Aim: to study the response of the bacteria to the gram stain.

Aim: to study the response of the bacteria to the gram stain.

Requirements:
Slide, 90% alcohol, crystal violet stain, iodine reagent, saffranin, wash bottle, distilled water, pipette, spirit lamp, needle, microscope, bacterial stain.

Sample:
Bacterial sample (curd water)

Theory:
Fresh curd water may be smeared to have a leptococci and leptobacilli. Gram stain was developed             by Christian gram, a physician. According to the response of the bacteria to the gram stain, the bacteria are classified as gram negative and gram positive bacteria.

The bacteria are treated with crystal violet (primary stain) and iodine (mordant), formation of crystal-violet complex takes place which gets impregnated to the cell wall of the bacteria. After treating the stained bacteria with the ethyl alcohol (which acts as a decolourising agent) some bacteria retains their colour and some loose their colour. The bacterium which retains the colour is called as gram positive bacteria and the bacteria which loose the stain are called as gram negative bacteria.
The differential response of the bacteria to the gram stain is due to the composition of the cell wall. The gram negative bacterial wall has high lipid content, and so when the bacteria is washed with the ethyl alcohol then the lipid layer of the wall is dissolved and so does the crystal violet-iodine complex. This makes the bacterial wall colourless, and when these bacteria are treated with saffranin then the bacteria takes red coloured stain.

In the case of the gram positive bacteria the cell wall has insignificant lipid content and when the cell is washed with the decolourising agent then the bacteria retains the blue colour due to the presence of the Christian violet-iodine complex. Thus even if the bacterial is stained with the saffranin then also there is no scope for the bacteria to gain the red colour of safffranin, thus they remain violet coloured and hence is concluded as gram positive bacteria.

Procedure:
The slide was cleaned with the ethyl alcohol to make it grease free. A thin layer of smear of bacterial sample was made on slide by sterilised needle. The smear was allowed to be fixed by passing the slide quickly over the flame. Few drops of crystal violet were dropped over the smear. Crystal violet was drained away and iodine solution was added over the smear for 30 seconds. Smear was washed by dropping ethyl alcohol until no more colour flows from the smear. Smear was washed with distilled water and saffranin was applied to the smear for 30-60 seconds. Again smear was washed with sterile water. The slide was strained and dried in air.


Observations:
Smear was examined under low power then under high power and finally under oil emersion. Colour of the bacterial cell was noted. The bacterial cell retained the violet colour and hence is considered to be positive stain.
Shape of the bacterial cell was sketched.

Result:
Since the bacteria retained the stain that is the violet coloured; hence it is gram positive.

Precautions:
The slide was sterilised and handled carefully. During fixing care was taken while passing over the flame. All the apparatus including the chemicals and the spirit lamp were handled carefully.

Real time PCR

Real time PCR

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The real time polymerase chain reaction is also called as q-PCR (quantitative PCR).

It is a laboratory technique based on PCR, which is used to amplify and simultaneously quantify the targeted DNA molecule.

Its key feature is that the amplified DNA is detected as the reaction proceeds in real time.

The common methods:

1.       Use of non-specific dyes: the nonspecific dyes are not complementary to any specific sequences of the DNA, rather it intercalates with the double stranded DNA.

2.       Sequence specific fluorescent probes: consists of oligonucleotides that are labeled with fluorescent reporters, which permits detection only after hybridization of probe with targeted DNA.

3.       The probe is tagged with fluorescent materials (reporter) along with is attached the quencher molecule in very close proximity with the reporter. The quencher quenches (absorbs) the fluorescence of the reporter.

4.       The probe is first hybridized with the DNA, then the polymerase is allowed to perform its task, as the DNA polymerase adds nucleotides to the template strand, the flurophore is released away from the quencher molecule and the fluorescence is produced, depicting the formation of double stranded DNA. The intensity of fluorescence is directly proportional to the quantity of the ds DNA formed.

AGAROSE GEL ELECTROPHORESIS (AGE) Theory before performing electrophoresis

AGAROSE GEL ELECTROPHORESIS (AGE)

Theory before performing electrophoresis
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       AGE is the most convenient and easiest way to separate DNA molecules. The DNA molecules are separated on the basis of their size under the influence of an electric charge applied to the electrophoretic apparatus. The smaller molecules will travel faster and farther as compared to the bigger molecules which travel slower.

Agarose gel is obtained from Red Algae (Porphyra umbilicalis). The IUPAC name of agarose gel is

(1 4)-3,6-anhydro – α – L – galactosepyranosyl – (1 3) – β – D – galactopyranan

In general agarose gel is prepared between 0.2 – 0.7%. The 0.7% gel is ideal for the separation of larger molecules. And the 0.2% gel is ideal for separation of smaller molecules.

EAUIPMENTS AND MATERIAL NECESSARY FOR AGE:

1.       Electrophoretic apparatus:

2.       Gel casting trays:

3.       Sample combs: to form wells for samples to be loaded in the gel.

4.       Loading buffer: it allows the sample to be filled in the wells.

5.       Tracking dyes: it migrates in the gel with the DNA fragments and provides visualization for the proceeding status of the electrophoresis.

6.       Ethidium bromide: it is a fluorescent dye which can be seen under ultraviolet rays. It incorporates with the DNA fragments and when the gel is visualized under the UV rays. The ethidium bromide fluoresces to mark the location of the travelled DNA fragments in the gel.

7.       Transilluminator: it is an ultraviolet box. Which is used to visualized ethidium bromide stained separated DNA fragments after electrophoresis..