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I hope yesterday’s lecture was very inspiring, you were able to understand the basics. As we know today, we are going to discuss a little bit about research materials and methods in relation with research methodology all of you must have already passed your post graduation or some of you may already be in your PhD studies. So, you have gone through the process
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of
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these exciting synopsis writing
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research paper writing or at least you go through those things on a regular basis right. So, materials and methods seem to be quite a common subject, but we will try to incorporate some new features to it, maybe that will be helpful.
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Okay. So, let us start
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first thing,
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when you go through the format of any research article or be it thesis be it a synopsis Firstly, you have to go to introduction before that even you sometimes have to write abstract along with key words then you have to go for introduction, then comes materials and methods.
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So, this thing forms a very important part
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for getting the insight of the results whatever results you obtain that depends on how are you able to handle the materials and methods
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alright.
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So, studies methods are one of most important parts used to
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judge the overall quality of the people. So, even if you are sending your research paper for the publication purpose, the editor is firstly going to look into the material and methods parts, okay, after finding the relevance of this research paper with his or her journal, he’s going to identify
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how carefully you have
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undertaken the study okay. So, method section should give readers enough information so that they can repeat the experiments because same thing has been done by us as well we as a student as a researcher have already gone through some previous articles resources. So as to make our understanding firm about that subject of interest, we will have also use some of their protocols, we may have modified those a little bit as
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well. Right.
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So,
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we will just discuss few steps, how you can write an effective materials and methods in your research manuscript did
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a research paper be it a thesis, okay,
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same concepts apply to everything,
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especially the thesis part, because that is a bigger manuscript, and it has to be perfect in
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all regards.
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So, we will start with the general discussion and then we will make it
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specific.
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So, first, the very first thing is you should start collecting whatever data you are having regarding the materials and method before you start writing the thesis or the research paper in reality, for example, the data when you decide that this is the topic of your
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interest, you are going to work with this and the day you
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you start arranging for the experiment itself, you should start noting down the important points Okay, you can have a separate register for that where you can find out what has to be written this is going to save a lot of your time when you are ultimately writing the overall manuscript because whatever results you may get again that will depend on how carefully you have managed your experiments. What are controls which were being used, how you compare these two things, and everything counts believe me it is basically the details the finer details, which count whenever you look at the research papers of the best journal like nature and you
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compare the research journal of any
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recently started publication house, you will find the basic difference here lies in the details more detail paper you will find the major okay. So, try to grab all those details. For example, you should know the compositions exact compositions like if you’re going to make KH2PO4 buffer with ph 7.6 what should be the exact composition into water or HCL or whatever it may be, what is the
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person solution of HCL that
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was being used to add
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what gram of K2HPO4. So, these details you should know because whatever experiments you are going to perform each and every experiment is going to be different from the previous one
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in some or the other regards.
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Obviously, if this is the alpha amylase test and second one is beta glucosidase test obviously, even if the buffer is similar, it is not going to be exactly the same, because the activity of enzyme will depend on the surroundings All right. So, you should pay attention to individual compositions, you can get a register for yourself and start writing all these things for you. Then quantities same as composition in what quantity what thing was used was it 100
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micrograms or it was hundred milligrams. If you go through some
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low standard low quality papers, you will see that they do not give that complete details of materials and methods and it is somewhat very difficult for you to understand what exact procedure they have followed okay. So, in order to avoid that thing your own work,
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you should give proper attention Okay,
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along with that instrumental details models
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even like you can see in this particular
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experiment, he has also mentioned the
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size of dialysis tube which has been
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used
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he has given proper details about what
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percentage of available
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sodium hypochlorite solution was used it was 0.9% which was weight to volume not volume to volume you know na differences between weight to volume % and volume to
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volume percentages
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Okay, similarly exact timings
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with for example, you have collected this particular sample Suppose, you have collected the sample from certain trees. So, what was
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the time of the day maybe the
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timing of
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Suppose, this is the study where you want to isolate the microorganism from various trees and you want to find out how the microbial activity in this rhizospere is getting affected by its exposure to temperature change in pH on a rainy day or
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during the various phases of the day itself. So, you
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will have to give exact timing Also, if you are a student of zoology and you want to find out where the birds are coming,
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how they are coming. So,
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exact timings exact details you should know So, that there is no
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confusion with the ultimate results which you are applying okay.
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So, if you have to collect the pictures, collect all sort of pictures like you obtain the sample from this particular tree, get this picture of that tree because more like what you do for your Instagram picture or what you do for your Facebook picture, you take like five or 10 selfies out of which you select the best one do the same for your experiment. So, take all sort of pictures select the best out of those and then put it into your thesis then sometimes you can find
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the researchers also go for flow charts.
