Question

b. 1 ml 1ml 「-I Final dilution 4(1 point) This represents a serial dilution to determine viable bacterial counts. Indicate the final dilutions in each of the two tubes. Note that two different volumes of the cells intube #2 are plated onto agar plates A and B. Indicate the dilution factor that will be used to report the original cell concentration on a per mL basis. 1 ml 1 ml Final dilution in Tube #1 = Final dilution in Tube #2- ml ml Multiply number colonies on Plate A by:-- #1 #2 Multiply number colonies on Plate B by:- 0.1 ml 0.5 ml ピ Plate B ー Plate A 5. (1 point) Describe two ways in which you could make a solution with a final dilution of 1:1000. Give specifie volumes you would use. You may suggest single and/or multiple step procedures.

Answer 1

To determine viable bacterial serial counts serial dilution is performed in 2 tubes #1 and #2

final dilutions in each of the tubes will be

#1 - Final diltuion will be 10^-1 i.e 1ml from original inoculum and 9ml broth

#2 - Final dilution will be 10^-2 i.e 1ml from tube #1 and 9ml of broth.

Now from tube two plating of sample has been performed but with two different amount of samples

Plate A- 0.1 ml of tube 2 sample Plate B-0.5 ml of tube 2 sample

To calculate the no, of colonies formed in plate formula which can be used is

no. of colonies x dilution factor(the inverse of the dilutions) /volume on a culture plate

How to indicate the dilution factor that will help to report the original cell concentration .

-To answer this question, you’ll need to know a bit about bacteria and how they grow. Many, but certainly not all, bacteria reproduce by binary fission where one cell replicates its DNA, grows in size, then splits into two bacteria. If these progeny cells disconnect after cell division, the culture generally contains mostly single cells. If you serially dilute this culture, plate the dilutions, and count the colonies which form after incubation, most colonies represent a single cell. In many species of bacteria, the cells do not disconnect, but stay connected forming chains of cells or clusters or packets of cells. If you serially dilute these types of cultures, plate the dilutions, and count the colonies which form after incubation, most colonies represent more than one cell. Therefore, it is common practice to report the final number of your dilution and plating protocol in Colony-Forming-Units per ml (CFU/ml).

Example-what is the number of Colony-Forming-Units (CFU) per ml of original stock?

We will report concentration in CFU per milliliter. You have diluted upto 10^-2 dilutions in the dilution scheme no furthur is diluted from that tube .So you can calculate the total effective dilution from 10^-2 dilution.x 10 ^-1

colony count x dilution factor (the inverse of the dilutions) = CFU/ml of original culture

ex. X Colonies x 10 ²x 10 ¹ = X10³ Colony forming unit/ml

To make a final dilution of a solution to 1:1000 methods used are

1. Serial dilution

serial dilution a simply a series of simple dilutions which amplifies the dilution factor quickly beginning with a small initial quantity of material .The source of dilution material comes from the diluted material at previous.In series dilution the total dilution factor at any point is the product of the individual dilution factor in each step upto it.

ex-To dilute this by a factor of 1:1000, remove 1 μl of the 1:100 dilution and place it in a tube containing 999 μl of ddH₂O or media. so the tube from which sample has been taken i.e (1:100) has been diluted by a factor of 1,000 i.e  1:1000.

2. Making fixed volumes of specific concentration from liquid reagent

V1C1=V2C2

If there is limited availability of liquid material ,so of that known concentration specific volume should be identified.

V=Volume C=concentration

V1=Volume of stock you will start with

C1=Concentration of stock solution

V2= Total volume needed at new concentration

C2= The new concentration

now by doing algebraic rearrangement in formula V1C1=V2C2

you can calculate the specific amount of original sample that should be used to form the solution of final dilution  1:1000.