protease production and assay

PROTEASE ENZYME PRODUCTION AND ASSAY

AIM: To isolate the protease enzyme producing organism from the environment and assay the enzyme production

PRINCIPLE: Proteolytic enzymes are ubiquitous in occurrence found in all living organisms and are essential for cell growth and differentiation. These enzymes not only play an important role in the cellular metabolic processes also gained considerable attention in the industrial community. Commercial proteases are mostly produced from various bacteria. Culture conditions play significant role on growth and production of protease by bacteria. Based on their acid-base behaviour, proteases are classified into three groups i.e. acid, neutral and alkaline proteases. The majority of commercial alkaline proteases are produced by bacteria, especially Bacillus sp. Several Bacillus species involved in protease production are B. cereus, B. sterothermophilus, B. mojavensis, B. megaterium and B. subtilis. Identification and characterization of microbial proteases are pre-requisite for understanding their role in pathogenesis. Proteases are also useful and important components in biopharmaceutical products. Currently, Protease performs numerous varieties of activities in detergent, food, pharmaceutical, leather, laundry, food processing etc.

MATERIALS REQUIRED:

Media and reagents: Bacterial or Fungal culture, Skim milk agar, Casein agar, SD Agar, Potato Dextrose Agar, Lacto phenol cotton blue &Gram’s staining kit, Folin’s reagent, IMViC Media, TSI Agar, phosphate buffer, 1% casein solution, 20% TCA (trichloroacetic acid).

Glassware: Petri plates, Conical flasks, Pipettes, Glass slides

Instruments: Spectrophotometer, Microscopes, Centrifuge, Colony counter, Waterbath.

PROCEDURE: (For bacteria)

1.      Sampling: The test soil samples were collected from the different sites of DRNGPSC Campus garden soil with aid of sterile spatula from 4-5 cm depth in to sterile plastic bags.

2.      Soil samples were air dried at room temperature.

 Isolation and purification:

3.       Isolation of bacteria from soil carried out by serial dilution method and isolated bacterial colonies were purified by sub culturing and stored as slants at 4±2⁰ C.

4.      Characterization of bacterial culture Various biochemical tests were performed for the identification and characterization of isolated bacteria viz Gram staining, Catalase test, Casein hydrolysis, IMViC test, Urease test, Nitrate test.

5.      Morphological, cultural, physiological and biochemical properties of the isolated strains were studied according to the methods given in Bergey’s manual of systemic bacteriology.

 Enzymatic study:

6.      Qualitative screening of bacteria (for protease)

The bacteria were streaked on casein hydrolysed medium and plates were incubated at 35±2⁰ C for 24 hours. A clear zone around the growth indicates proteolytic activity of the strains and visible difference in the extent of zone of clearing was recorded for proteolytic activity

7. Standard curve Prepared different concentration of protein (10¹ - 10¹⁰) in different test tubes by taking 0.1ml of protein and 0.9ml of distilled water and so on.

8. Added 1mlof protein and 1 ml of 1N NaOH in test tube and kept in water bath at 100^o C for 10 min. Tubes were cooled at room temperature and after 10 min add 1ml Folin’s reagent and leave it for 30 min and absorbance was measured at 750 nm.

     9. Quantitative determination (Proteolytic activity): Casein-yeast extract-dextrose broth were used for the enzymatic activity.

    10. Protease Assay The Protease was assayed by 3ml of bacterial supernatant (centrifuged 24hr old culture at 5000 rpm for 20 min) take in sterile test tube add 3ml phosphate buffer and 3ml 1% casein solution and place it on water bath at 35^o C. now add 5ml 20% TCA (trichloroacetic acid) added in reaction.

     11. Content was immediately mixed after adding Folin Ciocalteu Reagent in the mixture after 30 min 6 ml distilled water was added. Now absorbance was measured at 650 nm in every 24 hr till 72 hrs.

PROCEDURE: (For fungi)

1.      Organism and inoculums preparation fungal strains were isolated from soil of DRNGPASC garden soil, Coimbatore, by serial dilution plate method.

2.      Fungus were isolated from 10^-3 - 10^-4 dilutions by plating into Potato Dextrose Agar (PDA) medium. Isolated fungal cultures were screened for protease enzyme production.

