what is Gram Stain Technique with animation

Aim:

  To differentiate between the two major categories of bacteria: Gram positive and Gram negative.To understand how the Gram stain reaction affects Gram positive and Gram negative bacteria based on the biochemical and structural differences of  their cell walls. 

principle :Staining is an auxiliary technique  used in microscopic techniques used to enhance the clarity of the microscopic image.Stains and dyes are widely used in the scientific field to highlight the structure of the biological  specimens, cells, tissues etc.The most widely used  staining procedure  in microbiology  is the Gram stain, discovered by  the Danish scientist and physician Hans Christian Joachim Gram in 1884. Gram staining is a differential staining technique that differentiates bacteria into  two  groups: gram-positives and gram-negatives. The procedure is based on the ability of microorganisms to retain  color of the stains  used during the  gram stain reaction. Gram-negative bacteria are decolorized by the alcohol,  losing  the  color of  the primary stain, purple. Gram-positive  bacteria  are  not  decolorized by alcohol and will remain as purple. After  the decolorization step, a counterstain is used to impart a pink color to the decolorized gram-negative organisms.

Importance of a Gram Stain:

The Gram stain is a very important preliminary step in the initial characterization  and classification of bacteria.  It is also a key procedure in the identification of bacteria based on staining characteristics, enabling the bacteria to be examined using a light microscope.  The bacteria present in an unstained smear are invisible when viewed using a light microscope. Once stained, the morphology and arrangement of the bacteria may be observed as well. Furthermore, it is also an important step in the screening of  infectious agents in clinical specimens such as direct smears from a patient.

The Gram stain procedure enables bacteria to retain color of the stains, based on the differences in the chemical and physical properties of the cell wall.

     1. Gram positive bacteria: Stain dark purple due to retaining the primary dye called Crystal Violet in the cell wall.

                           Example: Staphylococcus aureus

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COMPETENT CELL PREPARATION AND TRANSFORMATION

AIM: To prepare competent cells which are capable of taking up foreign plasmid.

PRINCIPLE:

Transformation process allows a bacterium to take up genes from its surrounding environment; that is transformation involves the direct uptakes of fragments of DNA by a recipient cell and the acquisition of new genetic characteristics.

There are two major parameters involved in efficiently transforming a bacterial organism. The first is the method used to induce competence for transformation. The second major parameter is the genetic constitution of the host strain of the organism being transformed. Competent cells are capable of taking up DNA from their environment and expressing DNA as functional proteins. If a bacterium is said to be competent, it has to maintain a physiological state in which it can take up the donor DNA. Competence results from alterations in the cell wall that makes it permeable to large DNA molecules. 

Transformation & Storage Solution (TSS) enables researchers to prepare competent E. coli in a single step and to transform the cells without heat-shock. TSS has been reported to be faster and easier than other methods of producing competent cells, such as the traditional CaCl₂ method or other high-competency protocols.

The CaCl₂ method initially produces highly competent E. coli cells, but cell competency decreases rapidly after storage at -70°C for several weeks. Other protocols produce highly competent cells that have a long storage life, but the procedures are time-consuming, requiring several transformation buffers or heat-shock steps. In contrast, TSS is a simple, one-step procedure.

Advantages of using TSS Single-step preparation of competent cells. Store prepared cells at -70°C with little or no loss in transformation efficiency. Transform cells without heat-shock. Transformation efficiencies of 106-108/µg DNA are typically obtained.

In the process of transformation, the competent cells are incubated with DNA in ice. Then it is placed in a waterbath at 42ºC and further plunging them in ice. This process will take up the DNA into the bacterial cell. Then it is plated in an agar plate containing appropriate antibiotic.

The presence of an antibiotic marker on the plasmid allows for rapid screening of successful transformants. Blue –white selection (Alpha complementation) can be used to determine which plasmids carry an inserted fragment of DNA and which do not. These plasmids contain an additional gene (lac Z) that encodes for a portion of the enzyme β – galactosidase. When it transformed into an appropriate host, one containing the gene for the remaining portion of β –galactosidase, the intact enzyme can be produced and these bacteria form blue colonies in the presence of the chromogenic substrate X – gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside). 

These plasmids contains a number of cloning sites within the lac Z gene, and any insertion of foreign DNA into this region results in the loss of the ability to form active β –galactosidase. Therefore colonies that carry the plasmid with an insert (Transformants) will remain white and the colonies without the foreign DNA (Non-Transformants) will remain Blue.

We can also calculate the efficiency of transformation by using the concentration of DNA and number of transformed colonies. 
 

