Dark Field Microscopy

Hello readers ^_^

Hope you all are studying well.

Today let's discuss Dark field microscopy. There are lot of different microscopes and microscopic techniques. We will try covering most and if possible all of them in coming time.

Scanning Probes Microscopes (SPM)

Hello to all my struggling, sinking, brooding yet amazing Microbiologists.
Smile up, coz Staph is here with a new instrument today.

We will be learning, what we are learning is there in the title. So with no wastage and saving our precious time we will start with it.

Yeah right, saving your precious time to watch Pokemon, Naruto and Kick Buttowski later on.
Shut up.

So SPM is not a single time of microscope like SEM or TEM, but it is a term given to a family of microscopes which consists of Scanning tunneling microscope, atomic force microscope, scanning near field optical microscope.

If there are any other then please notify me because I've noticed you noticing this and I am just giving you a notice that I have noticed you and thus you notice it down to bring any other type into my notice.
What???

Girl get yourself diagnosed for ADD.

SPM are type of microscopes which make use of a very sharp fine tip of metal to scan across the metal surface in a raster mode to produce the images of the samples even at subatomic resolutions. These microscopes are an excellent combo of spectromicroscopies serving us dual benefits of microscopy and spectroscopy so that spectroscopy of the same sample area is performed of which the high resolution image is obtained. With this, one is able to get the details of morphology and structure of the sample added with the knowledge of it's chemical nature and it's thermal, optical and magentic properties too.

No special sample preparation is required in this microscopy.
Liquid samples and live biological samples can also be studied.

Probes used are fine metal tips obtained by etching the metal wires in some specific chemicals. In some cases tips are mounted on a piezo drives which vibrates and it's function is to scan the sample surface. PZT (Lead Zirconium Titanate) is commonly used piezo crystal.

The needle scans the entire image and digital images are collected at several points. In order to obtain a high resolution images it is important to shield the microscope from any mechanical vibrations and external magnetic fields.

Different types of SPM will be discussed separately in detail in coming posts.
As of now, let's end with a high six.

-Staph <3

Fluorescence Microscopy

Hello everyone,

Today I will be talking about some important point about Fluorescence Microscopy
 Before going to the main topic i would like to discuss some important points

After irradiation with Ultraviolet, certain substance have property of emitting radiation of their own at a wavelength longer than that of the exciting source.

If the luminescence has capability of persisting for an appreciable period of time, the luminescence is called phosphorescence, But if the luminescence has capability of persisting only while the exiting source acts a on the substance  the luminescence is called Fluorescence. 

Also fluorescence is observed in specimen that have not been treated with fluorochrome , the emission is called Autoflourescence. The fluorescence acquired after treatment of a specimen with a fluorochrome is called Secondary fluorescence 

In fluorescence microscopy, the sample you want to study is itself the light source. The technique is used to study specimens, which can be made to fluoresce..The "fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a more simple set up like an epifluorescence microscope, or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescent image.

Principle

The fluorescence microscope is based on the phenomenon that certain material emits energy detectable as visible light when irradiated with the light of a specific wavelength. 

A suitable exiting light source is employed which passes through exiting filters. Four main types of light source are used, including xenon arc lamps or mercury-vapor lamps with an excitation filter,lasers, super continuum sources, and high-power LED's. 

The basic task of the fluorescence microscope is to let excitation light radiate the specimen and then sort out the much weaker emitted light to make up the image. First, the microscope has a filter that only lets through radiation with the desired wavelength that matches your fluorescing material. The radiation collides with the atoms in your specimen and electrons are excited to a higher energy level. When they relax to a lower level, they emit light.

To become visible, the emitted light is separated from the much brighter excitation light in a second filter. Here, the fact that the emitted light is of lower energy and has a longer wavelength is used. The fluorescing areas can be observed in the microscope and shine out against a dark background with high contrast.

How specific areas detected

If a fluorescent dye can be made to interact with specific cellular components - attached to an antibody that binds to a cellular protein, for example - then it can be used as a probe for microscopy. A specimen stained with this probe may be illuminated with pure, filtered light corresponding to its excitation wavelength and then viewed through an emission filter which is opaque to all other light except for its emission wavelength. The structures tagged with the fluorescent probe will appear to light up against a black background in a (hopefully) high contrast image.

Mounting Mediums

• Xylene or toluene solution of isopbutyl methcrylate is satisfactory mounting medium for temporary mounts.
• Liquid petroleum or glycerol
• Auramine stained with dry lenses

Fixatives

Formalin,alcohol, acetic acid Formalin, Regaud's fluid. the most common in use is Picric acid

What can you do with a fluorescence microscope?