3.      The organisms were identified using lacto phenol cotton blue mounting method.

4.      The isolated culture (Aspergillus flavus) was purified by routine sub-culturing and stored at 4^oC for further use.

5.      Enzyme assay by plate zone method:  The activity of alkaline protease was also measured by casein and gelatin plate technique. One unit of enzyme activity was defined as the amount of enzyme in 25µl of enzyme solution that produced a clear zone of 1mm² at pH 8 and 30°C for 18 hours. 25µl - 1mm² 1U 50µl - 20 mm² 10U 100µl - 30 mm² 15U The 10 U for 50µl and 15U of activity for 100µl were achieved by alkaline protease.

Figure: Casein and gelatin was hydrolyzed by alkaline protease showed a clear zone.

Coli phage isolation from sewage and titration

ISOLATION AND TITRATION OF COLI PHAGES

AIM: To isolate and titrate of coli phages from the given sample

INTRODUCTION:

Bacteriophages, they are “bacteria eaters” and are infectious agents that replicate as obligate intracellular parasites in bacteria. A typical phage contains head, neck and a protein tail.  Bacteriophages are classified into two major groups on the basis of their mode of propagation:

1.      Virulent (Lytic phage): Growth of virulent phage in susceptible bacteria destroys the host cells and produces many copies of themselves.  e.g.  T2 and T4 phages of E. coli.

2.      Temperate Phage: phages which are followed in lysogenic cycle.

Plaque assay is one of the widely used approaches for determining the quantity of infectious virus in a sample.  Only viruses that cause visible damage of cells can be assayed in this way.  Plaque assay was first developed to calculate the titers of bacteriophage stocks. Currently, its modified procedure is being used for the determination of titer of many different animal viruses too.

PRINCIPLE: 

When a suspension of an infective phage (e.g. T4 phage) is spread over the lawn of susceptible bacterial cells (e.g. Escherichia coli), the phage attaches the bacterial cell, replicate inside it, and kills it during its lytic release. Lysis of the bacteriophage is indicated by the formation of a zone of clearing or plaque within the lawn of bacteria. In the absence of lytic phage, the bacteria form a confluent lawn of growth.

Each plaque corresponds to the site where a single bacteriophage acted as an infectious unit and initiated its lytic cycle. The spread of infectious phage from the initially infected bacterial cell to the surrounding cells results in the lysis of the bacteria in the vicinity, eventually forming the plaque that is large enough to be visible to the naked eye. Plaques do not continue to spread indefinitely. The size of the plaque formed depends on the virus, the host, and conditions of culture. The number of plaques that develop and the appropriate dilution factors can be used to calculate the number of bacteriophages i.e. plaque forming units (PFU) in a sample. 

Isolation of a Bacteriophage from Sewage Sludge  

1.      Bacteriophages were isolated from sewage samples by using enrichment cultures. A total of five sewage samples were collected from sewage near our college campus.

2. 35.0 ml of a filtered (Prefiltered to remove debris) sample was mixed with 35ml  of 10× Nutrient broth and with 5.0 ml of pure cultures of isolated E.coli. After proper mixing the enrichment cultures were incubated for 24 h at 37°C to allow amplification of lytic Coliphages.

3. 10.0 ml of sewage bacteriophage culture was transferred into a centrifuged tube and the sample was centrifuged at 2000 rpm for 5 minutes.  

4. Most of the remaining cells were pelleted. The supernatant was transferred to a 10.0 ml syringe barrel fitted with a 0.45 micron filter.

5. The supernatant was filtered to remove bacteria from the phage sample. The filtrate (lysate) was stored at 4^oC.

Procedure for Bacteriophage Plaque Assay:

Preparation of Stock Solution by serial dilution

1.      Place six sterile saline tubes (4.5 ml each) in your test-tube rack.

2.      Label one tube “control” and label the remaining five tubes consecutively from 10^-1 through 10^-5.

3.      Label six nutrient agar plates the same as the tubes.

4.      Using a sterile 1 ml pipette, aseptically transfer 0.5 ml of the bacteriophage suspension provided to the saline tube labelled 10^-1.

