Bioprocess engg. & technology

Experiment 1

Principle : 1(A)

Effective storage means that the organism is being maintained in a viable state without contamination and changes in genotypic or phenotypic characteristics. The organism must be easily restored to the same condition it was in prior to preservation. Several methods are available for the preservations of the microorganisms but there are two criteria for selecting a preservation method for a given culture. They are:

1. period of preservation desired and

2. nature of culture to be preserved. For different physiological groups of bacteria, specific maintenance requirements exist.

Preservation techniques are broadly classified into two types: short term preservation methods and long term preservation methods.

• Short Term Preservation Methods

1. Direct Transfer to Subculture: The simplest method for maintaining short term viability of organisms, most often used for bacteria, is periodic subculture to fresh medium. The medium used should support survival of the organism but minimize metabolic process and slow the rate of growth. A medium with too high a nutrient content will induce rapid replication that requires more frequent transfers. In this method, transfer organisms into screw –top test tubes and to store them in an organized location away from light and significant temperature changes. Storage at low temperatures slows metabolic processes and maintains viability for longer periods. Usually, 5 to 10 representative colonies were used when performing transferring process.

2. Freezing at -20 ºC: Refrigeration or freezing in ordinary freezers at -20 ºC is sometimes used to preserve organisms for longer than can be accomplished by repeated transfers. The media used for storage appear important, since preservation times vary from a few months.

3. Drying: Soil should be autoclaved for several hours on two successive days. It is then transformed into sterile glass tubes. Commercial silica gel can also be used in small cotton plugged tubes after heating in an oven to 175 ºC for 1.5 to 2 h. Alternatively, a suspension of 108 organisms can be inoculated onto sterile filter paper strips or disks. The paper is dried in air or under vacuum and is placed in sterile vials. These vials can be stored in the refrigerator for up to 4years, and then single strips or disks can be removed as needed. This method is commonly used for quality control organisms.

• Long Term Preservation Methods

1. Ultra Low Temperature Freezing: Microorganisms can be maintained at temperature of -70°C or lower for prolonged periods. Systems for achieving these temperatures include ultra low temperature electric freezers and liquid nitrogen (-196 ºC) store units. Storage vials must be able to withstand very low temperature and maintain a seal for their concerns. Polypropylene or glass tubes may be used. There are two types of cryoprotective agents: one that enter the cell and protect the intracellular environment and others that protect the external milieu of the organism. Glycerol and dimethyl sulfoxide (DMSO) are most often used for the former; sucrose, lactose, glucose, mannitol, sorbitol, dextran, polyvinylpyrrolidone and polyglycol are used for the latter. Combinations of agents are sometimes used. Cryoprotectants that enter the cell usually provide better protection for bacteria. Once prepared, it can be stocked at room temperature for months. Organisms are inoculated in a medium that adequately supports maximal growth. Cultures are allowed to mature to late growth or stationary phase before being harvested.

2. Freeze –Drying (Lyophilization): Freeze –drying is considered the most effective way to achieve long term storage of most bacteria. The term “lyophilization,” which means “to make solvent loving,” Freeze drying is defined as a controllable method of dehydrating labile products by vacuum desiccation. Better preservation occurs because intracellular ice crystallization contributes greatly to organism loss in the frozen state. Removal of water from the specimen effectively prevents this damage. Among bacteria, the relative viability with lyophilization decreases from spore formers to gram positive bacteria to gram negative bacteria. In addition, dried organisms take up little space, large numbers of vials of organisms can be stored, and organisms preserved in this way can be easily transported long distances at room temperature. Glass vials are used for all freeze-dried specimens. Cryopreservation is a storage method to preserve structurally intact living cells and tissues at very low temperature.