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First they start the principle of their
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experiment, then they give basic details about the experiment and sometimes they also make a very nice flow chart right. So, this is what makes it easy for the observer to understand what you are trying to say and how at which particular stage which reagent is taking part okay. So, you can take help of flow charts as well,
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then formulae should be clearly mentioned at the start itself
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like after each and every experiment, you should mention what is the formula for finding that particular result if there is a common formula for all those experiments, which you are explaining here, you can give that at the start and mention that using the above mentioned formula I calculated this particular result ok. So, same thing for three locations, I got it from this particular part of the village from this particular house or something exact biogeographical locations are also needed something for example, I got this sample from this particular mango tree which was located exactly at
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30 degree north
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altitude this
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longitude this latitude.
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So, all of these things can also be measured carefully.
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So, this will help you to
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prevent forgetting important details when ultimately you are going to write the overall manuscript like this is a good example how the author has given all the details of his or her expiry date you can see he has a told you the size was this this was the composition and what was the exact timing what was the exact temperature So, every detail he has tried to give along with proper references, okay, how it is not necessary that every time we are going to follow the reference step by step you may make certain modification. So, try to decide that that
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this particular
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reference was used with a bit of modification. And then after you can give your details like here you can see the sample was taken samples of shoot tissue were collected from a randomly selected healthy tree of SU Mini from Ludiana Punjab 30.9 degree North 75.85 degree east and this much elevation, but this gives the exact location also this is going to help the reader or the second person who is trying to follow your procedure from which location you got the sample and thereafter how you got the results. Okay. So, second step here becomes start with general information that applies to the entire manuscript. Like I said,
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if you are going to use only one formula for
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all those experiments, which are going to conduct mention that at the start or you can also get the list of all those results which you have used
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throughout your experiment at the start,
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you can give the list name of the chemical,
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the supplier, percentage purity and so many other things, especially it becomes important when you are submitting your thesis because then you will go for viva each and every member of your panel is going to ask you
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several questions from detail steps.
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So, if you are confident about the details, you will be confident that you know the procedure you will know the principle behind that and you will be easily able to defend yourself. So, that is why detailing is important. For example, characteristics of the study population suppose you are a student of biodiversity and you want or zoology and you want to find out the types of birds which are present in that particular region. So, you can go for that these were the characteristics of that bird which I was looking for into this particular region or community or village and this information can be given at the start itself because if again and again in each and every experiment you keep writing I got this bird from here I got this bird from here it is going to cause confusion and it will be repeatability. So, it may not be accepted in a very good quality journal
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okay like the
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sources genotypes of bacterial stains or description of samples or sample sites like if you’re collecting suppose hundred trees or hundred thousand specimens from hundred different spaces, make a general table at the start itself serial number 123 400 and name of the sample name of the city or village exact sample size and all those things you can put it here then you can start being specific about your experiments.
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There comes the third point
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match the order in which methods are described to the order of results that was so, this comes into force when you are really writing your complete manuscript when you have already record the results. So, what you can do here first, you find out what what the results are there because you may do experiments in a seriess, but you may not get results in that particular series. So, in that first particular series support you
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let me give you an answer like you are having
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10 types of microorganisms which you obtain and out of which you want to look for the presence of phosphates or solublization, the presence of zinc mobilization and so many things okay, our resistance mechanism. So, if you are going to publish a paper using this data, don’t give details of this thing or this thing
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automatically you will get to arrange
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your materials and methods with regard to the results which are going to apply okay. And also be sure that each method you use is described even if it is a weak sentence like I was giving you the example here. Yeah, you can see this one it says he has told you that the mode of inhibition by EAEP was conducted using the modified method described by this particular researcher Okay, then he has given complete details Okay, even if it is taking around
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250 words
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or even if it is consuming more space, he has given all the details. Now, you can see here it is EAEP we do not know what is this, but the author must have cited this EAEP at the start it says okay. So, this practice is helpful for
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transparency as well as reproducibility. So, if there is someone who wants to
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take your reference and go for this, he can reproduce the results easily. fourth point is always include citations for procedures that has been described previously like this, this is the citation obviously, this you are going to give at the back in the form of bibliography or references right. Now, that will depend on the type of Institute or the type of journal but should be the exact
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format of that reference but that is a separate thing.
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So, if you are going to
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keep details of all those processes, at the start of the
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experiment itself, it is going to
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help you later on for example, here you can see the name is Kazim et al suppose there is some one similar suppose it is Kabir et al and also the year seems to be same.
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So, you may get confused later on.
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But initially if you are having the details along with the rest of the procedure, you will not get confused and reference will not get missed. because later on whenever someone is going through your manuscript be the thesis be it sybopsis be it research paper or review article, they are going to look the first thing that all the references that have been cited at the back should also be present in the
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text. So, these references you will not
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miss out okay.
