bacteriologist in 1884.
  • The procedure includes staining of bacteria and observation under microscope.
  • The organisms are differentiate on the basis of holding of stain, the organism called to be gram positive if it retain crystal violet after decolourization and appear purple while the gram negative bacteria after decolourisation appear pink to red in colour because of the safranin counter stain.

Principle:

  • The differences in gram positive and gram negative is due the difference in the cell wall composition of the bacteria.
  • Cell wall of a gram positive bacterium consist of a thick layer of peptidoglycan layer with numerous teichoic acid this complex structure resist decolourization.
  • Cristal violet (primary stain)  penetrates cell wall and cell membrane after that iodine is used as moderant, crystal violet interacts with iodine and peptidoglycan layer and makes a complex called cv-I complex. Even after decolourizer is applied CV-I complex retains in the cell and making it to appear purple to dark blue.
  • In gram negative bacteria there is a thin peptidoglycan layer with loose cross linkage. The peptidoglycan layer is distributed loosely in inner and outer cell membrane. After the application of cristal violet and iodine on the cells, CV-I complex is not trapped in the peptidoglycan layer and decolourizer makes hole in the layer which allow to wash out the CV-I complex from cell and leaves the cell colourless. To make colourless cell visible a counter stain is used called safranin which gives pink to red colour.

Material required and Reagents:

  • Glass slide
  • Bunsen burner
  • Microbial strain (fresh 24 hours incubated)
  • Crystal violet
  • Gram’s Iodine
  • Safranin
  • 70% ethyl alcohol
  • Dropper
  • Microscope

Procedure to follow:

  • Clean a microscope slide and make a smear of distilled water.
  • With the help of an inoculation loop pic a minute portion from the isolated colony and mix it with the smear on slide to make a thin smear.
  • Air-dry the slide at room temperature.
  • Ignite a Bunsen burner and heat fix the smear by heating the slide over the flame (not overheat).
  • Pour some drops of crystal violet over the smear and wait for 1 min
  • Slowly pour cold-water on the slide from the edges to wash out excess crystal violet .
  • Flood the slide with iodine wait for 1 minute and then wash if off.
  • Decolourize with the help of ethyl alcohol until colourless slide is appear.
  • Now add few drops of counter stain i.e. safranin to the smear and left for 30 second, then wash it off.
  • Dry the slide with bloating paper
  • Observe the slide under microscope at 40X or 100X.

Observation:

  • Examine properly the slide an identify the gram reaction, describes morphology and arrangement of the microbial cell

Results:

  • Bacteria which appeared blue colour under microscope are referred as gram negative and those cells which show pink colour are called gram negative .

Limitations:

  • Over decolourization may lead to identification of false gram negative cells and under decolourization lead to identification of false gram positive cells.
  • If the smear is too thick or viscous it may retain too much primary stain and makes identification difficult.

Examples:

TypeExample
Gram-PositiveStaphylococcus, Clostridium, Listeria, Bacillus
Gram-NegativePseudomonas aeruginosa, Shigella spp, Yersinia pestis, E. coli

AUTOCLAVE

autoclave: principle, working and construction

Part-1

Content:

About:

  • Autoclave is a machine used in industrial and scientific process which require high pressure and high temperature
  • Autoclave provides a physical method for sterilization by killing bacteria, virus even spores by the means of steam under pressure.
  • It sterilizes the materials by heating them up at a particular temperature for a specific period of time.
  • It is considered as more effective method of sterilization based on moist heat sterilization,

Principle and working:

  • Autoclave work on the principle of moist heat which uses steam and pressure inside the chamber for sterilization.
  • The boiling point of water is 1000 C under atmospheric pressure (760 mm of Hg), high pressure in autoclave increases the boiling point of the water to achieve proper sterilization.
  • In addition high pressure facilitates the penetration of heat into the microbial cell and moisture helps in the coagulation of the protein cause death of the microbes.
  • Working temperature of autoclave is 1210 C at the pressure of 15 psi or 775 mm of Hg.
  • Steam comes in the contact with the surface of the material inside the chamber and kills the microbes by giving latent heat.
  • After completion of the sterilization the pressure is released from the whistle and the chamber is restored back to the ambient temperature.