5.      Mix the tube well by rolling it between the palms of your hands.

6.      With another 1 ml pipette, transfer 0.5 ml from the 10^-1 tube to 10^-2 tube. Mix the tube as in step 5.

7.      Using a fresh pipette for each transfer, transfer 0.5 ml of the suspension from the 10^-2 tube to the 10^-3 tube, and continue this diluting procedure consecutively for the remaining saline tubes. Do not forget to mix each tube well before and after diluting. 

Overlaying Plate with Phage-Agar Mixture 

1.       Obtain six tubes of melted soft overlay agar from the waterbath. Pipette 0.3 ml of a broth culture of E.coli into each of the soft agar tubes. Mix each tube well by rolling between your palms.

2.      Remove one inoculated tube of soft agar from the waterbath. Using a 1 ml pipette, aseptically transfer 0.1 ml of the 10^-1 saline phage dilution into the soft agar tube. Mix the agar tube by rolling it between your hands.

3.      Immediately, aseptically pour the soft agar onto the surface of the nutrient agar plate correspondingly labelled as 10^-1. Replace the lid and without picking up the plate, rotate it gently in a 6-to 8-inch circle on the surface of the table to evenly distribute the agar.

4.      Using a fresh 1 ml pipette each time and working quickly, repeat steps 1 and 2 for the remaining saline phage dilution tubes and for the saline control tube.

5.      For each dilution tube, use its correspondingly labelled nutrient agar plate.

6.      Allow the soft agar to solidify.

7.      Invert and incubate plates at 35°C to 37°C for 24 hours.

Results

1.      After incubation, each plate was examined and the number of plaques was counted on each plate that has clearly differentiated plaques.

2.      The plaques were counted and recorded.

3.      The number of lytic phages was calculated per millilitre that was in the original bacteriophage suspension using formula mentioned above.

Results of Bacteriophage Plaque Assay

If 48 plaques are observed in 10^-5 dilution factor, as the 0.1 ml virus is added, Plaque forming units/ml will be 4.8 X 10⁷. In your practical you can count the plaque forming units, calculate and tabulate is as follows: 

Dilution of phage	10-1	10-2	10-3	10-4	10-5
Number of plaques
Calculations of plaque units/ml

Ames test

Ames test

 Introduction

Ames test is used to measure the mutagenic potential of a chemical and also check can cause mutation in the genome of bacteria. The bacterial reverse mutation assay detects point mutations, both frame shifts and/or base pair substitutions. Strains of Salmonella typhimurium auxotroph is unable to synthesis histidine When this histidine (his-) dependent cells are exposed to the minimal media with trace amount of histidine and biotin only those cells which revert to (his+) independence are able to form colonies. The trace amount of histidine in the media allows all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. The his+ revertants are readily discernable as colonies against the limited background growth of the his-cells. By using several different tester strains, base pair substitution mutations and frameshift mutations can be detected. Spontaneous reversions occur with each of the strain, which will be considered as background level. Mutagenic compounds cause an increase in the number of revertant colonies relative to the background level.

Aim:

To check the mutagenic potential of chemicals by observing whether they cause revert mutations in sample bacteria.

Principle:

Ames test uses several strains of bacteria (Salmonella, E.coli) that carry a particular mutation. Point mutations are made in the histidine (Salmonella typhimurium) or the tryptophan (Escherichia coli) operon, rendering the bacteria incapable of producing the corresponding amino acid. These mutations result in his- or trp- organisms that cannot grow unless histidine or tryptophan is supplied. But culturing His- Salmonella is in a media containing certain chemicals, causes mutation in histidine encoding gene, such that they regain the ability to synthesize histidine (His+). This is to say that when a mutagenic event occurs, base substitutions or frameshifts within the gene can cause a reversion to amino acid prototrophy. This is the reverse mutation. These reverted bacteria will then grow in histidine- or tryptophan-deficient media, respectively.

A sample’s mutagenic potential is assessed by exposing amino acid-requiring organisms to varying concentrations of chemical and selecting for the reversion event. Media lacking the specific amino acid are used for this selection which allow only those cells that have undergone the reversion to histidine / tryptophan prototrophy to survive and grow. If the test sample causes this reversion, it is a mutagen.

Procedure:

I ) Isolate or procure an auxotrophic strain of Salmonella typhimurium for histidine (ie. His-ve) or mutant strain of E. coli for tryptophan. (ie. Trp-ve)

II) Prepare a test suspension of his-ve Salmonella typhimurium or E.coli in a plain buffer with test chemical (eg. 2-aminofluorene). Also add a small amount of histidine or tryptophan

III) Also prepare a control suspension of His-ve Salmonella typhimurium or E.coli but without test chemicals.

