#microbiological techniques

Hello everyone,Today's topic of discussion is "Negative Stain"

You can also find information about Gram staining here

Negative staining

is an established method,  easy, rapid, qualitative method often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, and thus visible. This contrasts with 'positive staining', in which the actual specimen is stained.The negative stain is particularly useful for determining cell size and arrangement. It can also be used to stain cells that are too delicate to be heat-fixed.

Eg of negative stain used :

nigrosin, ammonium molybdate, uranyl acetate, uranyl formate, phosphotungstic acid, osmium tetroxide, osmium ferricyanide and auroglucothionate

Principle of Negative Staining:

Negative staining requires an acidic dye such as India Ink or Nigrosin.India Ink or Nigrosin is an acidic stain. This means that the stain readily gives up a hydrogen ion (proton) and the chromophore of the dye becomes negatively charged. Since the surface of most bacterial cells is negatively charged, the cell surface repels the stain. The glass of the slide will stain, but the bacterial cells will not. The bacteria will show up as clear spots against a dark background.

Reagents of Negative Staining

India ink, Nigrosin (Nigrosin 100 gm/L, Formalin 5 ml/L in water)

To read the procedure , Advantages and limitations of Negative stain vist our site http://microamaze.blogspot.in/2015/11/negative-stain.html

https://www.blogger.com/blogger.g?blogID=5065214763789161932#allposts

With love 

Hello everyone,      Today's topic of discussion is " Phage Typing  of Salmonella typhimurium ".  

I have already covered  " Phage Typing  of Escherichia coli " in my earlier post .

 Principle:-

Phage typing is a method used for detecting single strain of bacteria. It is used to trace the source of outbreaks of infections. The viruses that infect bacteria are called bacteriophages and some of these can only infect a single strain of bacteria. These phages are used to identify different strains of bacteria within a single species.

 Phage typing is based on specific bacteriophage lysing bacteria. Phage typing of Salmonella is a useful typing tool for subcategorising the more common Salmonella enterica serotypes, i.e.S typhimurium and S.enteritidis. 

This can be useful to determine whether isolates, which come from different places at different times, are similar or different in their reactions with specific sets of phages used for typing.  Phage typing can show possible linkages between seemingly sporadic cases which could help in recognizing and tracing a common source outbreak.

Requirement :-

        Media:-

  Double strength nutrient broth:- 20 grams of Bacto dehydrated nutrient broth (Difco) ,8.5grams NaCl,  Distilled water to 1000 ml.

  Difco Nutrient agar: Bacto dehydrated nutrient broth (Difco)

The plate are prepared after autoclaving of the medias, and incubated overnight and examined  for contamination.Contaminated plates are discarded. The plates are further dried open at 37°C  for 1.5 hours.          

          Procedure:-

Very dry agar plates containing a rich nutrient source ( NA agar are flooded with a liquid culture of the bacterial isolate.

The liquid culture is removed, the culture film allowed to dry, and a set of phage  suspensions are inoculated onto the surface of the plate by means of a multipoint    inoculators.

 Phage spots are left to dry, and plates are incubated inverted overnight at 37oC.

The next day, phage lysis reactions are recorded, and compared to a set of standards

http://microamaze.blogspot.in/2015/11/phage-typing-of-salmonella-typhimurium.html

Though we all know what is replica plating technique practically, theortically it gets under my nerves a lot of times.

The reason being...

Do I need to isolate mutants or wild type?

What will I obtain on this plate?

Will be prototrophs will get selected or auxotrophs??

And many more similar doubts.

So here, we will try to simplify and answer all these doubts, and get the concepts clear for life.

Oh by the way...Did I say a hi to you guys??

I guess no.

So, hello everyone. the topic that we will be discussing today is RPT.

Let's start...5 6 7 8.

Concepts first.

What is RPT?

It is a microbiological technique which involves us to first isolate the culture on a solid media and after growth, it is replicated using a velveteen assembly onto a secong solid media to obtain the similar colony pattern.

Whether these medias will be nutritionally complete or deficient will depend and vary according to the requirement. This technique is very useful and probably the only one to identify the mutants and wild - types from a mixed culture.

What are auxotrophs and phototrophs?

Auxotrophs are the micro - organisms which due to some mutations have lost the ability to synthesize an XYZ compound or utilize and XYZ compound. Prototrophs are the wild - types which can synthesize all the components required for it's growth and also utilize all the nutrients. RPT makes use of this property of auxotrophs and phototrophs for differentiation.  

Is any other property used for differentiation?

Yes, apart from auxotrophy any other mutations can be used for differentiation. Mutations not always result in loss of function. Sometimes due to certain mutation, there can be a gain of function in mutations like resistance to certain antibiotics or chemicals etc. which also helps in classifying them from wild - types.

How should the media be selected?

Now this is a really tricky question.

But let's try making it simpler.

