Wednesday, April 25, 2012

Viva Voce #2

As I said in my previous post Viva Voce #1, my university practical exams are going on, out of the 4 practicals, now I completed 3 :) Today was a day of E.coli, as I got E.coli with all experiments :)
Microbio is my favorite favorite subject, and the lab, I love to be in microbio lab with my microbial buddies :) :P Here we go with my microbiology practical exam :)

I prepared well for my lab exam yesterday night. I made myself clear with all the concepts behind lab techniques and experiments. Had good confidence :) as this is my favorite lab :)
I was given with a question paper saying:

Major experiment: 
Inoculate 10% of E.coli culture in nutrient broth of ph5 and check the growth rate by measuring turbidity and plot a graph.

Minor experiment: 
Perform gram staining of the given culture, observe shape, color and interpret the results.

:) :) :)))) I got a biggggggggg smile when I saw this easy question paper and started with my experiment :)
I cleaned and sterilized the laminar air hood, took my pH5 nutrient broth, cuvettes, pipette. For accounting the turbidity spectrophotometer is used generally by us. So, I took blank as the broth with out any inoculation in a cuvette and I inoculated the broth with given E.coli culture.

After inoculation, I took that inoculated broth in a cuvette for taking OD at 0 minute. I kept the broth flask in shaker at 37 degree celsius. Oh, gosh... :( I forgot to take two blanks :( I realised this only when I started towards the spec :(  What to do.. I was blinking for a minute, then I checked the cupboard for sterile nutrient broth of pH5, to my goodness I found one more flask of sterile pH5 nutrient broth. (Again I got a biggg smile...)

I took another blank from that sterile broth :) and took O.D. I took O.D of the inoculated broth for every 30 minutes, say at 30,60,90,120,150 minutes.

E.coli normally grows at optimum pH of 7. So, in my pH 5 broth, I expected that the growth would decrease with time. But, to my surprise it increased. Growth was increasing with time. I was confused :(
Then, side by side, in the gap of 30 minutes in taking O.D, I completed gram staining too. I was given with E.coli for Gram staining too. Got, beautiful pink rods :) interpreted that E.coli is a gram negative, hence it stains pink :)  ( What a great experiment :P :P )

In between the external examiner called, "Register number 05..."
oo.. I'm that 05.. I rushed to him with my answer sheets. He got my sheets and started probing it.. He started shooting questions then, Here we go with my Viva voce #2

External examiner: Your getting increased growth with time, do you think, optimum pH of E.coli is 5?
Me: No sir, optimum pH of E.coli is 7.

External examiner: Then, your getting increased growth? Justify this? 
Me: (Thought for a while) Sir, it might be due to contamination with some other species which grows at pH5.
( He was convinced with this answer)
E.coli - Gram staining

External examiner: List some preservation techniques?
Me: Stab culture, Glycerol stock, Lyophilisation.

External examiner: What's the purpose of Safranin in Gram staining?
Me: It is a counter stain. Gram positive retains crystal violet while decorising with ethanol, while Gram negative loses it. So, to give a contrast , Safranin is used. Gram positives won't take up safranin.

External examiner: So.. Crystal violet is a basic dye or acidic?
Me: It's a basic dye, sir.

External examiner: Why a basic dye for staining bacteria? why not an acidic?
Me: Sir, normally bacteria have negative charge, so, using an acidic dye which is also negative charged, there will be repulsion and there won't be staining.

External examiner: okay, that's it. you may go.

He was satisfied with my answers :)  An easy Viva voce once again :)

Then I continued with my experiment, got increase of growth, drawn graph and gave inference as " The increase in growth might be due to contamination of the culture with other species which grows at pH5"

And we were given with some 4 questions:
Here are my 4 questions:

1) Interpret the spotter 1. ( Mam gave an EMB - Eosin methylene blue agar plate) 
My interpretation:
The given plate is an EMB - Eosin methylene blue agar plate - divided into two halves.
In one half, there was growth showing metallic zinc color - indicating that it was E.coli culture.
In another half no growth was obtained, indicating that half was inoculated with a gram positive bacteria.
Principle: EMB agar is a selective media, it allows only gram negatives to grow and E.coli gives metallic zinc with EMB agar.

2) How will you prepare 1ml of 1:5 dilution culture? 
Ans: 0.2 ml of culture in 0.8 ml of broth

3) You are isolating 5 different S.aureus cultures, whether it is necessary to check susceptibility for all the five? Why?
Ans: It is necessary to check susceptibility for all the 5, because one may have resistance and other may not have (  when Isolated from different sources)

4) A simple calculation for calculating phenol coefficient was given. I did it right. I don't remember the numbers given, so, I share just the formula: 

Phenol coefficient = (Greatest dilution of disinfectant that kills microbe in 10 mins but not 5 mins) /
(Greatest dilution of phenol that kills microbe in 10 mins but not 5 mins)

Finished successfully and submitted my paper, came out with satisfaction :) :) :)
Just one more practical exam tomorrow, just it is Communication skills practicals! :) :) Yaaaiii.. yaaii.. did well in all the practicals :) Have a good time :) :)) 

Tuesday, April 24, 2012

Viva voce #1

Hello! Here I go with my practical experience :) I'm doing my university practical examination this week. Completed 2 out of 4 practicals :) My mood got spoiled in my lab because of the moody chart!
 But, successfully, did the viva voce  good for the 2 exams :) I shared some of my viva voce questions with answers as it might help someone searching for some answers (as I do every time, the day before exam)

Chemical Engineering laboratory:
I was given with a question :

" Draw Moody chart for fluid flow through non circular pipe" with some specifications of pipe diameter.

