Exam Practical Biochemistry 2nd Year

Assalamualaikum warahmatullah
InsyaALLAH disini saya akan cuba mempermudah
cara2 utk mendetect and melakukan experiment
* Tapi ingat ini adalah tutorial semata2
dan jangan lupa juga membaca buku praktikal utk teori
kerana bnyak soalan mumkin keluar dari buku "biru cair" itu
dan jangan lupa juga utk membuat latihan dalam buku itu :D


Experiment 1 - titration

In Gastric juice, we have 3 types of acid
- Free Hcl (for free acidity)
- combined HCl
-Organic acid(all of them -total acidity)

Free acidity - Amount of 0.1 NaOH to combine with Free Acid (HCl) to neutralize the solution

STEPS
1. Put 5 ml (i think -according to the pipette) in the beaker
2. put 3 drop of topfer indicator (yg warna merah tu)
3. And put only One drop of Phenophtalein (this is what prof told me b4)
4. stir the mixture well
5. add one-by-one drop of 0.1 NaOH until salmon pink acquired
6. salmon pink = free acidity (pH 3)- take the reading (X1)
7. put more drop until the colour become pink again
8. Pink - Total acidity (pH 8)-take the reading (X2)

* Do the experiment twice - to take mean (if possible)

Comment on calculation
Free acidity = Mean X1 x (100/5) = .............. 0.1 N-NaOH %
Total acidity = Mean X2 x (100/5) = .............. 0.1 N-NaOH %



Ni adalah salmon pink yg disahkan oleh para doktor
tp dalam gambar ni x berapa jelas sebab lighting masa tangkap gambar haritu
x beberapa betul
kalau ikut gambar ni maybe 2 drop before ni adalah salmon pink

CANE NAK DAPAT WARNA PINK MCAM IKAN SALMON (salmon pink)?
-first skali bayangkan sushi yg selalu org jepun makan 2 (warna dia lebih kurang camtu la)
-Kalau x yakin sama ada pink @ salmon pink senang je
-->add one more drop
kalau bertukar jadi salmon pink alhamdulillah
kalau bertukar jd warna oren terus
kita tolak je 0.1ml dari reading (X1) kita
kan senang gitu
sama jugak dengan (X2)
gunakan taktik penipuan yg sama k


Inilah ikan salmon yg dikatakan merah-keorenan sket
(atas permintaan Angin bayu Iskandariah kot)
Sedap kan?
mmg sedap pun


Experiment 2 - Billirubin

Normal value
1. Total = 1 mg/dl blood
2. Direct = 0.25mg/dl blood

STEPS
For total Bilirubin
1. Add 1ml Total Bilirubin reagent
2.Add 50 microlitre sodium nitrate
3. Add serum 100microlitre SERUM

* Lepas tu sorok dalam poket @ mana2 tempat gelap (dalam laci) selama 10min
*pakai automatic pipette dengan cara yg betul
* jgn kene cahaya sgt, bg mendapatkan result yg tepat

Calculation
Total bilirubin = Absorbant of Sample x 14

Experiment Haemoglobin
Normal Value
Adult Male : 14-18 g/dl blood
Adult Female : 12-16 g/dl blood

STEPS
1. Add 2.5 ml Haemoglobin reagent
2. Add 10microlitre (i guess b'coz i forget-tp ikut automatic pipette yg disediakan) of sample
3. Store in the dark for 10 minutes
4. Find the reading

Calculation : (Absorbant of sample/Absorbant of standard) x conc. standard
= conc. haemoglobin

Experiment Total Lipid
1. 1ml of H2SO4 + 25 microlitre of plasma
2. Boil them until boil (apa punya ayat la nie)
3. After boiled + 1ml of vanillin phosphate
4. after 5-10 minutes in dark, the solution will turn into Pink (a little)
5. Find the conc. of total lipid using colorimetre

calculation = *same as usual (tgk kat atas)

Blood Haemolysis

-warna terang n cahaya boleh menusuk kedalam test tube
maybe due to
1. shaking
2. Change in pH
3.Increase in temperature
4. osmotic pressure (hypo @hypertonic)
5. Disturbance of membrane integrity (protein @ lipid)

How to prepare ?
1. addition with H2O
- High Osmotic pressure inside the cell will direct the water to move inside the cell
- causing Burst of the cells (Hypotonic)

2. add with urea solution (0.9%)
- Urea is freely permeable to membrane of the cell
-lead to increase in osmotic pressure inside the cell
-water comes in
- Burst of the cell

3. addition with NH4Cl
-NH4+ will enter the cell while Cl- are not..
- Lead to increase in osmotic pressure inside the cells
- Water comes in
-Burst of the cell

Non-Haemolisis

How to prepare ?