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Then, statistical details also are very important. For example, if you just say that t test was performed, the person will not understand
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okay. So, if you are using some particular
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software as well for that t test
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, you can
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also tell him that thing you can properly mention that software and you can also write what was the origin of this software suppose you are doing your PhD in some particular university that a statistical department made itself have made a software for these type of tests, which is very common nowadays, okay. So, you can cite from where you obtain this software and how did you proceed with it. So, you can make a flowchart here then avoid discussing the pros and cons of methods, because discussion part should be taken for discussion section only. So, just give the details of the materials and methods and protocols
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give the pictures
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sometimes it is recommended that you give the picture of spectro photo meter or electron microscope it says it is somewhat sometimes demanded otherwise it may not be demanded
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okay. So, it depends on the
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advisor, the student the committee.
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So, you can put
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all those details, but don’t go for discussion even if you find certain research papers, who have compared to different methods
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for the same
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sample, they are not going to give you the details of the comparison in material and method sectionn. For example, there is a person who is trying to look for the
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This is your Oh yes. So, if there is a person who is trying to
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assess his protein or you can say cell structure, this is a particular type of cell which he has obtained and he wants to see the structure of cell in details using suppose sem and TEM.
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So, what
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he is going to do is going to give a title abstract keywords basic introduction and then he will go for Materials and methods,
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where he will tell you
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how he proceeded with the sample isolation then how he conducted sem, where did he conduct what type of model of sem was used, he can also provide you with the flowchart of the steps then he will go for TEM in the same way, but the discussion part will remain there for either the result and discussion portion or sometimes a separate result and separate discussion. Okay. So, you have to discuss ultimate results over there itself, where you can also compare it with some previous researches by some other researchers, okay. So, next step is to save the space by
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in order to save the space,
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first thing what you can do is do the listings make the tables right for example, if you’re putting out a research paper for publication, there is always a word
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limit. So, you have to give each and every detail but you also
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have to go for
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minimum number of words. Again, if it is a paid journal, based on the number of pages, they are also going to charge you okay. So, to save the space be concise, give all the details, like in this particular thing you can see within one line he has given so many details, so, try to give all the details, but don’t repeat something. So, to cause that what you can do, you can and list all the things all the materials for those particular experiments at the start of material and Methods section is you must have seen in the research papers after the title material and methods they give two or three lines about the chemical those sorts of chemicals like it is hi media merck or they also give the list okay. So, that thing can be
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because that will prevent repeatability.
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Then there comes few other points it should be clear from the method section how all the data in the results section were obtainede and I said if the first step here in the result is the isolation of microorganisms. So, first step in the material and method should also be isolation of microorganisms or at least it should be second step after the first step which should be source of trees from where you obtain your microorganisms and then you isolated correct similarly
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the study system should be clearly described
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give all the specifications and make it also the controls should be included. Now, how many of you have done PCR polymerase chain reaction regular routine practice in the labs? So, tell me what different types of controls Do you go for during the
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PCR reactions?
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What are the type of PCR controls that you have undertaken? Now the thing is let me give you a brief about the procedure
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just like in any the action
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PCR reaction also should be given controls
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Okay, let me give you an example of again. rhizobium, you know rhizobium is the one which carries out nitrogen fixation in plants right. So, rhizobium. So, this is
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supposed the rhizobium species which you have
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obtained from some test organism okay. So, from some tests species you have got your
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rhizobium organism
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and you want to find out that its DNA is similar or dissimilar to standard positive rhizobium
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leguminosum
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DNA or not with respect to nod region which is responsible for nodule formation okay. So, you are having performing a test into the test you have
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obtained a rhizobium specie a rhizobium
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culture from some unknown plant or suppose soil sample or some organism maybe okay. So, this you have obtained and you want to look for it nod genes okay via PC
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r multiplication.
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So,
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first thing you will do is you will take a positive control
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okay. So, positive control always take that particular sample which you are sure about that it is going to have that particular gene for example, rhizobium leguminosarum is definitely going to have nod gene right. So, in one of the PCR tubes you will multiply its DNA ok.
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So, the test as well as positive
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control they will be using
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same or different primers
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they will use a more different primers,
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different primers, now listen No Why
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should we go for different primers
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we should not go for different primers because
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the same reason we are trying to look for into these two species So, we are going to check for the presence of nod region. So, primary should also be specific to nod region only na and it is also rhizobium which is also rhizobium we just don’t know whether it is leguminosarum or not. So, species same that means same primers can go for this as well and this as well.
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So, we are going to take same
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set of primers for test as well as positive template okay and also it will show us that this is also rhizobium leguminosarum and your test sample is also rhizobium leguminosarum, if it turns out to give you positive test
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then you have negative control
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where
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other than rhizobium any specie you can be for example, you can go for a streptococcus which is also a microorganism, but that does not give rise to nodule formation. So, again you’re going to take same set of primer,
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which is not going to bind here and it is not going to give any result. So, it is going to give you the negative control for the identification then you have an amplification control. So, what does amplification control does? It is going to tell you whether the PCR situations are sufficient
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to carry out the
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replication process or not.
