Class started immediately today without any briefing about the project and Dr Wan asking about the bacteria that Fadhly (also known as Choy) had shared in our group's Edmodo which is about Bacillus thuringiensis (Bt).
Bacillus thuringiensis is a natural bacterial disease of insects. Bt has an unusual properties which some of Bt strains produce proteins that kill certain insects with alkaline digestive tracts. Thus, it is widely used as the main active ingredients in insecticides to kill insects.
And the class went on with the remaining of Topic 2 Microscopy which covers on the preparation for light microscopy. The smear-steps involve preparing smears, fixation and staining.
Kindly note that fixation is important so that we do not break the internal or external structures of the cell. Fixation consists of two steps where firstly the organism is killed and next, it is preserved. This can be done in two ways which is heat fixing where the overall structure is kept preserved but not the internal structures. And another method is by chemical fixing where it is used for more delicate organisms.
After we're done 'fixing' it, it's time to stain!
They are three types of staining :
- Simple staining
- Differential staining (Gram staining, acid-fast staining)
- Special staining (Negative staining, endospore staining, flagella staining)
Before we proceed on the three types of staining, it is important to know what is stains or dyes. And why we have to use dyes? Why can't we use hmm simple things like.... water colour? Hehe. That would look so unprofessional though ʕʘ̅͜ʘ̅ʔ or it's just not suitable, scientifically. I guess. (///◔(ｪ)◔///) However, Dr Wan told us that the functions of dyes are they help give colour for the staining process, easier to identify the structure, bind to the cell wall (that's why the organism is coloured/stained!!) and help to highlight the internal structure (like I mentioned before that some organelles are colourless and need to be stained in order to be observable when using the microscope).
But before that, though we know what dyes are used for, do you know what actually this thing called "DYE" is? （・θ・） <-- that's a bird.
Dyes are salts composed of a (+) and a (-) ion, one of which is colourful (which is called "chromophore")
--->> Chromophore is part of molecule that's responsible for its colour. The colour arises when the molecule absorb a certain amount of wavelength of visible light and reflects others.
Dr Wan told the class today that the difference between simple staining and differential staining is simple staining requires only one staining agent while Gram staining requires two staining agent which is crystal violet and safranin.
However, other difference that I found out based on the net and netizens is that simple staining tends to colour all the organism the same colour while differential stain (Gram staining is included under differential stain, remember~) is used to spot difference between microorganisms. Thus, since Gram staining is a differential staining, it is able to determine which is gram positive and gram negative bacteria while if we use simple staining, since it would be in the same colour, the difference couldn't be detected.
GRAM STAINING
As the lesson about the Gram staining went on, Dr Wan asked us what must we do for bacteria, as if what test must we do when handling a bacteria. Firstly! Most importantly! We isolate the bacteria first. Next, do Gram staining on the bacteria! This step is important in order to be able to classify the bacteria into their specific characteristics,that is, based on their cell wall structures.
And next, Chew was the one who explained to the class how is the procedure for Gram staining. Chew did a good job explaining it though! :D
From what I understand is Gram positive bacteria tend to have thicker peptidoglycan cell wall. And when the Gram positive bacteria is decolourise with the alcohol, the thick peptidoglycan cell wall is able to retain the crystal violet (the primary stain) molecule. But the truth is it is because the decolourizer actually dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate the tightened peptidoglycan layer, thus they're trapped in the cell of Gram positive bacteria. The violet/purple colour remains.
On the other hand, Gram negative bacteria with a relatively thinner peptidoglycan cell wall is degraded when added with decolourizer, hence it is unable to retain the crystal violet-iodine complex and the colour is lost. No violet colour for Gram negative bacteria.
Lastly, when the Gram positive and Gram negative bacteria is stained with safranin, as a counterstain. Gram positive bacteria is not affected by it because safranin is lighter than crystal violet, hence the red colour of safranin does not disrupt the violet/purple colour. Note that safranin is a weak water soluble molecule. As for the Gram negative bacteria, it is stained red when added with safranin.
ACID-FAST STAINING
Acid-fast staining is used on bacteria that are fastidious or like Dr Wan said very pussy. ⁀⊙﹏☉⁀ An example of a fastidious bacteria is Neisseria gonorrhoeae because it requires blood or haemoglobin and several amino acids and vitamins in order to grow.
While Gram staining used crystal violet stain, iodine as the decolourizer and safranin as the counterstain, acid-fast staining used Carbol Fuchsin and phenol, acid alcohol and methylene blue.
An acid fast organisms will be stained red while a not acid fast organisms will stained be stained methylene blue.
As for SPECIAL STAINING, there are negative staining to detect colourless bacteria. The capsules will become colourless instead of the body of the bacteria because capsule repels stain thus they appear colourless against a stained background.
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| Negative stain of rotavirus (most common virus causing severe diarrhea in infants and young children) |
FUN FACT (Though it's a bit scary for me, but it's fun, in a way, to know about this)
The helicobacter pylori usually causes ulcers and commonly found in the stomach. This bacteria infect humans due to the bacteria's shape and the way they move allow them to penetrate the stomach's protective mucous lining, where they will produce substances that weaken the lining and make the stomach more affected to damage from gastric acids.The bacteria can also attach to cells of the stomach, causing stomach inflammation and can stimulate the production of excess stomach acid. Over time, infection with the bacteria can also increase the risk of stomach cancer.
One of the way to detect the presence of helicobacter pylori is by staining it with Warthin Starry's Silver Method where the bacteria is stained black against a dark-yellow background.
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| A section of a stomach with Helicobacter pylori infection. |
Other than negative staining, there is also endospore staining which is used to differentiate organisms that can produce endospore and those that cannot. Spores have a resistant outer coating that is composed of the protein keratin. Keratin resists staining. Thus, in order to stain a spore with the primary stain (malchite green), the keratin must be heated to drive the stain into the spores. Vegetative cells are then decolourised with water and 0.5% of safranin (red stain) as counterstain. In the end, the endospores will appear green within a red or pink cell (which is the vegetative cell).
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| The keratin is heated in order to stain the spore. |
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| Endospore staining procedure. |
Lastly, flagella staining where the stain will thicken the flagella of the microbe.
After we finished with the Topic 2 Microscopy lesson. Dr Wan assigned a group activity to all of us that have to be submitted on that day which is... Making a mind map for the next topic! Using any one of the useful mind map website that she has recommended. And this is what we managed to do that day!
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