Friday, August 30, 2013

Testing for Presence of Biological Molecules - Carbohydrates, Lipids, Protein, and Nucleic Acids

CARBOHYDRATE TESTS:

1) Benedict's Reagent - ONLY tests for "reducing sugars" like glucose, galactose, and fructose. Will NOT detect non-reducing sugars like sucrose. This reagent must be mixed with the solution you are testing and then HEATED in a hot water bath for about 3 minutes--enough time to produce a visible color change IF the solution contains even a small concentration of reducing sugar(s).

Your NEGATIVE CONTROLS (-) should be: (a) DI water and/or (b) sucrose solution. DI water can be your negative control for any of the following tests, since we know pure water does not contain carbohydrates, lipids, protein, or nucleic acids (DNA, RNA). However, a BETTER negative control for Benedict's Reagent would be a sucrose solution, as sucrose is NOT a reducing sugar, and thus should NOT produce a positive indication (a color change) when the Benedict's Reagent is heated.

Your POSITIVE CONTROL (+) should be a pure glucose solution (or a solution containing a different reducing sugar).

COLOR CHANGE INDICATION (when the solution tests "positive" for reducing sugars): green (low content), orange (moderate content), red (high content), brown (very high content).


2) Iodine (i.e. IKI, I2KI, or Lugol's Iodine. Gram's Iodine will also work, but it tends to be a more expensive reagent) - ONLY detects starch (strings of glucose monomers joined together). Iodine will NOT detect individual glucose monomers.

Your NEGATIVE CONTROLS (-) should be: (a) DI water (deionized water) and/or (b) a pure glucose solution.

Your POSITIVE CONTROL (+) should be a pure starch solution.

COLOR CHANGE INDICATION (when the solution tests "positive" for starch): brown.



PROTEIN TEST:

Biuret Reagent - ONLY detects protein (amino acid chains). The Biuret Reagent WILL NOT detect individual amino acids (monomers of proteins/polypeptides).

Your NEGATIVE CONTROLS (-) should be: (a) DI water and/or (b) a pure amino acid solution.

Your POSITIVE CONTROL (+) should be a pure protein solution.

COLOR CHANGE INDICATION (when the solution tests "positive" for protein): purple or black.



LIPID TEST:

Sudan III or Sudan IV - ONLY detects lipids (i.e. fats, oils, triglycerides).

Your NEGATIVE CONTROLS (-) should be: (a) DI water

Your POSITIVE CONTROLS (+) should be: (a) pure lipid/fat/oil MIXED WITH a little bit of soap/detergent (this helps to prevent separation of any water content in the Sudan III/IV Reagent from the lipid/fat/oil), (b) whole or reduced-fat milk (emulsified fat).

COLOR CHANGE INDICATION (when the solution tests "positive" for lipids/fats/oils): red.



NUCLEIC ACID TEST:

Dische diphenylamine test - ONLY detects DNA.Will NOT detect RNA. Requires heating/boiling then cooling (ice bath).

Your NEGATIVE CONTROLS (-) should be: (a) DI water and/or (b) pure RNA solution.

Your POSITIVE CONTROL (+) should be a pure DNA solution.

 COLOR CHANGE INDICATION (when the solution tests "positive" for DNA): blue.


For photographic examples of these tests, go here.


Friday, August 23, 2013

How Do I Measure Liquid Volumes? Proper Lab Equipment Choice

In your basic biology lab (college-level course), it is very likely that you'll be required to know how to use various common tools used to measure volumes of liquids.

Rule 1: If the tool isn't calibrated (if it is the kind of tool that needs calibration), then your measurements are NOT RELIABLE (won't be accurate).

BAD equipment for measuring accurate and precise volumes of liquid:

These are ACTUALLY TERRIBLE FOR MEASURING ACCURATE AND PRECISE VOLUMES OF LIQUID (but are very useful in other functions!)

a) Beakers - usually made of glass or plastic. DON'T USE THIS when you need to measure an accurate and precise volume of liquid. DO use a beaker for storing or mixing things together (when appropriate) or when the measurement of volume does not have to be very exact (like how you would use a measuring cup for baking).




b) Erlenmeyer flask - same problem/use as the beaker (see above).




VERSUS

GOOD tools/glassware for measuring accurate and precise volumes of liquid:

For large-ish volumes (1 mL - 1000 mL):

a) Graduated cylinder - usually glass or plastic. Pretty good for measuring large-ish volumes (around 1 mL - 1000 mL) of liquid. BE SURE to avoid the "paralax error" by reading off the volume of liquid in the graduated cylinder at the "meniscus". A graduated cylinder is nice when you need to measure a bunch of different "something" milliliter volumes, like 5mL, 7 mL, and 9mL, for example.