Autoclave components/ parts:

Chamber:

  • It is the main part of an autoclave consist of inner chamber and outer jacket
  • Inner side of the chamber is made up of stainless steel or gunmetal and the outer side is made up of iron
  • A healthcare laboratory uses the autoclave which has an outer jacket filled with steam to reduce the time taken for the sterilization.
  • Size ranges from 100 L to 3000 L .

Lid:

  • It is the next important part of an autoclave. A lid is used to seal the chamber and create sterilize condition inside the autoclave.
  • Lid having rubber seal to make chamber airtight, screw clamps are also used.
  • It consists of various other components

Pressure gauge:

Pressure gauge is used to indicate the pressure created inside the chamber. It ensures the safety of the autoclave while operating.

Whistle/ pressure releasing unit:

It is present on the lid, work of the whistle is to control excess pressure by releasing it.

Safety valve:

It is important part because it is used in case when autoclave failed to work properly and continuously increasing pressure. It contains a thin layer of rubber inside it that burst itself to release pressure and avoid and damage.

Electric heater:

  • An electric heater in present underneath the chamber which is electrically operated and boils the water and produce steam inside the chamber.
  • Water level in the chamber is vital as if chamber doesn’t have sufficient water the heating element might be burnt and if there is too much water in chamber it could interact with trays and material kept inside it.

Waste water cooler:

  • Many autoclaves come with cooling system which is used to cool the hot effluent into the drainage.
  • Used to prevent any damage to the drainage pipe from hot water.   

Procedure of running:

  • Before every operation autoclave should be checked for any item left from previous operation
  • Water level should be checked if needed add sufficient water.
  • All the material to be sterilized place inside the chamber.
  • Close the lid and tightened the screw and safety valves  to ensure airtight condition and switch on the heater.
  • Once the water boils the chamber is allowed to release the mixture of air and water from the discharge tube to ensure only moisture inside the chamber. The complete displacement is observed once  the bubbles cease to come out from the tube
  • Now let the steam allowed to reach the desirable pressure (15 lbs).
  • Once the pressure reached to its desired level the excess pressure is released by the whistle. After the first whistle the autoclave run for holding period i.e. 15 min.
  • After the completion of holding period the heater is switched off and leave autoclave to cool down until the pressure gauge indicate the pressure inside the chamber equals to atmospheric pressure.
  • Discharge pipe is now opened to allow air inside the chamber and lid is opened, sterilized material are taken out from the chamber.

LABORATORY GLASSWARE

LABORATORY GLASSWARES

  • Petri dish
  • Conical flask
  • Beakers
  • Test tube
  • Pipette
  • Graduated cylinders
  • Funnels
  • Glass slides or Microscope slides
  • Centrifuge tubes
  •  Inoculating loop

Petri dish:

  • Is a shallow transparent lidded dish.
  • It is also known as petri plate or cell culture dish.
  • Petri dish or petri plate was discovered by Sir Julius Richard Petri.
  • It is used to culture cells of different microorganisms such as bacteria, fungi and small mosses.
  • Diameters ranging from 30-200 mm.
  • It is usually made up of borosilicate glass.

Uses:

  • In microbiology for culture of microorganisms.
  • In cell culture for cultivation of isolated cells.
  • In sample storage and display.

Conical flask:

  • It is a type of flask which have a conical body with flat bottom and a cylindrical neck.
  • It is also known as Erlenmeyer flask or a titration flask.
  • It is named after scientist Emil Erlenmeyer.

Uses:

  • In microbiology, it is used for the preparation of microbial culture.
  • In chemistry, it is commonly used for mixing by swirling during titration and other solvents.