Materials Required:

1. LB broth

2. LB agar plates

3. Micropipette and tips

4. Bunsen burner

5. Inoculation loop

6. 80% glycerol

7. -80°C freezer

Procedure: Practical 1(A)

• To create a Glycerol Stock:

1. Pick a single colony from a plate and grow an overnight in the LB broth.

2. Add 0.5 ml of the overnight culture to 0.5 ml of 80% sterile glycerol in the microfuge tube.

3. Vortex the mixture of overnight culture and glycerol slowly.

4. Store the glycerol stock at -80 ºC.

• To pick the Culture from a Glycerol Stock:

1. Take the microfuge tube from the -80 ºC freezer.

2. Scrape off a portion of stored culture from the top of the frozen glycerol stock and streak it onto an LB agar plate.

3. Invert the plates and incubate it for overnight incubation at 37ºC.

Opening Single-Vial Preparations

1. To recover the cell suspension from the glass ampoule, score the neck of the ampoule with a small, sterile file.

2. Disinfect the outside of the ampoule with freshly prepared 70% ethanol or dip it into a beaker of freshly prepared 70% ethanol.

3. Wrap the ampoule within several folds of a sterile towel or gauze to dry residual ethanol.

4. Working in a laminar flow hood, hold the vial upright and snap open the vial. Ensure that your gauze does not become too wet with ethanol, or alcohol could be sucked into the culture when the vacuum is broken. Rehydrate the material immediately.

Opening Double-Vial Preparations

1. Heat the tip of the outer vial in a flame.

2. Add a few drops of water on the hot tip to crack the glass.

3. Strike the end of the vial with a file or pencil to remove the tip.

4. Remove the insulation and inner vial with sterile forceps. Gently raise the cotton plug.

Initiating Frozen Cultures

1. Prepare a sterile test tube that contains the recommended medium for bacterial growth as listed in the supplied product sheet. Ensure that the medium contains all necessary reagents and is equilibrated for temperature and pH.

2. Thaw the sample vial via gentle agitation in a water bath that is set to the normal growth temperature of that strain. Thawing will be rapid; approximately 2 minutes or until all ice crystals have melted.

3. Remove the vial from the water bath and decontaminate the outer surface using 70% ethanol. Follow strict aseptic conditions in a laminar flow hood for all further manipulations.

4. Unscrew the top of the vial and transfer the entire contents to a sterile test tube containing the appropriate growth medium. Additional test tubes can be inoculated by transferring 0.5 mL of the primary culture to additional secondary cultures.

5. Incubate cultures under the appropriate temperature and atmospheric conditions as recommended on the product sheet.

6. Examine cultures after the recommended incubation period. The incubation period will vary between strains and is listed on the product sheet.

Handling Test Tube Cultures

1. Incubate the culture upon receipt under the appropriate temperature and atmospheric conditions recommended on the product sheet. Do not store the culture in a refrigerator.

2. Transfer the culture to fresh media as specified on the product sheet. When transferring a broth culture, aseptically withdraw approximately 1.0 mL of the culture and transfer into 5 mL of fresh broth, or transfer several drops of the suspension to an agar slant or plate. When transferring a culture from an agar slant, aseptically transfer a single colony to 5 mL of fresh broth or to an agar slant or plate.

3. Incubate the culture under the appropriate temperature and atmospheric conditions recommended on the product sheet.

Conclusion:

By performing the method of preservation (glycerol stock), we can preserve isolates for longer times.

Practical 1(B)

SPECTROPHOTOMETER

Spectroscopic techniques employ light to interact with matter and thus probe certain features of a sample to learn about its consistency or structure. Light is electromagnetic radiation, a phenomenon exhibiting different energies, and dependent on that energy, different molecular features can be probed. The interaction of electromagnetic radiation with matter is a quantum phenomenon and dependent upon both the properties of the radiation and the appropriate structural parts of the samples involved. This is not surprising, since the origin of electromagnetic radiation is due to energy changes within matter itself. The transitions which occur within matter are quantum phenomena and the spectra which arise from such transitions are principally predictable. Electromagnetic phenomena are explained in terms of quantum mechanics. The photon is the elementary particle responsible for electromagnetic phenomena. It carries the electromagnetic radiation and has properties of a wave, as well as of a particle, albeit having a mass of zero.

MICROPLATE READER

The microplate is the standard format of miniaturization and automation for bioassays (Biochemical and cell-based assays) associated with drug discovery. Within each microplate is a 2D array of wells with a limited volume for the experimentation to take place. The most common well densities for screening come in 96, 384 and 1536 wells per plate. Numerous manufacturers make microplates in a wide range of materials specific to equipment and customer needs. The footprint of the microplate and well locations has been standardized by ANSI (American National Standards Institute) and SLAS (Society for Laboratory Automation and Screening).