1. addition with Normal Saline
- Solution is isotonic, osmotic pressure outside n inside the cells are the same
- Rate of movement of water enter and leaving the tissure are the same

2. Addition with Sucrose (9%)
- Osmotic pressure inside and outside the cell are the same
- Due to high molecular weight of Sucrose (Number of molecule inside the cell)

3. Urea in Saline
- Saline is stabilizing agent
- as the urea move inside the cell, it increase the osmotic pressure inside the cell
- then it attract water to move inside the cell
- The saline act as stabilizing agent, pull again the water to move outside the cells
-so, haemolysis is not happened

Acid/alkaline haematin

How to prepare ?
Acid haematin - addition of HCl
- Acid cause denaturation of protein on membrane of the cell
- cause disintegration of membrane
-haemolysis

alkaline haematin - addition with NaOH (slow--> brown colour)
- sama macam acid haematin

Blood in Ether (cause haemolysis)
-due to disintegration of lipid part of the membrane
-by addition with ether

Blood in Saponine
- Haemolysis
-decrease the surface tension/disintegration of lipid part (just like bile salt)

BY USING SPECTROSCOPE

Oxyhaemoglobin
(haemoglobin yg oxigenated with O2 * bukan oxidized tau)
-Globin part will be normal
cane nak beze (haemochromogen)?
-kalau oxyhaemoglobin ada nampak 2 band sebelum gelap
-that's mean that dekat belah kiri sekali xde warna kuning (spectroscope)

Haemochromogen
-dari segi "naked eye" memang nampak macam oxyhaemoglobin
tp dalam spectroscope berbeza..
apa yg beze ?
ada nampak 3 bands (kalau oxy ada 2bands je)
-maksudnya nampak band warna kuning kat kiri sekali (ni apa yg kita nampak la)

How to prepare?
- 5ml alkaline oxyhaematin + sodium dithionite (reducing agent)

where we can found this haemochromogen ?
-very minimal amount inside mitochondria

Reducing Haemoglobin
Naked Eye - merah kebiru2an (kat dalam ni warna ungu sebab merah+biru=ungu)
-Hehe, bukan sebab ambil angle x betul (xblaja ngan Rahman D)
Haemoglobin warna Biru = That's why bila banyak reducing haemoglobin = cyanosis

How to prepare ?
5ml of oxyhaemoglobin + Few drop of Reducing Agent (Sodium dithionate)

Globin part = Normal

Alkaline Oxyhaematin
Naked eye - Beze sebenarnya ngan met-haemoglobin
- Dia jernih sikit dari met-haemoglobin (cahaya keimanan boleh tembus sikit2)
Kalau pakai spectroscope
= Nampak banyak gak la warna (bukan merah je)

How to Prepare ?
- 5ml alkaline alcohol (boiled) + 2 drops of blood

Globin part = Denatured (bacause boiled)

Met-haemoglobin
Naked eyes appearance - Gelap sangat (seperti malam yg menyinsing, betul ke?)

Spectroscope Appearance?
- Hanya warna merah kelihatan
- sebab frequency warna merah paling tinggi, so cahaya masih lg nampak
-macam pelangi la merah atas sekali sebab frequency...

How to Prepare ?
5ml of 1:10 diluted blood sample
(maybe 1 :20 / 1:100 xpe xpenting sgt pun ratio ni *kalau nak selamat amik 1:20 ikut dlm buku) + few drops of oxidizing agent (10% of potassium ferricyanide)

Applied Medicine
- occur when cardiac patient -taking nitrate(drug)


insyaALLAH ini je yg sempat dirakamkan
semoga bermanfaat

Jadi mintak jasa baik pembaca semua
SAMA-SAMA KITA DOAKAN saudara2 MUSLIM Falestin
semoga ditabahkan keimanan mereka

wallahu a'lam
-islahmujahidin-

"Tidakkah kamu memperhatikan bahawa
sesungguhnya ALLAH telah menciptakan langit dan bumi dengan haq(benar)?
Jika dia mengkehendaki nescaya dia membinasakan kamu
dan menggantikan kamu
dan mendatangkan makhluk yg baru(utk menggantikan kamu)."

"Dan demikian tidak sukar bagi ALLAH "

- surah Ibrahim 19-20