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Again you can go for
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any random target like you can again take the streptococcus DNA and put the primers which are not nod specific any primers you just put
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and check
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if streptococcus is growing or not like you can go for the housekeeping genes obviously, housekeeping jeans are going to be there in the streptococcus genome and if you give primers corresponding
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to that it is going to get multiplied okay
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if the reaction conditions if the PCR conditions are good enough Okay, then there is something called reagent blank reagent blank is something which is having only regular PCR ingredients like magnesium ions taq polymerase then your nucleotides and other things okay, but there should be no template it is going to tell you that if it is going to give rise to any type of result, any multiplication that means you are PCR, reagent buffer reaction conditions are contaminated and you should not consider any of the above mentioned results because we have not given any template
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but it is still getting multiplied. So, what is getting multiplied
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it is the contamination okay.
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So, you can
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you also have to give the
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primers, the controls, you also have to mention the controls properly the outcomes of the study should be defined you should know before the start of the reaction what should be my results. So, those outcomes you should know
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the outcome measure should be objectively validated that
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just like in this case, if you are getting results in this case also that means your sample is contaminated. So, you should objectively validate across several controls and with several other Test Options, the method used to analyze the data must be statistically sound as I said it should have minimum standard deviation. Now, the standard deviation is something which you should determine based on again those tests statistical tests, but how do you find out what is the appropriate statistical data what is the appropriate standard deviation which is considerable for example, online one hand you are having a result then hundred milligram sample is getting produced on the other hand you are having a clock which is giving rise to hundred quintles of grain So, what should be standard deviation here and why should we standard deviation here obviously since the quantity is small here standard deviation also needs to be smaller for example,
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hundred plus minus
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1.2 milligram
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here
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hundred quintile It is so it can be plus minus one quintil
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you do get me So, depending on the size of
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data you we’re getting standard deviation will also vary it because here if we’re going to give the result of hundred quintal plus minus 0.1 Quintal
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that is going to make your advisor suspicious
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that he has manipulated the results okay. So, also as I said previously, citations should be given in the desired format by that particular institution or that particular research journal all materials and instruments should be identified including the suppliers name and locations like hi media Mumbai okay. Few more tips include only what is necessary for only for one repeating the experiment to know but without the kind of unnecessary details that break the flow of write as I said, if you are telling that this is the plant that was obtained from this location, so, give that data at the start itself not after every experiment, not in every experiment, you should start telling. So, this was a microorganism that I obtained from this plant that was obtained from that particular site that is not required, instead at the start you can give a table give the location give the name of the plant and make a subtitle make a shorter name for this like as one as two or anything like this, okay.
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So, materials and
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equipment utilized during the experiment should be mentioned throughout the procedure as they are used. So,
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by this what he means is every time you should tell that
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this is alpha amylase test, this is alpha glucosidase test. So, here also I used this particular buffer here also I use this particular buffer, but here the pH was this much different here the pH was this much. So,
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the buffer composition you can give at the start,
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but buffer smaller detailing should be given at the middle. Also, like this is a spectrophoto meter which we’re going to use. So, at the start
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itself you can tell this is that particular spectro photo
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meter from that company, this is that particular
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model, but
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in the middle it self during the process itself you should keep telling
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the spectrophotometer was being used.
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So, finally, it is generally recommended at materials and methods section should be written in past tense okay. So, you you have already done the research you know that it has already been done. So, the result should also be in past tense
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Okay. Now, this was about the general
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materials and methods
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Okay, I hope these things are clear to you know,
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now, but more we need to know for example, you are student of microbiology or zoology or some other basic science, you need to classify the thing that you are having or even you are a student of
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biochemistry you have obtained a new protein
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again you need to classify it
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based on its structure based on based on it functions. So, classification and taxonomy forms a very important part during the materials and methods section itself. Now, you should know what is classification matters taxonomy taxonomy is something that is given rise by the classification. So, suppose you are having four different samples a one a two a three nd a four okay. So, based on certain criteria like hair color,
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like skin color
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or shape off here or whatever, maybe
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you are classifying them as
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different organisms. So, that is classification, but after this classification, how many groups have they formed? That is taxonomy.
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So, classification is evolved mechanism by which organisms are
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organized by taxonomy what we mean it is a branch of
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biology that names and
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groups organism according to similar characteristics. So, they have found four different groups suppose there is a fifth organism which is having similarity to this one. So, it will go into its or its taxa or domain or Kingdom phylum what ever
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So,
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classification is arrangement of supposed bacteria into various groups based on stereotypes anti microbial resistance or whatever you want to study nomenclature is the naming like we say physum sativum all bacillus subtilis. So, this is the nomenclature which is mainly binomial genus and strain genus and species after species there comes strain so, this is usually genus and species Pisum is genus sativum is species
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okay then there is sometimes one more thing like
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Okay lensculinaris
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and then it comes something like medik
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I say this is genus, this is species, What is this thing?
