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1. What is the final dilution factor for a suspension made from 10 ul of a 10-3 dilution diluted to 1000 ul with broth? 2. If your orignal culture had 6.7 x 106 cells/ml, what is the concentration of the final dilution made from a 10-2 dilution of a 103 dilution 3. How many cells are in 50 ul of a 2.2 x 104 cells/ml culture? 4. Calculate the original culture concentrations from the following counts: 112 colonies 0.1 ml plated 10-5 dilution 5. Calculate the original culture concentrations from the following counts: 121 colonies on 112 of the plate when 0.1 ml of a 10-8 dilution was spread over the whole plate 6. How would you prepare a 10 4 dilution of a bacterial culture? Assume you want 1000 ul total of the dilution

Solution

Ans. 1.

I. First dilution factor ‘a’ = 10^-3                           ; [of 10 uL aliquot]

II. 10 uL is diluted to 1000 uL

            Second dilution factor, b = Initial volume / Final volume = 10 uL / 1000 uL = 1/100 = 10^-2

Final dilution factor = multiplication of suitable dilution factors

= dilution factor a x dilution factor b

= 10^-3 x 10^-2 = 10^-5       

Thus, final dilution factor of the suspension = 10^-5         

Ans. 2. Total dilution factor of the suspension = 10^-2 dilution of a 10^-3 dilution

                                    = 10^-2 of 10^-3 = 10^-2 x 10^-3 = 10^-5           

Final Concertation = Initial concertation x Total dilution factor

                                    = (6.7 x 10⁶ cells/ mL) x (10^-5)

                                    = 6.7 x 10¹ cells/mL

                                    = 67 cells/ mL

Ans. 3. Given,

Concertation = 2.2 x 10⁴ cells/ mL

                        = 2.2 x 10⁴ cells/ 1000 uL                        ; [1 mL = 1000 uL]

                        = 22 cells/ uL

Now,

Number of cells = volume of aliquot x concentration of aliquot

                        = 50 uL x (22 cells/ uL)

                        = 1100 cells = 1.1 x 10³ cells

Note: The unit of volume shall be the same. Calculation can also be done by converting 50 uL to mL.

That is, Number of cells = 0.050 mL x (2.2 x 10⁴ cells/ mL) = 1100 cells

Ans. 4. Original culture concentration = Plate count x volume of inoculum x dilution factor

                                                = 112 colonies x 0.1 mL x 10^-5

                                                = 1.12 x 10^-4 colonies/ mL

Since each colony is assumed to arise from a single cell (also called- colony forming unit, CFU) in the inoculum, the original culture concentration = 1.12 x 10^-4 cells/ mL

Ans. 5. Total number of colonies on the plate = 2 x (number of colonies on ½ plate)

                                                            = 2 x 121 colonies = 242 colonies

Original culture concentration = Plate count x volume of inoculum x dilution factor

= 242 colonies x 0.1 mL x 10^-8

= 2.42 x 10^-7 colonies/ mL = 2.42 x 10^-7 cells/ mL

Ans. 6. A 10^-4 dilution means that the original culture is diluted 10⁴ (= 10000) times. For example, taking 1 uL of culture and diluting it to 10000 uL with broth gives a 10^-4 dilution.

However, we have to make 1000 uL solution. Required volume of bacterial culture to make 1000 uL of 10^-4 dilution = final volume x dilution factor = 1000 uL x 10^-4 = 0.1 uL. Since, measuring 0.1 uL may be difficult most often, we take an alternate route of multiple serial dilution as follow-

1^st serial dilution: Take 10 uL of bacterial culture in a sterile Eppendorf tube. Add 990 uL sterile broth or saline to it.

Now, final volume of the solution = 10 uL (culture) + 990 uL (broth) = 1000 uL

            Dilution factor = Initial volume taken / final volume made upto

                                    = 10 uL / 1000 uL = 10^-2

Thus, dilution factor for 1^st serial dilution = 10²

2^nd serial dilution: Take 10 uL of 1^st dilution (10^-2 dilution prepared above) in a sterile Eppendorf tube. Add 990 uL sterile broth or saline to it.

Dilution factor of 2^nd serial dilution= Initial volume taken / final volume made upto

                                                = 10 uL / 1000 uL = 10^-2

Total dilution = dilution factor for 1^st serial dilution x dilution factor for 2^nd serial dilution

                        = 10^-2 x 10^-2 = 10^-4

Therefore, the second serial dilution gives a solution of 10^-4 dilution.