There are two categories of media pairs that you can use as master plate and the replica plate.

Nutritionally complete medium and nutritionally complete medium minus one component that the mutant is not able to synthesize.

On the nutritionally complete media, both auxotrophs and prototrophs will grow, But in the medium which is devoid of a particular component which the auxotrophs are unable to synthesize (leu, proline, etc) only prototrophs will grow and auxotrophs will fail to grow. Thus by comparing both the plates you can determine auxotrophic colonies.

An alternative to this medium can be - minimal medium as the master plate and the minimal medium with the component that the auxotroph is not able to synthesize. Here, on the replica plate both auxotrophs and prototrophs will grow whereas on the master plate only phototrophs will grow.

    2.  Nutritionally complete medium and nutritionally complete medium with a selective agent that             the mutant is sensitive or resistant to (antibiotic, disinfectant, etc) (Also known as negative                   selection)

On the nutritionally complete medium, wild types and mutants will grow. But on the plate containing the selective agent, only the mutants will grow. Or vice versa, if the wild type was resistant and mutated to sensitive, then the mutants will fail to grow on the replica plate and only wild types will grow. By comparison of both the plates, you can determine the sensitive and resistant colonies.

After the replica plate, an efficiency plate which has the medium similar to master plate is also plated to ensure the efficiency of replication. Mostly, the results of replica plate are compared with the efficiency plate and not the master plate.\

Hufff, finally done.

I hope this helps you.

Feel free to ask any more queries if you have.

Lots of love, high six.

Dixy is back with new experience to be shared  :)

Today I will be writing few important points about SDS PAGE Electrophoresis,  as I have used this technique many time during my Post graduation  .

When I first performed SDS , that time I didn't knew  even to seal the apparatus , but now i can interdependently use it.  So don't be afraid of this technique , you can also do it easily by just following few points which I will be mentioning below.

You must have seen the apparatus

It consists of 2 plates, a comb,2 clamps, 2 side spacer, a buffer tank , power pack

Do you know you can prepare gel of different porosity by mixing  ingredients in different concentration

But the first thing is to do is sealing the apparatus with 1 % Agarose.

All the step should be carried out with Gloved hands.

All ingredient should be prepared fresh.

Also point should be noted , to mix the ingredients gently , ensuring no air bubbles forms.

Carefully pour the mixture of gel directly into glass plate assembly as soon the TEMED is added ,  not forgetting the fact that the mixture contains acrylamide and TEMED which are carcinogenic .

Add water above the Gel mixture which allows polymerization of gel

Once  comb is placed , don't move it , this way u will not get uniform wells.

Fill the buffer in buffer tank

Always add sample in small volume with micropipette

Cover the lid and connect the electrophoresis tank to the power supply.

Allow the sample to run till it reaches 3/4 of gel

while separating  gel  from plate , care should be taken not to tear the gel, or else u may lose separated  proteins.

The gel is stained with Coomassie blue stain or Silver stain ,and destained

Hope this may help you,

Thank you

With love ,

Hello everyone ,

DNA isolation is a process of purification of DNA from sample using a combination of physical and chemical methods. The first isolation of DNA was done in 1869 by Friedrich Miescher. Currently it is a routine procedure in molecular biology or forensic analyses.Purification or isolation of nucleic acids is the first step in most molecular biology studies and all recombinant  DNA techniques. Today I will be writing about Isolation of DNA from cell .

Source : the simplest source of DNA could be bacteria

The methods include

1) Cell lysis: It solubilizes Nucleic acid . This step include

                     addition of SDS /Alkali

                      Boiling

                      addition of Chaotropic agents

2) Enzymatic treatment:Addition of protease (Protinase K), inhibit DNAse activity

                                     Addition of Nucleases, removes unwanted nucleic acid (RNAse or DNAse)

3) Phenol extraction:  Here equal volume volume of phenol is added to the sample and centrifuged. This allows partition of phase . As proteins are hydrophobic, thus partition in organic phase. Also DNA seen in aqueous phase in upper layer .

4) Ethanol precipitation: Here 0.3M sodium Acetate/ Potassium acetate / Ammonium acetate( pH 5-5.5) is added to neutralize the highly charged backbone and promote hydrophobic interactions.then equal volume of Ethanol /Isopropanol is added and incubated overnight at 4 °C .the precipitated DNA is collected by centrifugation . The pellet is rinsed with 70% Ethanol to remove excess of salt, dried and dissolved in buffer .

5)Adsorption Methods: This step mainly separates RNA from sample.

The above sample as obtained above is mixed with a resin ( silica or diatomaceous earth). the DNA binds to the matrix but RNA do not. the DNA is eluted with low salt buffer.

ThankYou

with love

Enzyme-linked immunosorbent assay, commonly known as ELISA (or EIA), is similar in principle to RIA but depends on an enzyme rather than a radioactive label. An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product. Such a substrate is called a chromogenic substrate.