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What is medicus here Anyone
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sometime it is like crl sometime it is like LINNAE I’m giving you the hintsYes, it is not actually author it is actually the scientists who named it like Karlus manias medicus is also the name of the scientists actually, it is a big name, I think it was a Russian scientists, so they are having big names. So, this is the name of the scientist who had given this particular classification okay. So, this is not author and this is also not strain. So, first comes Genus then species then comes subspecies or they may be strain Okay. Then identification is of practical use to distinguish certain organisms from others, which gives rise to classification, nomenclature and ultimately the next one is science of classification, identification, nomenclature and making of a whole system okay. So, how do we do the classification? Like if any one of you is a student of microbiology he would go for bergis is manual right? But going through bergis manual is going to be a tedious task until unless you are having a software for that right. So, there are certain things called taxonomy
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taxonomy keys, do you know anything about these
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a simple and useful tool
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that was previously manual, but now again this automatically using these software’s You can find these software’s online. Okay. So a simple and one of the most useful tools available to scientists trying to identify an unknown organism, taxonomy keys are useful tools guiding researchers to whats the known name of an organism. So, this is an unknown organism, considering its various features and compare it with all those organisms, you will come to know to which organism does it belong. However, all taxonomy keys are not created equally. For example, we see that all the plants of the Great Lakes or argentinian mono, Argentinian monocot, but that doesn’t make any sense Instead, it is important to pick all those qualities which you want to find them. So, let me give you an example like I was telling these other different software’s which are available. So, the tools for getting these software’s are available on databases like this, you can say this is a database file bought from this particular University, you have to create username and password and then you can check your newly found organisms, based on already collected information in those databases. Like you have this one which is called expert three previously use or expert to So, all of these are having databases, which are having information from various scientists various sources, which you can use to compare your sample. Now, how are you going to look into each and every sample individually each and every research individually, so data bases Are there it is automated as well, like let me just explain a little bit. So, this is an identification key either print based or computer aided devices are there, obviously print based is going to be hectic, so Computer Aided automated systems are better like these software’s So, you can identify any type of organisms or any types of things even because you are having details okay. So, these are the certain keys keys keys to a lock okay. So, there are two basic keys dichotomous key and polychates . So, both of them are having their different strategies while in case of dichotomous keys what you do you get two options and based on two options, you compare yoursample with it and then you go for either one of them. However, in case of poly chaetes what you do, you take your samples, you take your sample, and you compare it with a lot of different informations about several different samples, okay. So, if you find this characteristic similar to this one, you discard these two and then you proceed with this similar type of identification, let me explain like dichotomous key as the name suggests two things are there, okay. So, it always gives two mutually exclusive choices in parallel statements, which allows the user to determine the identity of items using a sequence of alternative choices. For example, here you see you are having a suppose bean? So, you will look for the seeds, if the seed matches with round one the answer to your So, the question is that your seed is a soyabean seed well, if it does not match the round seed criteria, then the second criterion as well. So, if the seed is oblong again, you are having two choices, if they are white, then they can be northern beans or if they are black, they can be black. So, you have two options round or have long, if it is round, it is soybean if it is oblong, again, you are having two choices, either white seeds or black seeds, it is white seed your answer is your sample is northern
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similarly,
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so that you can see here, something is written as couplet, what is a couplet a pair of standard statements which is seed round seed oblong, that is called couplet and each half of the couplet is a lead. So, this is a lead and this is the second lead. So, it is leading you to some results. So it is a lead Okay, at each couplet of a dichotomous key the user is presented with two choices about a specific character present in the group of organisms right here in this lead, this is the character in this lead, this is the character. So, sometimes the characters are quantitative for example, measurements suppose 50 kgs 30 kgs, something like this units and sometimes the characters are qualitative, like oblong seed round seed white seed black seed. So, these are couplets and leads now what is this numerical key and alphabetical key when you write it like one two, then it is numerical, if you write it like a B, and it becomes alphabetical and the thing is, do not ever go for the answers based on only one observation, even if you are carrying off any lab experiment, you at least go for duplicate or teiplicates right. So, apply the same criteria always select a lot of seeds or lot of samples from your population and then try each and every key and get the result and doctors. So, based on that thing, as I said you can have new numerical key for the dichotomous keys or you can alphabetical keys as dichotomous. Similarly, you can also have other types like pictorial key where pictures are given and you can look at the pictures and come here they can be box type of keys where boxes are given like monera is having no nucleus protesta having nucleus plant is having autotrophicc mode and Annamalia and fungi are having heterotrophic mode. So, these are single cells, these are more than one cell these are mobile and these are immobile or you can have circular type of keys or you can have branching type of keys you can have these which are also having brackets or you can have any key which is not having any bracket it is one it is to it is three it is4 head is 1234. But again this one then for 2this first this second, first second and so on okay, so, there are various options indented is simply space a tab has been given. So, based on the looks, dichotomous key can be having either of these types and all these keys you can find online using those things.okay
49:31
Then
49:33
it is explaining the same thing as a user makes a choice about a particular characteristic of an organism, he or she is led to a new branch or a new couplet of the key each couplet provides characteristics that become progressively more specific until the final step is reached and identification is reached okay like here it was only two step here it can be multi step like once cell two cell autotrophic heterotrophic mobile im mobile.okay
