Isolation of Bacteria from different samples
[Water, Soil, Milk, Air]
hence, To prevent contamination and possible infection from the bacteria, make sure to properly dispose of all Petri plates, samples and pipettes. the procedure is only for cultivatable bacteria.
therefore we can only use it for the cultivatable bacteria.
Serial dilution of the sample
for instance, sample will be dilute with the ratio of 9:1.
Growth in agar
- Preparing agar
so, Nutrient agar is usually prepared. (normally about 1% w/v) we use it because it causes a higher growth of non-fastidious organisms.
Flask, Aluminum foil, Petri plates and cotton.
|Beef extract||0.3 g|
|Distilled Water||100 mL|
- Preparation of agar:
above all, agar is prepared by the recipe.
- Sterilization of agar and plates
bacteria are present everywhere therefore, Petri plates and agar (Properly lid and packed) are autoclaved for 2 hrs under 15 psi and 121oC (20 min at dynamic).
- Preparing plates
- Pour plate Method (for Milk, Water, Soil)
- Add any antibiotics or supplements (spirit)
- Pour the plates
(30 mL for in a 100mm, hold the bottle in the right hand; remove the cap with the little finger of the left hand. Flame the neck of the bottle. Lift the lid of the Petri dish slightly with the left hand and pour the sterile molten agar into the Petri dish and replace the lid)
- Allow the plates to set and If there are any bubbles in the plates so briefly pass the flame over to pop them.
- Dry the plates in the laminar flow hood with the lid slightly off for 15 minutes so that it is the germ-free.
- Pour sample (serially diluted) by micropipette (usually 1 µL) over the centre of agar on a plate so that it spreads nicely.
- By the help of spreader glide the sample by rotating it gently so that there is no destruction.
- For Air
after that Filled with agar, Petri dishes are placed in an open environment for 5 minutes so that the bacteria will enter in it.
Petri plates are let set in an incubator for 24 hrs in the incubator at 37oC, in addition, we can also provide some pressure. it causes the bacterial colonies to grow properly, in other words, they get optimum Temperature. Therefore, the optimum temperature is a major requirement.
- first of all, Sterilize the inoculating loop with the help of the flame or bunsen burner.
- Pick an isolated colony from the agar plate culture and spread it over the first quadrant (approximately 1/4 of the plate) using close parallel streaks or Insert your loop into the tube/culture bottle and remove some inoculum.
- You don’t need a huge chunk. because it will mess up the slide which causes a faded view of bacteria.
- Immediately streak the inoculating loop and very gently over a quarter of the plate using a back and forth motion.
- Any other motion causes the destruction of colonies and will affect the results.
- Make streaks on other 3, 1/4 parts so that you will get more colonies.
INCUBATE THE PETRI DISHES AGAIN FOR 24 Hrs so that bacterial colony can grow. In conclusion, we can use this procedure for cultivatable bacteria.
in addition to it, gram staining is used to observe bacteria under the microscope.
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