 

b) Volumetric flask - glass. Great for measuring EXACTLY ONE volume of liquid--that is, a single volumetric flask is designed for measuring only one specific volume, such as 5 mL, 100 mL, 1000 mL (1 Liter), etc. Wonderful for mixing/swirling components of a solution together, as well. With the volumetric flask, also read the "meniscus" as it coincides as closely as possible with the single line on the neck of the flask that indicates the "one specific volume" that flask measures.

http://www.monashscientific.com.au/FlaskVolumetric.jpg


For smaller volumes (1 mL or less):

c) Pipette - there are many subtypes, but they tend to be bulb and Pasteur pipettes (in old-school chemistry labs or classes), serological and mohr pipettes (graduated pipettes that are very similar to each other and are used in modern labs and some classes), or volumetric pipettes (modern labs and some classes). Pipettes of any kind usually require some kind of pipette "aid", as in some apparatus or add-on tool that helps the user to suck up/expel liquid. In the old days, people pipetted by mouth instead of using a pipette "aid"--and just so you know, pipetting by mouth is a super bad idea because you might inhale or swallow part of your experiment, which is dangerous, expensive, and anti-scientific. Just don't do it! Use a pipette aid.

 Bulb pipettes: 

 
Pasteur pipette (with balloon bulb)




 

d) Micropipette - lots of varieties due to different manufacturers, but they all do the same thing: allow you to measure and/or transfer insanely small volumes of liquid. Most labs have micropipettes that measure volumes in MICROLITERS. 1 microliter is so small, it's less than 1 "drop" of liquid (think of a single rain drop). Micropipettes come in variety of subtypes based on what range of "small volume" it is intended for measuring.


http://img1.tradeget.com/Satnam2Enterprise%5C3I566SKE1micro_pipette.jpg







Wednesday, July 31, 2013

ABO/Rh Blood Typing - A Simple Guide

Visual Example of an ABO/Rh blood test:




Quick explanation of ABO/Rh blood type testing: To understand how this kind of blood typing test works, you will need to (a) understand what antibodies are and how they function and (b) what agglutination is. The photo above shows a slide with 4 SEPARATE blood drops (from the same person). EACH of these four blood drops has been mixed with a liquid containing ONE antibody type (which are different for each blood drop).

Background:

(a) What is an antibody and HOW does it function in ABO/Rh blood typing?

Antibodies are Y-shaped proteins that are, as you probably know, important players in the immune system. In the immune system, antibodies "flag" foreign-looking molecules (called "antigens"). This flagging helps to signal ("recruit") certain other immune cells to destroy the foreign-looking thing, because that foreign-looking thing might be toxic, or be attached to something toxic/harmful.

Antibodies have a special physical property: they can be made in "batches" that are extremely specific to only be able to "flag" ONE kind of antigen (some small molecule), such as those on the surface of red blood cells in humans. In blood typing, we can take advantage of this physical "specificity" property that antibodies have. The common antigens found on red blood cells in humans are "A" antigens, "B" antigens, and Rh factor antigens. We can MAKE antibodies to EACH of these antigens, respectively, and use them for blood typing. (See Link 1 below for video and further details).


(b) What is agglutination?

 *To be added*


What is the basic set-up for blood typing?

The basic set-up FOR EACH DROP OF BLOOD is this: drop of blood from ONE person + ONE drop of liquid containing one type of antibody that only sticks to a certain, known antigen. For example, let's consider the first liquid drop in the photo, starting from the left (i.e. the drop labeled "anti-A"). What, specifically, is in that first liquid drop? This "anti-A" labeled liquid drop contains two things: drop of blood (hence, red blood cells) + antibodies that are *specifically* AGAINST human "A" antigen (little molecules that can be found sticking out in many places on the cell surface of red blood cells of people who have either type A blood or type AB blood).

 The story about agglutination here is that the anti-A antibody will ONLY "stick" to human "A" antigens that this antibody "sees"/runs into when type A or type AB blood is mixed with a liquid solution full of anti-A antibodies. This "sticking" of anti-A antibody to "A" antigen on the surface of red blood cells is another way of saying a direct agglutination reaction is occurring. In the case of blood typing, a common class of antibody called IgM is used because this class of antibody has a shape that allows it to "stick to" many red blood cells at the same time, thus bringing about VERY fast clumping/agglutination reaction if the correct antigen ("A" in this case) is present that corresponds to the antibody (in case, anti-A antibody). Thus, if no "A" antigens are present on the red blood cells in the presence of anti-A antibody, NO agglutination reaction will occur.


In short, if the blood typing set-up is what it normally is: drop of blood + ONE type of antibody to a certain, known antigen (e.g."A" antigen, "B" antigen, or "Rh" factor/antigen), then NO agglutination reaction (i.e. NO clumping) observed means that the person DOES NOT have THAT antigen (i.e. the antigen that the antibody is specifically "against", that is, "able to stick to") on the surface of their red blood cells. However, if clumping IS observed, then the person DOES have that antigen present on the surface of their red blood cells.  

For your flash cards/study guide:

YES clumping = blood sample HAS the antigen. 
NO clumping = blood sample DOES NOT have the antigen

What do I to determine blood type from the 4 blood drops each mixed with a different antibody?

So, determining clumping (agglutination) or not is pretty easy--it's either YES or NO. There is either no in-between in the case of blood typing using antibodies.