IV)  Incubate the suspensions at 37°C for 20 minutes

V) Prepare the two agar plate and spread the suspension on agar plate.

VI)  Incubate the plates at 37°C for 48 hours.

VII) After48 hours count the number of colonies in each plate.

Result Interpretation:

• The mutagenicity of chemicals is proportional to number of colonies observed.
• If there is a large number of colonies on the test plate in comparison to control, then such chemical are said to be mutagens.
• Very few numbers of colonies can be seen on control plate also. This may be due to spontaneous point mutation on hisidine or tryptopan encoding gene.

Left: Control without mutagenic chemical

Right: The colonies occurred on his+ or trp + containing media reveals reverse mutation. It results the chemical possess a mutagenic poential

Motility test- Hanging drop technique

MOTILITY TEST

HANGING DROP TECHNIQUE:

AIM:

 To observe the bacterial motility by following hanging drop technique

PRINCIPLE:

Hanging drop preparation is a special type of wet mount (in which a drop of medium containing the organisms is placed on a microscope slide), often is used in dark illumination to observe the motility of bacteria.

Hanging Drop Method Preparation

In this method a drop of culture is placed on a cover slip that is encircled with petroleum jelly (or any other sticky material). The cover slip and drop are then inverted over the well of a depression slide. The drop hangs from the cover slip, and the petroleum jelly forms a seal that prevents evaporation. This preparation gives good views of microbial motility.

 Materials required:

1.      Glass slides (glass slide with depression) or Normal glass slide with adhesive or paraffin ring, Paraffin wax, Loop, Coverslip, Microscope, Bunsen burner

2.      Young broth culture of motile bacteria (e.g. Proteus mirabilis)

Procedure:

1.      Take a clean glass cavity slide and hold a clean coverslip by its edges and carefully dab Vaseline on its corners using a toothpick.

2.      Place a loopful of the broth culture to be tested in the center of the coverslip.

3.      Turn the concavity slide upside down (concavity down) over the drop on the coverslip so that the vaseline seals the coverslip to the slide around the concavity.

4.      Turn the slide over so the coverslip is on top and the drop can be observed hanging from the coverslip over the concavity.

5.      Place the preparation in the microscope slide holder and align it using the naked eye so an edge of the drop is under the low power objectives.

6.      Observe the slide and Focus the edge of the drop carefully in the microscope.

Result: The movement of bacteria was observed under the 40x objective with optimum light background.

GRAM'S STAINING

Gram staining

AIM:

To stain the bacterial smear by following Gram’s staining procedure and observe their morphology and arrangements

PRINCIPLE

The differences in cell wall composition of bacteria account for the Gram staining. Gram-positive cell wall contains a thick layer of peptidoglycan. 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. The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of bacteria. The outer mem

brane 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. 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 of the gram-positive cell is dehydrated by the addition of ethanol. After decolorization, the gram-positive cell remains purple in color, whereas the gram-negative cell loses the purple color and is only revealed when the counter stain, the positively charged dye safranin, is added.

Materials required:

Inoculation loop, slide, Bacterial culture, Bunsen burner, Gram staining kit

PROCEDURE:

Gram Staining Procedure:

1.      Prepare a smear over a clean slide and heat fixed.

2.      Flood the smear with crystal violet and wait for 1 minute.

3.      Wash slide in a gentle and indirect stream of tap water for 2 seconds.

4.      Flood slide with the mordant: Gram’s iodine. Wait 1 minute.

5.      Wash slide in a gentle and indirect stream of tap water for 2 seconds.

6.      Decolorize the smear by Flood with decolorizing agent (Acetone-alcohol decolorizer). Wait 10-15 seconds or add decolorizer drop by drop on slide until decolorizing agent running from the slide runs clear.

7.      Flood slide with a counter stain, safranin. Wait 30 seconds to 1 minute.

8.      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.

9.      Observe the results of the staining procedure under oil immersion (100 x) of a microscope.

Results:

§  Gram-negative bacteria will stain pink/red and

§  Gram-positive bacteria will stain blue/purple.