Determine the localisation of specific (multiple) proteins

Determine the shape of organs, cells, intracellular structures  

Examine the dynamics of proteins

Study protein interactions or protein conformation

Examine the ion concentration etc.

 with love 
-Dixy

AGAROSE GEL ELECTROPHORESIS

Hello everyone,

Today I will be writing few important points about Agarose Gel Electrophoresis,  as this the basis technique employed in Microbiological laboratory .  All the student of Microbiology should know this technique , So are You all ready to learn about Agarose Gel Electrophoresis 

The term electrophoresis  describes a   migration of charged ions under  electric field. The charged particles of various biological molecules will migrate either  to the  cathode or to the anode depending on the nature  of  their net charge. The equipment  required  for electrophoresis  consist basically of  two items  a power pack and electrophoresis unit

A power pack supplies a direct current between the electrodes in the electrophoresis unit. Electrophoresis units are available for running  either vertical or horizontal gel systems.

Agarose is a linear polyacrylamide (average relative molecular mass about 12000)made up of the basic repeat unit agarobiose,which comprises alternating units of galactose and 3,6-anhydrogalactose

Agarose gels are made by suspending dry agarose in aqueous buffer (TAE),then boiling the mixture until a clear solution forms. This is poured and allow to cool at room temperature to form a rigid gel.The gelling properties are attributed to both inter -and intramolecular hydrogen bonding  within and between the long agarose chains. This cross-linked structure gives the gel good anticonvectional properties. The pour size in the gel is controlled by the initial concentration of agarose,the large pour sizes are formed from low concentrations and smaller pour size formed from higher concentrations.

Agarose is used in gel electrophoresis to separate nucleic acids (like DNA) by size, charge and other physical properties. Gel electrophoresis uses an electrical current to make particles move. For example, DNA is negative, so it will travel towards to positive electrode of the gel box.

Agarose has small pores through which a DNA can travel. Bigger fragments of DNA travel shorter distances, because it takes longer for them to navigate through the pores of the agarose gel. Identically sized pieces of DNA will travel the same distance, which is why we get bands (DNA with loading dye) after we run a gel

PROCEDURE

1)Pour the gel slowly into the tank,leave to set for at least 30 minutes, preferably 1 hour, with the lid on then add Ethidium Bromide.

2)Pour TAE buffer into the gel tank to submerge the gel to 2–5 mm depth. This is the running buffer

3)Add an appropriate amount of loading buffer with DNA sample which will be used as sample in this electrophoresis

4)Load the first well with marker,then other wells with different samples of DNA

5)Close the gel tank, switch on the power-source and run the gel at 5 V

6)Monitor the progress of the gel by reference to the marker dye

Advantage

Relatively inexpensive, non-toxic, does not cause the sample to denature, so at the end of the electrophoresis procedure, precious or limited samples can still be recovered and used for other experimental procedures. It can give very good resolution and separation of large from small molecules as the agarose gel's pore size can be specified by the user, and only requires small amounts of sample. The process is fast (minimum of 30 minutes), and the apparatus is easy to set up and operate.

Disadvantage

If there is an increase in temperature there is chances in genetic material can adopt the shapes which are not needed.If the molecules are very similar in size, then it may be difficult or impossible to resolve or separate them from each other. Quantification of the amount of a molecule by observing the size or intensity of fluorescence of a band may be difficult.

Applications

• Separation of RNA and protein molecules
• Forensic science
• DNA fingerprinting
• Recombinant DNA technology
• Cloning
• Used in southern and northern blotting

Incubator

Hello everyone,

Here I am with another  Instrumentation named Incubator

The microbiological incubator is deployed in research and industry in a wide variety of applications with living organisms.Profuse growth of microbes is obtained in the laboratory by growing them at suitable temperatures. This is done by inoculating the desired microbe into a suitable culture medium and then incubating it at the temperature optimum for its growth.

Incubation is done in an incubator , which maintains a constant temperature specifically suitable for the growth of a specific microbe. As most of the microbes pathogenic to man grow profusely at body temperature of normal human being (i.e. 37°C), the usual temperature of incubation is 37°C. The incubator maintains optimal temperature,humidity and other conditions such as the carbon dioxide (CO₂) and oxygen content of the atmosphere inside.The incubator maintains optimal temperature,humidity and other conditions such as the carbon dioxide (CO₂) and oxygen content of the atmosphere inside.

An incubator provides conditions for optimal growth of microbiology cultures. Natural or forced air convection maintains a constant, even temperature in a range from a few degrees above room temperature to as high as 100 °C. Microbiology incubators come in a wide range of sizes and shapes - even tiny benchtop incubators for microfuge tubes.

The incubator has a thermostat, which maintains a constant temperature, set according to requirement. The temperature reading on the thermostat is approximate. Accurate temperature can be seen on the thermometer fixed on the incubator. Exact temperature, as per requirement, is set by rotating the thermostat knob by trial and error and noting the temperature on the thermometer.

 They can be used for many applications beyond traditional bacterial culture, such as 

• Temperature dependent incubations,
• Growing cell cultures
• enzymatic reactions or for reagent storage. 
• Reproduction of germ colonies with subsequent germ count in the food industry
• Reproduction of germ colonies and subsequent determination of biochemical oxygen demand (wastewater monitoring)
• Reproduction of micro organisms such as bacteria, fungi, yeast or viruses
• Breeding of insects and hatching of eggs in zoology
• Controlled sample storage
• Growing of crystals/protein crystals
• Programmable controls and refrigeration are some of the additional features available in incubators on the market today. 