INDIRECT ELISA

Here, in Indirect ELISA steps are slightly different, though the basic principle remains the same.

Two different antibodies are used.

Primary antibody, let's consider this as Ab 1.

And a Secondary antibody, Ab 2.

A serum sample or any other sample containing the primary antibody is added to a microtitre plate containing the antigens.

Reaction is allowed between them. and any free Ab 1 is washed off.

Then a secondary antibody(Anti-isotype antibody) linked to an enzyme, specific to the primary antibody is added

Reaction is allowed between them. and any free Ab 2 is washed off.

A substrate specific to the enzyme is added and a chromogenic reactions occurs between the two.

This can be measured by spectrophotometery.

Uses: Generally, serum antibodies to HIV can be detected by indirect ELISA within 6 weeks of infection.

That's all

Hope it helps

Lots of Love and be Amazing!!

Hello Readers..

Today we learn something about Western blotting. I do not have an idea as to why they it is called western, but let's get an idea about blotting technique.

Western blot is a technique to detect specific protein present in any sample.

Well to identify the proteins, first they need to be separated which is carried out by gel electrophoresis.  You can check it out over here.

After separation of the protein fragments, they are transferred onto nitrocellulose membrane.

Nitrocellulose membrane is used because proteins bind well to it.

There can be two methods for this transfer :-

a) This is a newer method using electricity. Voltage is applied across the gel and the fragments are pulled on to the membrane and maintain their original organisation.

b) This is a traditional method in which capillary movement of the buffer is used. This method is a bit time consuming. The nitrocellulose sheet is placed above the gel , and a weight is kept at the top. the entire stack is kept in a transfer buffer. the buffer moves up due to capillary action and brings the proteins from the gel onto the membrane in the same organisation.

The next step is probing.

Antibodies specific for the target proteins are used as a probe. This will be our primary antibody. These probes will bind to the target proteins, and any unbound antibodies are washed off. Any non specific binding, like binding of the probe to the nitrocellulose sheet is avoided by blocking.

Blocking is a step done prior to addition of the probe. they membrane is immersed into a solution of Bovine Serum Album (Rich source of Protein) in buffered Saline. the proteins in BSA bind to the nitrocellulose membrane all over where the target proteins have not bound (as in they fill up all the empty space on the membrane), thus leaving no room for the probes to bind.

After incubation post probing for 24 hrs at 4oC, a secondary antibody which will be specific to the primary antibody is added to sort of amplify the signal. Our this secondary antibody, like in ELISA will be something that produces signal like production of a colored reaction or radioactivity. Reporter enzymes are widely used for production of luminiscence. After addition of secondary antibody, the membrane is incubated for 2-4 hours at room temperature.

Next step is detection:

Detection of the target protein will vary according to the secondary probe used.

For eg.

For a colored substrate a colorimetric detection will be used.

For a radioactive label radioactivity detection is required. this can be done simply by exposing a X ray film to the membrane and it will develop showing darker spots where the label must have bound the target protein, etc.

Western blotting is a very important technique in practicals as well as exam point in view.

Hope this post helps you all learn.

Keep learning and do not forget to be amazing <3

Lots of love

Important points to remember (And to impress the examiner :p )

First developed in 1960 by two endocrinologists, S. A. Berson and Rosalyn Yalow, to determine levels of insulin–anti-insulin complexes in diabetics.

Sensitive technique - measures hormones, serum proteins, drugs, and vitamins at concentrations of 0.001 micrograms per milliliter or less.

Antigen is generally labeled with a gamma-emitting isotope such as 125 I, but beta-emitting isotopes such as tritium (3 H)are also routinely used as labels.

Principle:

RIA involves competitive labeling of the labeled and the unlabeled antigen to the antibody.

Labeled antigen at a saturation concentration (don't worry,it means concentration at which all the binding sites of antibodies are occupied)  + Antibody

Then test samples of unlabeled antigen of unknown concentration are added in  larger  amounts.

Now here, the antibody is unable to recognize the difference between the labeled and the unlabeled antigen,so  the two kinds of antigen compete for available binding sites on the antibody.

As the concentration of the unlabeled antigen increases, the antibody just like a betraying friend or any douchebag boyfriend unbinds the already labeled antibody, and like a snatchy girlfriend the unlabeled antigen displaces the labeled one and occupies the site.

The decrease in the amount of radiolabeled antigen bound to specific antibody in the presence of the test sample is measured in order to determine the amount of antigen present in the test sample.

To determine the amount of labeled antigen bound, the Ag-Ab complex is precipitated to separate itfrom free antigen (antigen not bound to Ab), and the radioactivity in the precipitate is measured.

A standard curve can be generated using unlabeled antigen samples of known concentration (as a standard sample), and from this plot the amount of antigen in the test mixture may be precisely determined.

Thanks you.

Lots of Love <3