SERIAL DILUTION AND METHODS OF

INOCULATION

STREAK POUR AND AGAR STABS

PRESENTATION FOR VALENTINE MHUTE
ND1 PHARMACY 2020
Harare Polytechnic
Serial Dilution
Serial dilution refers to the methodical reduction of the
known or unknown entity or microorganism through
successive resuspension of an initial solution (solution0)
into fixed volumes of a liquid diluent.
 The diluents usually consist of 0.45% saline, though the
composition can be varied. An experimenter can choose
any volume he/she wants to use for each diluent, and it is
most often a multiple of 10, facilitating the logarithmic
reduction of the sample.

VALENTINE MHUTE ND1 PHARMACY 2020

Serial Dilution Continued….
For example, solution0 contains a total of 100 Staphylococcus
aureus cells suspended in 10ml of nutrient broth.
If 1ml of the solution0 is removed and added to 9ml of the
saline (diluent1), the new solution (solution 1) would contain
1/10th of the initial concentration of Staphylococcus aureus.
The new solution (solution 1) would contain 10 Staphylococcus
aureus cells.
This process repeated by removing 1ml of the solution 1, and
adding it to another 9mL of saline-diluent 2 would yield
solution2, containing only a single Staphylococcus aureus cells.

VALENTINE MHUTE ND1 PHARMACY 2020

Serial Dilution Continued….
Since each new solution (9ml of the diluent + 1ml of the
solution) contains a total of 10ml, one can conclude that
the dilution factor for this reduction is 10 or that it was a
10-fold serial dilution.
Since we only began with 100 cells during this example
and that we are diluting by an element of 10, only two
steps are required to succeed in absolutely the minimum
concentration of 1 cell.

VALENTINE MHUTE ND1 PHARMACY 2020

Serial Dilution Continued….
Aliquoting and resuspension continue during this fashion until
the ultimate tube is reached, diluting the stock concentration by
an element of 10 each with each step.
 Serial dilution is that the simplest technique for obtaining
manageable concentrations of the specified organisms.
 It is complemented by a petri dish streaking and spreading.
The advantage of this approach is that the experimenter can
harvest pure strains of one species or separate strains from a
mixed population.

VALENTINE MHUTE ND1 PHARMACY 2020

Serial dilution of a stock solution

VALENTINE MHUTE ND1 PHARMACY 2020

 
Procedures on how to carry out serial dilution:

i. Obtain the flask labeled "Nutrient broth" from the incubator and shake vigorously.
ii. Pipette 1ml of solution0 into the test tube labeled T1. 1ml is used here for
simplicity- smaller or larger volumes of diluent may also be used.
iii.Remove 1ml from test tube T1 and add it to test tube T2.
iv.Remove 1ml from test tube T2 and add it to test tube T3.
v. Remove 1ml from test tube T3 and add it to test tube T4.
vi.Remove 1ml from test tube T4 and add it to test tube T5.
vii.Remove 1ml from test tube T5 and add it to test tube T6.
viii.Remove 1ml from test tube T6 and add it to test tube T7.
ix.Remove 1ml from test tube T7 and add it to test tube T8.
x. Remove 1ml from test tube T8 and add it to test tube T9.
xi.Remove 1ml from test tube T9 and add it to test tube T10.

VALENTINE MHUTE ND1 PHARMACY 2020

Methods of bacteria inoculation
Streak pour and Agar stabs

All culture media must be carefully checked visually

before use for contamination, expiry date, and significant
physical imperfections such as the uneven distribution of
media, variable amounts of the medium in the Petri
dishes/tubes, colour, gross deformation of the surface on the
media.
 Culture media should have an identifiable batch or quality
control number and have passed Quality Control tests before
use.
VALENTINE MHUTE ND1 PHARMACY 2020
Streak Plating Technique

The aim of this procedure is to obtain well-isolated colonies from a

specimen or culture inoculum by creating areas of increasing dilution on a
single plate:
 Select an appropriate dilution of your target organism. For instance,
solution4 will yield a 1/10,000th dilution of your initial concentration.
Most of the time, dilutions of 1/1,000th (T3/Solution3), 1/1,000,000th
(T6/Solution6), and 1/1,000,000,000th (T9/Solution9) are evaluated to
enumerate microbes.
Using a plastic sterile disposable inoculating loop or a reusable metal
inoculating loop that has been under fire for no less than 10 seconds,
immerse in the desired solution from step 5. Calibrated inoculating
loops should transfer 0.01ml. (Caution: Do not allow the flamed loop
to contact bacteria immediately after removing from heat).

VALENTINE MHUTE ND1 PHARMACY 2020

Continued…
Slightly raise the Petri dish cover on one side so that only the
inoculating loop can access the agar. Slide the inoculating loop
across the top of the media in a zig-zag fashion being careful
not to compromise the agar. Lower the Petri dish lid.
Use a new disposable inoculating loop or re-sterilize your
reusable loop.
Rotate the plate by roughly 1/3rd (~118°) and reduce the
frequency of zig-zag motion.
Again, employ a new disposable loop or re-sterilize a metal
loop before rotating a final time, and reduce zig-zag frequency
once more. Lower the Petri dish lid.
VALENTINE MHUTE ND1 PHARMACY 2020
Continued…
Repeat steps 7.2 - 7.6 until a minimum of three Petri
dishes has been streaked for three different dilutions,
using a new disposable loop or by re-flaming a reusable
loop.
Replace the lid and then incubate the streaked agar plate
at the appropriate temperature in an inverted position to
prevent condensation. For anaerobic organisms, use an
anaerobic chamber.

VALENTINE MHUTE ND1 PHARMACY 2020

Plate streaking for bacterial enumeration and
strain isolation

VALENTINE MHUTE ND1 PHARMACY 2020

Agar Stab Technique

The method outlined below is used to prepare stab cultures to

observe motility or when inoculating certain types of solid medium
and to pick single colonies from a plate:
i. Using an aseptic technique, take up a single well-isolated colony
with a sterile inoculating stab needle and stab the needle several
times through the agar to the bottom of the vial or tube.
ii. Replace the cap and tighten loosely when incubating to allow gas
exchange.
iii.Incubate the stabbed agar plate/slant at the appropriate
temperature

VALENTINE MHUTE ND1 PHARMACY 2020

References
Allen, M.E., Gyure, R.A. (2013) An Undergraduate Laboratory Activity Demonstrating
Bacteriophage Specificity. Journal of Microbial Biological Education 14: 84-92.
Ben-David, A., Davidson, C.E. (2014) Estimation Method for Serial Dilution
Experiments. Journal of Microbiological Methods 107:214-221.
Goldman, E., Green, L.H. (2008) Practical Handbook of Microbiology.
Koch, R. (1883) New Research Methods for Detection of Microcosms in Soil, Air and
Water.
Lederberg, J., Lederberg, E.M. (1952) Replica Plating and Indirect Selection of
Bacterial Mutants. Journal of Bacteriology 63:399-406
Sanders., E.R. (2012) Aseptic Laboratory Technique: Plating Methods. JoVE 63:e3063.

VALENTINE MHUTE ND1 PHARMACY 2020