In HTS laboratories, microplates are generally categorized into “Compound Plates” and “Assay Plates”. Compound plates are for storage of the molecular library to be screened against and durable such the same plate can be used for extended periods and across several screens. Assay plates are where the experimentation takes place, selected based on the assay conditions and only used for the extent of an individual screen.

MICROSCOPE

With two lenses, the compound microscope offers better magnification than a simple microscope; the second lens magnifies the image of the first. Compound microscopes are bright field microscopes, meaning that the specimen is lit from underneath, and they can be binocular or monocular. These devices provide a magnification of 1,000 times, which is considered to be high, although the resolution is low. This high magnification, however, allows users to take a close look at objects too small to be seen with the naked eye, including individual cells.

Practical 1(C)

Bacteria in soil occur singly and in aggregates. To estimate the number of bacteria in a gram of soil, the soil must be both diluted and mixed thoroughly so that the aggregates are broken up such that a suspension of single cells is achieved. The cell suspension is then serially diluted so that from some dilutions a reasonable number of cells (30 to 300) are dispensed into Petri plates. The samples in Petri plates are then mixed with sterile, molten (liquid) agar medium which is then allowed to solidify. This method of plating is called pour plating. In a later experiment, we will use spread plating.

Upon incubation each cell will give rise to a colony either in the agar or on the agar surface. It is possible that a colony could have arisen from two or more cells that stuck together. Thus a colony forming unit (CFU) may have originated from one or more cells. The viable titer is determined by counting colonies (CFU's) and multiplying by the dilution factor. This method only counts living cells as dead cells do not reproduce to form colonies. The viable titer is determined from countable plates: plates from dilutions that yield at least 30 colonies (so that a statistically significant number has been counted) and less than 300 colonies. When a plate has more than 300 colonies, there is such crowding that fast growing bacteria overwhelm slow growers: the fast growers either remove nutrients or produce inhibitory end products before slow growers can form a visible colony.

Visible colony. Later, isolated colonies will be examined for the types of cells and one will be restreaked to obtain a pure culture. A pure culture is defined as the progeny from one cell. Actually we will be making an axenic culture from a clone (colony). Assuming that one cell could have given rise to the colony, we call these pure cultures even though we have no technical proof of that. Proof of pure culture involves showing that all the colonies on the restreak are identical and Gram staining these to demonstrate all the cells in the resulting colonies are identical and the same as those on the original plate.

Materials required

Practical 1 (c)

• 9 ml sterile dilution blanks (16x150 mm capped tube)

• 100 ml sterile dilution blank (square bottle)

• 6 sterile 1 ml pipettes.

• Molten (45^oC) Nutrient Agar

• 10 sterile Petri plates.

• Balances and weighing boats.

• Nutrient Agar plate (Day Two)

• Gram staining reagents, slides.

Practical 1(b)

MICROPLATE READER:

Microplates are typically offered in opaque white, opaque black or translucent. Compound plates are typically translucent such that compound volumes and color can be seen. Opaque plates are used to enhance detection technologies. Generally white assay plates are for luminance assays and black assay plates are for florescence assays. The bottom material of an opaque plate may be clear to support bottom reads and colorimetric (absorbance) assays. The microplate reader is designed to detect and quantify biological, chemical or physical events found within the well of a microplate. There is currently a wide range of detection technologies to suit specific assay requirements many of which can be combined into single multi-purpose instrumentation.

MICROSCOPE:

Light Microscope: With two lenses, the compound microscope offers better magnification than a simple microscope; the second lens magnifies the image of the first. Compound microscopes are bright field microscopes, meaning that the specimen is lit from underneath, and they can be binocular or monocular. These devices provide a magnification of 1,000 times, which is considered to be high, although the resolution is low. This high magnification, however, allows users to take a close look at objects too small to be seen with the naked eye, including individual cells.

Confocal Microscope: the confocal microscope uses a laser light to scan samples that have been dyed. These samples are prepared on slides and inserted; then, with the aid of a dichromatic mirror, the device produces a magnified image on a computer screen. Operators can create 3-D images, as well, by assembling multiple scans. Like the compound microscope, these microscopes offer a high degree of magnification, but their resolution is much better.