MATERIAL REQUIRED:

TSS solution (transformation and storage solution)

LB – 2.12gms

PEG – 10gms

100% DMSO – 5ml

1M MgCl₂ (pH 6.5) – 5ml

Weigh these components, dissolve it completely in 80ml of milliQ water and make up the volume to 100ml with milliQ water. Autoclave and store at 4⁰c (storage period: 1 week)

LB medium:

Weigh 2.5gms in 80 ml Milliq water in 500ml conical flask (head space is necessary for aeration). Dissolve it completely and make up the volume to 100ml with milliq water. Autoclave and store at room temperature.

Autoclaved dry 1.5ml eppendrof vials, sterile centrifuge bottles (250ml), dry ice with 100% ethanol.

PROCEDURE:

1.      A single colony (DH50 / sbl3) was inoculated into 5ml LB media and grown overnight at 37⁰C with shaking at 200rpm.

2.      1ml overnight culture was inoculated into 500ml conical flask containing 100ml of sterile LB broth.

3.      Culture was incubated at 37⁰c/200rpm till it reaches an OD of 0.3.

4.      The flask was removed from the incubator and kept on ice for 20 minutes.

5.      The culture was transferred to 250ml sterile centrifuge bottle and centrifuged at 1500rpm for 15minutes at 4⁰C

6.      The supernatant was discarded and pellet is restored.

7.      Carefully add 10ml of ice cold TSS solution and resuspend slowly.

8.      Aliquot 100µl of cells in 1.5ml sterile eppendrof vials and immediately flash freeze in either liquid nitrogen or dry ice containing ethanol(Note: make sure the cells are frozen immediately else the efficiency might go down).

9.      Store at -80⁰C IN cryobox with date of preparation and determine the transformation efficiency with any plasmid on the same day.

Preparing the competent cells:

Reagent: TSS (transformation and storage solution for chemical transformation)

         85% LB medium

         10% PEG (wt/vol, MW 8000)

          5% DMSO (vol/vol)

          50 mM MgCl₂ (pH 6.5)

Autoclave or filters sterilize. Store at 4⁰C for <2 weeks.

1.      Streak the cell stock on a LB plate (added antibiotic if cells have antibiotic resistant). Incubate the plate at 37⁰C overnight.

2.      Pick a single, well isolated colony and inoculate it into 5ml of LB broth (plus antibiotic). Incubate at 37⁰C overnight with shaking at 220rpm.

3.      Transfer 1ml of the standard overnight culture to a sterile 500ml flask containing 100ml of LB medium (do not add antibiotic at this step). Incubate the cells at 37⁰C with the shaking at 220rpm, until OD600 reach 0.5; this usually takes 2.0-2.5 hr. heck the OD frequently when it gets beyond 0.2 to avoid overgrowth.

4.      When the culture reaches an OD600 of 0.5, chill the flask on the ice for 20 min and the collect the cells by centrifugation at 1500rpm for 5min at 4⁰C.

5.      Resuspend the cells in 10ml of ice cold TSS solution. Now the competent cells are ready to be transformed.

6.      Aliquot 150µl competent cells to 1.5ml tube. If they are not immediately used, cells can be stored at 4⁰C for maximum of 6 hr. without significant loss of competency. The same competent cells can also be stored at -70⁰C for long term storage (pre-treat with liquid nitrogen or dry ice).

7.      Competent cells should give a minimum of 1x10⁶ tranformants per µg of plasmid DNA.

8.      Transformation frequency of frozen cells is 30% of that of the fresh cells, when used within two months.

Transforming the cells:

1.      Add DNA (20 µl) ice cold 150µl competent cells. Thaw -70⁰C competent cells first by hand temperature.

(You can transform smaller portions of cells (e.g. 75µl or 50µl), but you should decrease the volume of DNA and media proportionately.

2.      Incubate on ice for 30 min, with occasional mix.

3.      Heat shock at 42⁰C for 2 min.

4.      After heat shock, put on ice for 2 min.

5.      Add 0.8ml LB broth.

6.      Shake and incubate at 37⁰C for 60 min.

7.      Plate (<200µl) on the appropriate agar plates which contain antibiotic.

8.      Incubate plates at 37⁰C, overnight.

OBSERVATION:

The transformed cells were plated on medium containing ampicillin and the negative control plate didn’t show any growth since there were no transformed cells in that plate.  The test plate showed growth indicating the presence of transformed cells.

    

 

Positive control showed colonies

Negative control shows no colonies

Test shows few colonies

RESULT:

The organisms were transformed successfully with the plasmid inducing resistance towards ampicillin.

Troubleshooting:

Low efficiency of transformation was observed. It may be due to the improper handling of the cells. The cells should be thawed and used immediately. Refreezing of the cells can decrease efficiency. Non-optimized tubes were used. Usage of 17 x 100-mm polypropylene tubes is the best as the heat shock step is calibrated to the size and material of these tubes. If different tubes are used, the heat transfer to the cells may not be optimal. An excess DNA was used. No more that 1-10 µg of DNA should be used in 100 µl of cells. Electroporation must have being a better method.