50:05
like this
50:07
followed correctly keys will lead you to the correct name of an organism or object. Similarly, dichotomous keys can be developed to identify anything and any sort of classification. That means if you are a researcher who has years of experience on to the same thing, suppose you are a microbiologist and you are regularly working on the microorganisms So, you can also develop your own keys because it is also possible that when you are working in some particular environment, you may get those microorganisms which are not present any where else so, you have to develop a key for yourself, you need to devise your own. So that can also be developed based on similar manners. So, as I was saying second type is quality of keys, which is involving the process of eliminations like these are the tools which are used to help identify unknown objects or species just like previous keys these keys are generated using interactive computer programs. So, these are also so the user is presented with a series of choices that describe features of the species that they wish to identify for example, you are having a flower now, you will have to judge this flower again based on several criteria, but
51:36
suppose you are having a flower Now, first thing is select for flower character
51:45
now, there may be a lot of keys given at this step think red, yellow orange, you choose the flower color, suppose your flower color is yellow. So you will select this one and these three will be rejected
52:03
okay then write down the possible pollination system. So, you right here yellow
52:11
then you write second thing what type of pollination is present is it with the help of bee is it with the help of birds or some other source or maybe it is the water or maybe air So, then you will have to find out what type of pollination system your flower is looking for. Suppose it is via bees then you will search some other feature whether it gives you nector or not yes or no. So, nectar yes or no then similarly, you will keep on going for more than one characters for your sample one after the other and together they will give you the result that is the dandelion is there which is yellow in color and it is bee polinated. However, in the previous case, each observation was associated with something else here all the observations are separate from each other right. Like here we saw it is a seed which is either round or oblong if it is round then it is soyabean but if it is oblong then you have two choices, but here you just have two choices out of which you keep on selecting okay. So, basically for dichotomous you have to go in a series here all the observations are separate this or this this or this this or this this or this, you select this this this or this based on those four observations, what is the sample. So, again, the user is presented with a series of choices that describes features of the species they want to identify the user then checks off a list of characteristic present in the organism they wish to study, the program looks to match those characteristic with all the species they can possibly match like here I am just giving you two or three options, but there may be rain there may be air there maybe anything else. So, if a species does not have that character state, it is eliminated from the list the more characters states listed the most species that are eliminated okay. So, I was giving you only the example of one you can have multiple samples at once and you can keep checking them all togetther it is more like a high throughput mechanism, multiple options, multiple samples all okay. So, this thing continues until only one species is left if all went well and the keys fit your group of organisms that is the name of this species you have located even the best keys have their limitations. So, make sure you verify your identification using multiple tools. For example, like first thing what you do here is you are having four species ABCDOkay, now, what you want to do you want to first check them all for Flower colour. Now, these are the four species which you have to compare with your specimen is yellow in color right. So, first you have to check for the species which is yellow in colour okay it is red blue, green, yellow. So, these three are me right. So, this D may be one of its possible specie options, then you go for flower signs suppose this is now 1234 next set of species with which you are going to compare it. So, if first species is the one which is having large size second is even larger, third is small fourth is even smaller. So, if this is also large and this is also large then this one matches with this one and these three are eliminated So, second option you put here is first option. First is D second is one for these two features match with this, maybe these two species are similar to each other, Are you getting me or not. So, you have to look for a number of different species during polycle and compare their
57:06
characteristics with the characteristics of your own specimen there comes another important concept this was only about taxonomic classification. Now, whatt is the size of a sample that should be checked it should not be very small it should not be very large a small sample is not considerable, when it is not sufficiently powered to detect a difference between the groups and the study may turn out to be false in negative suppose okayso, I’m saying that this is a city into the city, but what do you want to find out you want to find out what is the major of occupation of the citizens. So, if you are taking sample only from this population and not from these regions, so, that is not going to give you a fair idea of the of the occupation of all the citizens in this whole big city small sample size is not going to be valuable here instead you should take if you are going to take only small sample size again You should go for this region this region this region this region this region and this region. So at least six samples should be collected from similar from similar dimensions about five meters square hear five meters square here and five meters okay, but a very large sample is also not recommended sometimes because sometimes if a small sample is giving you enough information, why should you ask for a bigger sample? Why should you up for so much wastage of time and money right then, if you are a physician and you are trying to look for volunteers, that means more and more subjects you will try to take out suppose there is a disease suppose there is a okay somebody there is a doctor who is trying to look for alternative therapy for cancer suppose there 100 people hundred volunteers are coming to Him To find out the treatment. So, what he does, he’s just randomly dividing them into 10 groups of 10 each and he is trying 10 different doses or 10 different treatments on all those 10 . The thing is if you are taking only 10 people or five people that is a small group, okay, if one of the treatment does not go well of course, those one group of people which may be five or 10 people, they may face side effects, at least you will be able to help at least those 10 people now, but if you’re going to have a sample size of suppose 500 people for each group, if someone if that dose is lethal, all of them are going to die, how are you going to tackle the situation right. So, in those conditions, very large sample size is not recommended. Similarly,
1:00:32
okay.