Beaker:

  • It is a laboratory equipment or glassware.
  • It is generally container with flat bottom and cylindrical in shape and also have small beak to aid pouring.
  • They are made up of glass [Borosilicate glass] or certain plates.

It is of three types:

  • A low from or Griffin from beaker.
  • A tall from or Berzelius beaker.
  • A flat beaker or Crystallizer.

It is commonly used for diluting concentrated chemicals make buffers or catch products during an experiment.

Test tube:

  •  It is a finger –like length made up glass or plastic, which is closed at the bottom and open at the top.
  • It is also referred as Culture tube or Sample tube.
  • It is generally made up of Borosilicate glass.

Uses:

  • In Bioscience for handling and culturing of different organisms.
  • In chemistry commonly used for handling of chemicals.

Pipette:

  • It is a laboratory tool, sometimes also termed as pipet
  • It is commonly used in biology, medicine and chemistry labs for transfer of measured volume of liquid.
  • Pipettes ranges from 1-1000 µL are distinguished as Micropipettes and Macro pipettes.
  • Heinrich Schnitger was first to patent micropipette in 1957.

Graduated Cylinder:

  • It is commonly laboratory glassware which is used to measure the volume of liquid.
  • It has a narrow and cylindrical in shape.
  • Graduated cylinder also known as mixing or measuring cylinder.
  • Graduated cylinder are made up of Polypropylene.

Funnels:

  • It is a tube that is narrow at the bottom and wide at the top.
  • It is commonly made up of stainless steel, aluminum, glass, or plastic.

Uses:

  • used for pouring liquids or powder.
  • used for holding filter paper in filtration.
  • used in transferring liquid in small container.

Glass slides or Microscope slides:

  • It is flat piece of glass.
  • It is commonly used for the examination under microscope.
  • A standard microscope slides ranges from 75mm by 25mm and 1mm thick.

Centrifuge tubes:

  • It is generally plastic or glass tube which is used for containing liquid during centrifugation.
  • It is usually made up of Polypropylene.

Inoculating loop:

  • It is simple laboratory tool used by microbiologists.
  • It is also known as smear loop or, micro-streaker.
  • It is generally made up of metal wire [such as Nicrome, platinum or tungsten].
  • Inoculating loop used for transferring a small sample or inoculum from a culture of microorganisms.
  • It is also used in streaking on a culture plates.

STERILIZATION

Sterilization by autoclave, its principle, procedures, application and precautions.

Objective

Sterilization by using autoclave.

Principle

Sterilization is the process of removal or killing of microorganisms from the object. In the laboratory it is done by the use of an instrument, the autoclave.

Autoclave is a cylindrical vessels having double wall around all parts except the upper side. It builts to withstand the steam pressure of at least 15 LBS per square inch.

The principle used here is saturated steam under pressure. Saturated steam is the water vapour at the temperature saturated steam is the water vapour at the temperature at which it is produced.

The water molecules become more aggregated that increase their penetrating power. Autoclave is usually operated at 15 LBS per square inch pressure for 15 minute which raises the temperature to 121°C.

Procedure

  • Sufficient amount of water is placed inside the autoclave.
  • Pack the material properly before putting inside the autoclave for sterilization.
  • The steam outlet is kept open till  air from inside autoclave has been evacuated and then close the steam outlet.
  • The  pressure is allowed to remain at 15 LBS per square inch for 15 to 30 minute is done by controlling the steam.
  • Now, off the plug leave the autoclave for cooling down and thus the pressure is reached down to zero mark.
  • Then open the lid and take out the materials.

Application

  • Autoclave used to sterilize usual  non- carbohydrate media, broth and agar media, contaminated media ,etc.
  • This type of sterilization is also used in the commercial canning of fruits and vegetable.
  • In, Hospital Also to maintain hygiene and contamination free clothes and instruments.

Precautions

  • Ensure there is sufficient water in the autoclave before operating .
  • The lid should be closed tightly.
  • The air should be completely evacuated from the autoclave and the steam must have to material to be sterilized.