I got shocked, as I don't know what's a Moody chart! :( :( Non circular pipe?!!! Still again a great confusion!

But, I managed to guess what was that non circular pipe! It is nothing but, "Annulus". We use to say " Fluid flow through annulus" , so, I got little confused with the twisted question! Then, the moody chart.. I didn't bother about that. I did the experiment, drawn graph between friction factor and reynolds number as we did in our previous lab classes. But, to my goodness, I was right, Moody chart is nothing but, the plot between Friction factor and reynolds number! :D :D With out knowing that, I did it right! :D :P

 External Examiner :  What's the application of flow meters in biotechnology industry?
Me: For knowing and adjusting the behavior of nutrient broths or other solutions while they are transported via pipes.

 External Examiner: Which heat ex-changer type is efficient and why?
Me: Counter flow - as the temperature difference is maintained, efficient heat exchange takes place. whereas in parallel flow, the temperature difference gets reduced as the fluid reaches the end of the pipe, hence there will be no efficient heat transfer. ( Temperature difference is the main criteria for heat exchange )

 External Examiner: In fluidised bed, when porosity increases, what happens to pressure drop? 
Me: Pressure drop decreases. When there is more space for the fluid to flow freely, there will be no hinderance for the fluid flow, hence, there will be no drop in pressure. ( I thought for a minute and reasoned like this, I don't know whether I was right :P )

 External Examiner: Application of heat exchanger in biotechnology?
Me: Sterilisation and cooling during fermentation.

 External Examiner: Partition coefficient in liquid-liquid extraction? Discharge coefficient? 
Me:  Partition coefficent is ratio between amount of solute in extract to amount of solute in raffinate.
Discharge coefficient in head flow meters is the ratio between experimental discharge to theoretical discharge.

Hoping to get good grade - the highest "s" grade - above 90% :) :D :)

Instrumental Methods of Analysis: 

Question I got,

Major experiment: Estimation of sulphate concentation.
Minor experiment: Determination of pKa value of p- nitrophenol.

I enjoyed doing this, as we took the readings for the standard of sulphate estimation in groups :D :) Only for the unknowns we took the readings individually :)
pKa value determination is very simple using titrations and pH meter :) Got pKa value as 9.3 for
p-nitrophenol ( the exact value we gor during our previous lab classes )  Viva voce was also simple :) as follows :

 External Examiner: What's the principle behind this sulphate estimation?

Me: Based on the turbidity developed, readings are taken in terms of NTU for standard as well as unknown. By plotting graph, we could determine the concentration of unknown.

 External Examiner: Why nephlometer for this estimation why not spectrophotometer? 
Me: Spectrophotometer is generally preferred for non -turbid solutions. Nephlometer is specifically used for turbid solutions. Hence, we use nephlometer.

External Examiner: significance of pKa? 
Me: At the pKa, the compound will be more stable. to be short, pH at which the compound remains stable or the compound has more stability.

 External Examiner: What kind of components could be separated using TLC? 
Me: Colored pigments, oils and components with variation in molecular weight.

 External Examiner: Colored pigments could be examined easily after separation, but, how you will examine the separation of oils? they are not colored? 
Me: Using Visualizing agents like iodine vapor.

 External Examiner: Other possible methods for visualizing? 
Me: I don't know sir, (but, now I found one another method) If the sample contains any chromophore substance, it could be identified by visualizing under UV rays for sometime. It will develop color.

Successfully completed my 2 lab exams with good satisfaction. Hoping for "s" in both labs :) :D :P

Note: I don't know whether the above answers are right, but, I always believe that I would be right for at least 80 % .  :) :) (Confidence boss.. confidence.. :P ) If your reading this for your educational purpose, just cross check my answers. I might go wrong at times.
Have a good time :) 

Saturday, April 21, 2012

Aluminium and my day

I got interest with this aluminium :) I ran for half a day for finding this Aluminium sulphate container in my department. Let me explain the lovely experiment with Aluminium and alizarine :)

( Don't continue reading if your not interested in biotech or chemicals, this is a damn boring post)

Lowermost detection limit of Aluminium-Alizarine complex. 
Aluminium Can :) :) :D :P

An experiment, we did in our IMA (Instrumental Methods of Analysis). A sooper dooper experiment which I loved. This is because, me and one of my friend did it together while all others were doing record works.
Mam explained me the procedure, I explained my friend the procedure and we did the experiment.
I feel happy most times, when I'm provided with the responsibility. :) :D :P

Initially, I found  very difficult to understand the principle behind the experiment, but, mam made it clear by explaining twice :) The principle is very simple as follows:

Alizarine red dye and aluminium sulphate when mixed together, aluminium absorbs alizarine red dye and hence the intensity of the dye gets reduced, therefore, the OD(Optical density) after addition of aluminium to the solution will be less comparing the OD of pure alizarine red dye.