-Dixy

Media sterlization

Hello everyone,

You must have seen food get spoiled within 1-2 day  when kept outside. but why this happens ?

The most probable reason is presence of spoilage microorganisms in air . It is very important that such organism should not be present in medias , reagents etc , which can lead to error in analysis . hence all these materials  should be sterilized before use .

It is very important than every material used in microbiological laboratory whether it be water, media , glassware etc  should be sterile. Hence every microbiological laboratory contains an equipment known as AUTOCLAVE. So in this section I will be dealing with basic principle and working of Laboratory autoclave .

An autoclave is a pressure chamber is used to sterilize equipment and supplies by subjecting them to high pressure (15 atmosphere) steam at 121  °C for around 15-20 mins. Typically the chambers in autoclaves are cylindrical, because cylinders are better able to withstand extreme pressures than boxes, whose edges become points of weakness that can break.

 Principle of autoclave 

You've probably heard of pressure cookers? They were all common in use until microwave ovens became popular. They're like over-sized saucepans with lids that seal on tightly and, when you fill them with water, they produce lots of high-pressure steam that cooks your food more quickly .

Autoclaves work in a similar way, but they're typically used in a more extreme form of cooking: to kill all the microorganisms on things with steam long enough to sterilize them. 

You can try at home by boiling one potato in pressure cooker and other on open pan , and the interpret which cooks potato early, obviously the the answer is pressure cooker .

The extra pressure in an autoclave means that water boils at a temperature higher than its normal boiling point—roughly 20°C hotter—so it holds and carries more heat and kills microbes more effectively. A lengthy blast of high-pressure steam is much more effective at penetrating and sterilizing things than a quick wipe in ordinary hot water!

Working of autoclave :

most autoclaves contain a sterilizing chamber into which articles are placed and o steam jacket where steam is maintained. as steam flows from from steam jacket into steam chamber, cool air is forced out and a special valves increases the pressure above the normal pressure .The super heated water molecules conduct heat to microorganism.

Uses of autoclave :

In a microbiology lab an autoclave is used mainly to sterilize  medias, buffer , reagents etc. we can also sterilize small apparatus , certain glassware , heat resistant plastic containers , micro pipette tips etc.

Thank you,

Hope you understand 

with love 

-Dixy

Zeeman correction (Background error)

Heya everyone :D

So today I am talking about the Zeeman effect normally seen in absorption spectrometry.

Zeeman effect!! more like He man effect LOL.

Remember guys, He Man had a sword which was divided in half and when joint together became a true power sword.

Well in Zeeman effect the light passing from the source gets divided into 2 sigma and 1 pi component and when combined gives the true value of the absorbed light.

Area wise: 2 sigma components = 1 pi components.

Polarization : Sigma components are perpendicular to the magnetic field.

                      Pi component is parallel to magnetic field.

Hence this background error can be corrected based on the principle of polarization of light in the magnetic field. 

A magnetic field is superimposed on the radioactive source in method 1. As pi bands are absorbed by the analyte and background interference and sigma bands are absorbed only by the background interference, the difference between the two signals is the background correction.

In method 2, the magnetic field is superimposed on the cell containing analyte. The radiation first passes the rotating polarizer before entering the field the cell.The absorptive lines are split into pi and sigma bands. the pi bands have the same wavelength that of the emitted radiation. 

Therefore when radiation of parallel polarized light enters the cell, both the analyte and background interference absorb. But when the light is perpendicularly polarized,only the background interference absorbs. The difference between the two alternating signal is due to the analyte alone and thus gives the background corrected reading.

I know understanding this is a bit difficult, I have tried my best to simplify it though.

Hope it helps you all.

Lots of love

-Staph

Sodium Doedecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE)

Hello everyone , 

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 ,

-Dixy

Difference between TEM and SEM.

Here's a difference between Scanning and Transition Electron Microscope.

              SEM                                                                                               TEM
Based on scattered or back scattered electrons             Based on capturing the transmitted electrons
that hit the sample and revert back.                              once they enter the sample.

Gives information about the sample surface                 Gives information about the morphology,
and composition, elemental analysis, etc.                      crystallization, stress, nanoparticles, etc.

No specific sample preparation is required.                   Sample preparation is tedious as thin films are                                                                                         required

Low resolution and magnification                                  High resolution and magnification.

Large amount of sample can be analysed at a                 A very small amount samples is analysed at
time.                                                                                 a time.

Used for surfaces, powders, polished and etc-                Used for imaging of dislocations, tiny
hed micro-structures and chemical                                  precipitates, grain boundaries, etc.
segregations.

Images are seen on computer monitor.                             Images are seen on fluorescent screens.

Gives a 3D image.                                                             Gives a 2D image.

Hope it helps :D

Thanks

Lots of Love

-Staph