Throughout the month of June, I was interning at the Research and Development section of a company called Care Pro Biosciences Pvt. Ltd that uses microbiological principles to isolate useful microbes for agricultural development. Over there, I learned several basic cell culture techniques, bacterial isolation and laboratory etiquette. Here is attached a copy of the experiment which I performed over there.
Isolation, study and cultivation of soil microorganisms
Aim: To isolate, study and cultivate microorganisms from various soil samples using techniques of serial dilution, spread, pour and streak plate using PDA (potato dextrose agar) and various kinds of microscopes.
Material Required: -
For the PDA (500ml): 100g finely chopped potato, 10g dextrose powder, 500ml distilled water, 10gm agar, muslin cloth
For the experiment: Potato dextrose agar, soil samples collected from various areas, an autoclave, 15 peptone-agar petri dishes, micropipettes, 200ml saline water containing 20g of sodium chloride, 20 hard glass test tubes.
Procedure:
1. To prepare the PDA: take the potato and boil with distilled water in a hard glass beaker until the potato is soft to the touch- this means that the potato extract has been prepared.
2. Filter the potato extract through a muslin cloth into another beaker and add the dextrose and then the agar. This completes the PDA.
3. To prepare for serial dilution: Take the test tubes and fill 9ml of saline water in each of them.
4. Dissolve one gram of soil sample in one test tube. Before the soil has settled, take 1ml of the test tube contents and dissolve that in another test tube. Keep doing so for about six test tubes.
5. Autoclave the test tubes and PDA with proper technique. This sterilizes the sample and PDA so that no external organism influences the data gathered.
6. Using micropipettes, pipette out 25ml of PDA into five peptone-agar petri dishes, as well as the diluted sample. Start with the weakest dilution, i.e the sixth test tube, and use progressively stronger dilutions later on.
7. Use spread plate technique to isolate several microorganisms, and streak plate for a single microorganism.
8. Incubate in an incubator for 24, 36 and/or 48 hours.
Spread-plate technique: Using a sterile plastic spreader, spread the inoculum over the petri dish in a lawn-like fashion. Upon incubation, several microbial colonies are established.
Pour-plate technique: Using larger samples and mixing them with growth medium (in this case, PDA), pour each of them over petri dishes. This method is not viable for heat-sensitive microbes.
Streak-plate technique: Spread the inoculum in stripe-like fashion over a portion of the petri dish using a sterile inoculating loop, then from the initial inoculum, spread some more of the culture. Re-sterilize by holding over a lit flame until red-hot, wait for it to cool, then start streaking from the previous streak. Do this several times. This will result in a single colony being isolated.
Precautions:
• The inoculating loop and spreader used must be sterile. Do this by holding them over a flame until it is red hot, then cool it by pressing it over the petri dish (don’t touch the inoculum whilst doing so!). If the spreader/inoculating loop are not made of metal, do so by autoclaving.
• Autoclave the PDA and the test tubes meant for serial dilution before use.
• Disinfect the work area before and after use.
• Wash hands before and after work.
In my case, I prepared ten different petri plates, five of PDA and five of NA, and each of the five having dilutions of 10^-6, 10^-5, and all the way till 10^-2. After the colonies were established, colony counting procedure was executed.
Number of colonies per dilution
|
10^-6
|
10^-5
|
10^-4
|
10^-3
|
10^-2
|
PDA
|
41
|
128
|
351
|
TNTC
|
TNTC
|
NA
|
3
|
5
|
10
|
20
|
TNTC
|
Note that while PDA is mainly for fungal cultivation and NA is mainly for bacterial cultivation, every one of the colonies (at the time of counting) was a bacterial colony in both PDA and NA.
Then, from these petri plates I took up five unique colonies and performed the streak plate procedure with each of them on NA medium (streaking each of them five times to obtain a 10^-5 diluted strain of the same bacteria), after which I took the diluted strain up for a Gram Staining procedure.
Gram Staining procedure
This procedure differentiates different kinds of bacteria depending on whether they are Gram-negative or Gram-positive bacteria. These are differentiated on the basis of their cell wall thickness; Gram-positive bacteria have a thick cell wall and Gram-negative bacteria do not.
1. Make a slide of cell sample to be stained. Heat fix the sample to the slide by carefully passing the slide with a drop or small piece of sample on it through a Bunsen burner three times. (Cell samples were obtained from the petri dish
2. Add the primary stain (crystal violet) to the sample/slide and incubate for 1 minute. Rinse slide with a gentle stream of water for a maximum of 5 seconds to remove unbound crystal violet.
3. Add Gram's iodine for 1 minute- this is a mordant, or an agent that fixes the crystal violet to the bacterial cell wall.
4. Rinse sample/slide with acetone or alcohol for 3 seconds and rinse with a gentle stream of water. The alcohol will decolorize the sample if it is Gram negative, removing the crystal violet. However, if the alcohol remains on the sample for too long, it may also decolorize Gram positive cells.
5. Add the secondary stain, safranin, to the slide and incubate for 1 minute. Wash with a gentle stream of water for a maximum of 5 seconds.
If the bacteria are Gram positive, they will retain the primary stain (crystal violet) due to the thickness of their cell walls and not take the secondary stain (safranin), causing them to look violet/purple under a microscope. If the bacteria areGram negative, they will lose the primary stain due to the cell wall being very thin and take the secondary stain, causing them to appear red/pink when viewed under a microscope.
In my experiment, I used each of the diluted morphologies and performed Gram Staining, after which I viewed them under a microscope at 40X and 100X magnification.
Isolate
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Colony Morphology
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Gram Reaction
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Colour
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Margin
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Elevation
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CPB1
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Pale cream and opaque
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Circular and undulate
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Flat
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Gram negative
|
CPB2
|
Whitish-cream and opaque
|
Circular and entire
|
Flat
|
Gram negative
|
CPB3
|
Cream and opaque
|
Circular and entire
|
Raised
|
Gram negative
|
CPB4
|
Orange-red translucent
|
Irregular and undulate
|
Raised
|
Gram negative
|
CPB5
|
Darkish cream and opaque
|
Irregular and undulate
|
Flat
|
Gram negative
|
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