We tried using two series of solutions. One series of test tubes containing only alizarine red and another set containing alizarine red and aluminium sulphate i.e., testing with and without alumiium, to check the absorption of aluminium. We checked the absorption by decrease in OD value (using Spectrophotometer)
First, we tried for different concentrations of alizarine red (1,2,3,4,5 ml), keeping aluminium concentration as constant (0.1 ml).
We found that absorption was good with 3ml of alizarine. So, next we tried varying aluminium concentration (0.025ml, 0.050 ml, 0.075, 0.1 ml, 0.125 ml) with fixed alizarine red concentration (3ml). We found that the absorption was very good from 0.1 ml onwards. Below that, 0.025ml, 0.050ml, 0.075 ml didn't give decrease in OD ( indicating no absorption).

We calculated the amount of aluminium alone in 0.1 ml of aluminium sulphate and it was 34.5 micro-grams.
Approximately 34.5 micrograms of aluminium absorbs 3 to 4 micrograms of alizarine red dye (calculated from our experiment)


34.5 micro-grams of Al was the lowermost detection limit by using Al- Alizarine red complex method.

Why this experiment?
Because calculating the amount of Al is very important in the industrial waste. Hence depending on the absorption of alizarine red ( the reverse of the above mentioned experiment) by our sample, the amount of aluminium present in the sample can be easily detected. But, the minimum amount of Al must be above 34.5 micro-grams to use this detection technique.

I'm very happy with this experiment, I enjoyed doing this one, but, I don't know the reason for the happiness, may be I was made responsible to do this experiment :) :D :P

After a long week... gonna have a cool sunday in hot sivakasi :) Good night tata.. bye.. :) 

Friday, April 6, 2012

Hero villan

Every story has a hero and a villan (enemy). My story too have them. I love my hero, he is too smart and acts smart and good. But, this villan, he always troubles me, annoys me :(

You wanna know who are they? If you imagined hero as a guy and villan as another guy, am sorry, as usual, I meant it related with my subject. The hero is antibody and villan is antigen :D :D :D

My dad told me a story when I was too young ( hope I was doing 3rd class ) I got an wound in my right leg, I got fever because of the pain :( So, I didn't attend my classes that day. I was lying in my bed and dad was cleaning  and  dressing my wound. I was wearing a gown , it had a rope near the hip portion , I was chewing that rope.

Dad started telling a story , " appaa kutty (dad's pet) , you should not chew your dress , your dress might have lots of germs, they are all villans (enemies) and they will fight against the soldiers in your body.

I started wondering and asked him , " apaa (dad) whether my body has soldiers??!! " .
Dad said " yes, your body has little soldiers, they will assemble near your wound and fight against the villans, the medicine you take in will support your soldiers, the medicine is also like soldiers, but, if you chew your dress, germs will get inside your body, they will go and support the villans, then your wound will get more worse, so, don't bite, okay?"

As an innocent kid, I nodded and was imagining little little soldiers with weapons in their hands fighting against villans :) :)

But, now, I know, the heroes are the antibodies, villans are the antigens :) Now, I know things better than my dad , and I started explaining him stories of immunology  and drug resistances :)

A short note on drug resistance : " Take your medicines as prescribed by your doctor "

Whenever you take some course of medicine, do your course complete. When your doctor prescribes you some medicine and ask you take for a week, take it for a week. You may feel better within 3 days, because, your medicine might have destroyed 99% of your disease causing germs. So, you will stop taking your medicine by the 3rd day. But, after that, the remaining 1% will start multiplying, and, again it will make you diseased. Then, you will start blaming your doctor, and take your medicines once again. But, your medicines won't work this time that much quickly. Because, your disease causing microbes now recognized the mechanism of your medicine and it became resistant to your medicine. So, even when you take the same medicine after that, it won't affect your microbes. You have to again try with different medicine. 

That's why, doctors suggest you to take your medicine course completely. Tuberculosis(TB) causing Mycobacterium tuberculosis has developed resistance against several drugs, only because the patients stopped taking drugs when they felt little relieved (but, they are not cured completely). So, take your medicine course completely as per your doctor's advice. If you drop in the middle, then your medicines can't work over your microbe. You have to wait for a new medicine then. "Share this info with your friends and spread awareness. Never forget to follow your doctor's advice"

Hope, I made it clear? Got my point?

I started gaining interest with Immunology :)
How brilliant these microbes are!!! Very smart...! Gonna read much on Immunology :)
Gonna do something against this bad smart microbes :) :D :P  Okay  Taataa.. (Bye..)