1:00:34
Similarly, like the same situation to take is if you are taking 10 people in each group and one or two treatments are not working fine. Maybe later on you can shift the same patients to some other Those are some other drug that will do, but there are 500 people and they are treated in the same way their drug is not working properly. So, they will have faith issues they will lose faith in you and they will consider that you are treating them as objects okay. So, lower population size you can somehow managed but large population size large sample size you will not be able to manage in this particular situation like
1:01:22
Okay, so, we will quickly go across certain methods, how you carry out the
1:01:30
protocols for isolation identification of microorganisms, insects, animals and how you manage all that microbiology is the study of microorganisms which may be algae, fungi virus bacteria, right. So, how are they growing?
1:01:52
Like you can have either
1:01:56
isolation from this some particular sample maybe soil on you may be having air or you may have to test water or some liquid. So, similar protocols are adopted I’m giving you very basic idea like there are I said you are having viruses but viruses cannot be directly cultivated. So, I’m not taking them into consideration, but you are having bacteria which grow well nutrients you are having fungi like aspergillus nigeriawhich grow on PDF which is potato dextrose agar then there is sabrode agar which is good for yeast like Candida okay. So, what you do whatever be the medium be the sample be it soil air or water ultimately, if we’re trying to look for the presence of overall bacterial size, overall fungal size and overall candidal size you can go for a general protocol again what you will do like let me compare the three methods you are going to first make three types of activities obviously, you are going to make them in triplicates only, so three plates of this three plates of this three plates of this for yeast dilution and how are we going to make the dilutions if your sample is soil you are going to take 0.1 gram of soiland put it into saline solution or whatever buffer solution that is required or it may be water itself sometimes okay. So, that is going to give you a dilution of 10 days to power minus two why 10 raised to minus two because already it was one gram It was 0.1 gram which is equivalent to one into 10 rasised to power minus one right when you put it into another 10 ml of sample it is going to be one into 10 raised to power minus two sample that from that ten raised to minus two again, you are going to take 0.1ml and put it into 9.9 that is going to give you 10 raised t power minus four again you’re going to take 0.19 ml from ten raised to minus four and put it into 9.9 m I of water saline solution or buffer that is going to give rise to ten raised to minus six dilutionOkay, this is the case of soil. Similarly, you can take liquid sample 0.1 ml, which is already one into 10ten raised to power minus one dilution, you put 0.1 ml into 9.9 ml to make total 10 ml of the solution which is going to have 10 raised to power minus two concentration of your liquid. Similarly, again you are going to go for ten raised to power minus four to ten raised to power minus six dilutions okay. So, overall you are going to make the dilutions of up to ten raised to power minus six. And
1:05:20
you are going to put individual dilution
1:05:26
in the form of 0.1 ml solution to three different types of petriplates PDA agar, nutrient agar, sabrode agar That means, if you are having ten raised to minus six dilution, that means you’re having 10 ml of this out of this 10 ml you’re going to take 0.6 ml into nine right. So, you are going to take 0.1 ml and put it into three petriplate so 0.1 here 0.1 here 0.1 here of PDA P NA 0.1 mlhere for a second replicationof NA and third replication 0.1 here for sabrode 0.1 ML for second sabrode and 0.1 ML Of third sabrode so out of 10 raised to minus six dilution you are going to make nine plates for bacteria three for fungi and three for Candida Okay, say you do here same you can go for ten raised to power minus four and ten raised to power minus two and you should know what are the outcomes what should be the number of colonies which you are going to count now, if I state generally in general a plate should not be having more than 300 colonies of bacteria to count otherwise it is rejected okay then why is it discarded is washed away or simply thrown away simply similarly a plate should not have more than 300 colonies of Candida because Candida also looks like bacteria only it is very small pin drop size colony dew drop type of colony this is then for yeast also for the fungus you should have not more than 30 colonies per plate standard Plate I amtalking about you have seen Petri plates I suppose that I am talking about So, obviously after closing the lids you have to incubate it there and look for the results the next day or the third day. So, similar things happen for your sample of water as well or liquid as well. Now, if you want to go for air samples what you have to do again make the triplicates triplicate plates for these three and when the agar is solidified then open the dish in that particular environment in which you want to check microbial load okay for 20 minutes allow the microorganisms to sit on to the surface after that you just cover the lid put it in an inverted
1:08:18
position into the
1:08:21
incubators BOD at 37 degree which is going to provide it appropriate humidity appropriate temperature and within 24 hours you will get count of bacteria and within 48 to 72 hours yeast and fungi will be clearly visible in fact yeast also sometime close within 24 hours okay by what is bacteria and what is the plural of bacteria
1:08:49
what is the plural of bacteria
1:08:57
no bacteria is itself plural
1:09:01
bacterium is the singular when you have one type of thing that is bacterium one cell plural is itself bacteria same confusion is there with media medium is singular media is plural okay
1:09:28
Okay. So, this is how you can isolate. Now, obviously after isolation you’re going to get a lot of different types of micro organisms on the surface of petri plate. So, you will have to distinguish on your own based on other taxonomic keys which we have discussed previously or whatever information you are already having right. Suppose this is the one that you desire pick it up in the presence of sterilize condition which is laminar airflow in front of Bunsen burner using sterilized loop and streak it on to another plate where only one type of colony will grow from one typical cell. So, that is the purification
1:10:11
later on
1:10:12
that can be transported to low temperature conditions like refrigeration in a tube or at low temperature freezer or in liquid nitrogen or you can also go for lyophilization okay
1:10:39
. Let me just complete this one
1:10:44
Just let me just explain this one rest we will discuss tomorrow along with the next topic okay.
