Culture suspension preparation

• Microbiology laboratory culture suspension used for decreasing the culture concentration.
• This technique widely used and simple technique.
• Serial dilution purpose used 0.9 % sterile saline.
• 0.9 % sterile saline is best diluent for cell count.
• Cell numbers need to be reduced, which is done by repeatedly the amount of bacteria you have in your sample.
• A sample amount of bacteria sample is mixed with a diluent solution.
• First prepare inoculum is called stock solution or initial suspension..
• Next continue the dilution as per picture.
  
• Every dilution gently vortex.
• Prepared culture plating within 30 minutes time period.
• Incubate the bacterial culture plates at 32.5 ± 2.5⁰C for 18-24 hours (for except anaerobic bacteria).
• Incubate anaerobic organism plates in anaerobic jar or anaerobic incubator at 32.5 ± 2.5⁰C for 48-72 hours.
• Incubate fungal culture plates at 22.5 ± 2.5⁰C for 3-5 days.
• After the completion of the incubation period.
• Observed the colonies with the help of colony counter.
• Select the dilutions which are having cells between 10-100 CFU/mL.
• Count every colony on the plate.
• Preserve the 10-100 cells refrigerator 2-8⁰C for further test.
• By working backwards using multiplication with the " Dilution factor". you will be able to make a determination of the numbers of bacteria in your original sample.

Calculation :

CFU/mL = observed CFU  /  Volume to be analysed  X Dilution factor

Example:

Say you counted 38 CFU on the 10-5 plate with an inoculum of 0.1 mL

= 38 / 0.1  x 10-5 

= 380 x 10-5 

S.No	Serial Dilution	Plate-1	Plate-2	Avg
1	10-1	TNTC	TNTC	TNTC
2	10-2	TNTC	TNTC	TNTC
3	10-3	TNTC	TNTC	TNTC
4	10-4	TNTC	TNTC	TNTC
5	10-5	37	39	38
6	10-6	5	3	4
7	10-7	Nil	Nil	Nil