IF the blood sample CLUMPS in the presence of "anti-A" antibody, you know the person has the "A" antigen. But that's it, you still don't know if they are type A or type AB blood if you haven't looked at the result for the "anti-B" antibody reaction with their blood. On the other hand, IF the person's blood sample does NOT CLUMP in the presence of "anti-A" antibody, then FOR SURE you can say that the person does NOT have type A blood, NOR do they have AB blood--however, you don't know their definitive blood type yet because they could be EITHER type B or type O. You still need to look at the reaction of their blood in the presence of the "anti-B" antibody. As you can see, you can't blood type using just one type of antibody, you must use all three antibody types used in blood typing: "anti-A" antibody, "anti-B" antibody, and "anti-Rh" antibody.

IF the blood sample clumps in the presence of "anti-B" antibody, then you know the person has the "B" antigen. If you DIDN'T already get the result from the "anti-A" antibody test, then you'll only be able to say that the person's blood is either type B or type AB. If you DID already do the "anti-A" antibody test, and the person's blood HAD CLUMPING in the presence of "anti-A" antibody, then you can determine their ABO blood type (but not the Rh factor--yet, i.e. you can't say whether they are "positive" or "negative" for Rh at this point). For example, if the person's blood CLUMPED in the presence of "anti-A" antibody AND CLUMPED in the presence of "anti-B" antibody (again, these are separate reactions--one never mixes two different kinds of antibodies together for any step of blood typing), then the blood is type AB. However, if there was clumping in the presence of "anti-A" antibody but NO CLUMPING in the presence of "anti-B" antibody, then the blood is type A. The reverse of the last example follows logically: if the blood did NOT clump in the presence of "anti-A" antibody, but DID CLUMP in the presence of "anti-B" antibody, then the person's blood is type B.

What if there is NO clumping in the presence of "anti-A" antibody AND NO CLUMPING in the presence of "anti-B" antibody? Well, then that blood type is type O, because type O blood means the red blood cells have NO antigens, thus there is physically NOTHING for the "anti-A" antibody to stick to, just as there is NOTHING for the "anti-B" antibody to stick to.


I know my ABO type (A, B, AB, or O), but is my blood type A/B/AB/O "positive" or "negative"? This is a question of determining Rh factor.

IF the blood sample CLUMPS in the presence of "anti-Rh" antibody, then the blood is Rh POSITIVE (Rh +).

IF the blood sample does NOT CLUMP in the presence of "anti-Rh" antibody, then the blood is Rh NEGATIVE (Rh -).

When "reporting" one's FULL ABO/Rh blood type, write the ABO blood type (A, B, AB, or O) and then a superscript of either "+" or "-" to indicate either the presence (+) of Rh factor or the absence (-) of Rh factor.


Why is the Rh factor important for medical purposes?

Rh factor is important because of blood transfusions, some organ transplants, and pregnancies in Rh - women.

 Rh factor is another type of antigen that determines blood type (overall blood type), and it's important because just like the ABO blood-type matching for blood transfusions, the Rh factor can cause potentially fatal problems for the blood recipient if there is a mismatch. That is, it is a potentially problematic mismatch to transfuse a person with type A (for example) Rh+ (positive) blood to a person with type A Rh- (negative) blood. The problem arises from the fact that the person with Rh - blood may have an immune system that has already made natural, permanent, circulating antibodies to the Rh factor (where Rh "positive" indicates the "presence" of the Rh factor or "antigen" on the surface of red blood cells in that person's blood). In the case of this Rh factor transfusion mismatch, the recipient's "anti-Rh" antibodies would attack the donor's red blood cells due to the presence of the Rh factor/antigen on the surface of the donor red blood cells, which are seen (from the recipient's immune system's perspective) as "foreign", and thus must be flagged for destruction by "anti-Rh" antibodies. This leads to a transfusion reaction in which the recipient's Rh antibodies stick to the just-donated red blood cells the recipient desperately needs, resulting in agglutination and eventual destruction by other immune cells (thus causing "suffocation" at the cellular level through the recipient's progressive loss of oxygen-carrying red blood cells)--if the recipient doesn't FIRST die from the blocking-off of blood vessels all over their body due to the agglutination reaction brought about by the Rh incompatibility in the blood transfusion. On the other hand, it is perfectly safe to transfuse JUST the red blood cells of the a donor with type A Rh - blood into a recipient with type A Rh+ blood. This is safe because the recipient's immune system "doesn't care" if the donor's red blood cells LACK the Rh factor (antigen). Transfusing blood from a donor with type A Rh+ to a recipient with type A Rh+ is safe, of course, BECAUSE the recipient, by virtue of HAVING the Rh factor (antigen) present on their OWN red blood cells, has NO antibodies to Rh factor--thus, there is no possibility of a transfusion reaction based on the Rh factor.

Besides blood transfusions, organ transplants, etc, the other medical implication of Rh factor erythroblastosis fetalis, or the "hemolytic disease of the newborn".


Links:

1. A cute, concise explanation of ABO/Rh blood typing (~15 minute video):
ABO/Rh blood typing explanations easier to process when a proper analogy is used. Here is a link to a great video, that, albeit a bit cheesy, is very appropriate for those who want to learn the fundamentals about ABO/Rh blood typing for the first time.

2. Agglutination of red blood cells by IgM antibodies

3. ABO blood type table