1:10:52
So, I was going to discuss this
1:10:55
how do you culture microorganisms
1:11:01
culture part first you have seen the isolation part from soil water and air then I told you you can store it here there and their culturing is again getting multiple copies of the same
1:11:18
right, So, you can see that whatever
1:11:20
culture is there, when you are transferring it from one plate to another place or one plate to a test tube or test tube to plate, you have to prevent contamination for which you are having standard protocols, then genetic changes also have to be prevented like mutation, you should not keep basically placing it under the UV light right. Then along with all these things, you can also store your sample in
1:11:54
the form of dry spores
1:11:56
do you know what is a dry spore?
1:11:59
So, reproduction and survival both are assisted by spores. So, in the form of spores also you can store them like fungus can be stored actinobacteri and bacteria can be stored and then mineral oil like you are having a slant into which you are having a culture of microorganisms. So, over this you pour the sterilize mineral oil
1:12:24
up to hour
1:12:27
support this is the end of your slant here to here it should be one centimeter
1:12:34
above one centimeter
1:12:37
to the slant height, you should pour the sterlized mineral oil and then you can put
1:12:42
it there into the low freezer liquid nitrogen it self okay and there is something called lyophilizer Have you gone through this lyophilization is freeze drying where suppose this is the sample This is a sample of your microorganism or anything else that is put into a round bottom flask suppose it is 200 ml culture how it is 200 ml culture suppose you are having a sample which is Present in petri dish you want to mass multiply it. So, in the petri dish it was growing on solid medium, but then you will transfer it to liquid media
1:13:07
suppose 200 ml medium is there
1:13:23
into which you put the sample and allow it to grow under sterilize conditions for few days.
1:13:33
it is very common nowadays. So, you should try to use better equipments and instruments that will give you a hand on expertise and that will also be added to your
1:13:44
CV. So, that may help you later on during your research career or maybe Academic carriers itself.
1:13:52
So, you are getting a suppose 200 ml culture you directly put this culture into
1:14:02
the water. So, you put
1:14:07
this sample suppose it’s 200 sample along with 200 ml sample you also mixed 200 ml off your
1:14:16
ok leave this part I will just give you this thing
1:14:20
suppose this is your sample which is 200 ml out of which you want to take out only the bacteria.
1:14:29
So, what you can do you just
1:14:30
directly put this sample here in the round bottom flask
1:14:34
and put put it fix it
1:14:37
tightly to the lyophilizer where the temperature is really low at least minus 130 degree Celsius is there. So, whatever liquid is there it will ultimately go for
1:14:49
drying actually
1:14:52
liquid will get converted to water will get converted to ice and because of the sucking action of lyophilizer all that ice will be sublimated to gas from solid to liquid condition it will get converted solid to gass condition it will get converted and that gas will be set up by this instrument apparatus itself. So, you will be left with only the cells those cells which is basically the spores can be store in this manner okay.
1:15:30
So,
1:15:33
okay also you can so, this is called lyophilization or freeze drying other than that you can go for spray drying that is you take your sample and spray it on to sterilize surface where your sample is undergoing dryness because of the air okay and then you collect the specimen which is inside there is something alsocalled fluid bed drying
1:16:01
which is complicated to explain So,
1:16:03
I have given a link here this is a YouTube
1:16:06
video which will show you how fluid bed drying helps to save the cultured microorganism to preserve
1:16:16
the cultured microorganism
1:16:17
okay. So all of these are preservation techniques at this you can check the fluid by drying here
1:16:25
using the same okay so
1:16:27
since we are out of time I think I should stop it here itself.
1:16:34
Okay, then Okay, then see you tomorrow. Good evening.