Scanning Electron Microscope: The scanning electron microscope, or SEM, uses electrons rather than light for image formation. Samples are scanned in vacuum or near-vacuum conditions, so they must be specially prepared by first undergoing dehydration and then being coated with a thin layer of a conducive material, such as gold. After the item is prepared and placed in the chamber, the SEM produces a 3-D, black-and-white image on a computer screen. Offering ample control over the amount of magnification, SEMs are used by researchers in the physical, medical and biological sciences to examine a range of specimens from insects to bones.

Transmission Electron Microscope: Like the scanning electron microscope, the transmission electron microscope (TEM) uses electrons in creating a magnified image, and samples are scanned in a vacuum so they must be specially prepared. Unlike the SEM, however, the TEM uses a slide preparation to obtain a 2-D view of specimens, so it's more suited for viewing objects with some degree of transparency. A TEM offers a high degree of both magnification and resolution, making it useful in the physical and biological sciences, metallurgy, nanotechnology and forensic analysis.

practical 1 (C)

1. Mark the sterile dilution blanks in the following manner: the 100 ml dilution blank is 10^-1 and the 9 ml tubes sequentially are 10^-2 ,10^-3, 10^-4, 10^-5, 10^-6.

2. Go outside and with a weigh boat obtain some soil.

3. Weigh one gram of soil out in another weigh boat. Add that gram to the 10^-1 dilution blank and shake vigorously for at least 1 full minute. Make sure the cap is securely tightened during the shaking.

4. All the 10^-2 dilution to sit for a short period. Then aseptically transfer 1 ml from this dilution to the 10^-3 tube. The instructor will demonstrate how to make an aseptic transfer. Mix thoroughly.

5. Using a fresh, sterile pipette for each succeeding step, transfer 1 ml from the 10^-3 dilution to the 10^-4 dilution blank, then from the 10^-4 to the 10^-5, then from the 10^-5 to the 10^-6. Each time the sample transferred must be thoroughly mixed with the dilution fluid before being transferred to the next tube. Mark two plates for each tube dilution on the bottom with the dilution it will receive. From each dilution tube (but not the 10^-2) place 1 ml of dilution fluid into each of two sterile Petri plates. Be sure to use aseptic technique.

6. Take a flask of Nutrient Agar (that you made in Experiment 3) from the 45^oC water bath and aseptically pour molten agar into each Petri plate for that set. Pour in 15 ml, the exact amount is not important: you need to get enough to cover the bottom of the plate and mix with the 1 ml inoculum in the plate. Rapidly but carefully pour all plates of that set. Then gently swirl each plate on the bench so that the inoculum gets thoroughly mixed with the agar. It is important to do this thoroughly, but not too vigorously so as to get the molten agar on the Petri plate top.

7. Allow all the plates to stand without moving so that the agar may solidify and set completely.

8. Invert the plates and stack into pipette canisters and place in the incubator or at room temperature until next period (2 days).

Result

Practical 1 (C)

By using isolation method we have isolated colonies of amylase enzyme producing microorganism as seen in figure 1.

practical 1(b)

Reference Material

Preservation of bacterial culture: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC374436

Isolation of Microorganism: en.wikipedia.org/wiki/Isolation_(microbiology)

Questions

1. Why must you cool the melted agar to 45^oC before pouring into the Petri plates.

2. Are all the bacteria in soil counted by this procedure? Is it possible that this medium could not support the growth of certain bacterial types? Thus, is the Nutrient Agar count of the bacteria in soil an overestimate or underestimate of the actual number of viable bacteria in soil?

3. How would you design an experiment to test the answers to Question 2?

4. Suppose someone gave you a slant containing bacteria and said: "here's a pure culture". How do you prove that this is really a pure culture?

5. Why is it important to obtain pure cultures?

6. What is Magnification and Resolution?

7. Explain the Beer-Lambert Law.

8. Applications of the microplate reader in the field of research.

9. What is the difference between Fluorescence Microscope and Confocal Microscope?

10. What is the difference between Scanning Electron Microscope and Transmission Electron Microscope.