VAN MHUTE - Microbiology and ID

HARARE POLYTECHNIC
SCIENCE TECHNOLOGY DIVISION
COVER SHEET
FIRST NAME(S): VALENTINE . SURNAME: MHUTE .
COURSE: ND1 PHARMACY DATE: 23-11-2020 .
NAME OF LECTURER: MR P MOYO .
SUBJECT: Microbiology and Infectious Diseases .
ASSIGNMENT/TEST/PRACTICAL: ASSIGNMENT 2 .
MARK: MODERATE MARK: .
COMMENTS:
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. .
. .
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LECTURER’S SIGNATURE: DATE: .

Serial Dilution
Serial dilution refers to the methodical reduction of the known or unknown entity or microorganism
through successive resuspension of an initial solution (solution 0) 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. For example, solution 0 contains a total of 100 Staphylococcus
aureus cells suspended in 10ml of nutrient broth. If 1ml of the solution 0 is removed and added to 9ml of
the saline (diluent1), the new solution (solution1) would contain 1/10th of the initial concentration of
Staphylococcus aureus. The new solution (solution1) would contain 10 Staphylococcus aureus cells. This
process repeated by removing 1ml of the solution1, and adding it to another 9mL of saline-diluent2 would
yield solution2, containing only a single Staphylococcus aureus cells.
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.
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.
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.
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.
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:
i. Select an appropriate dilution of your target organism. For instance, solution 4 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.
ii. 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)
iii. 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.
iv. Use a new disposable inoculating loop or re-sterilize your reusable loop.
v. Rotate the plate by roughly 1/3rd (~118°) and reduce the frequency of zig-zag motion.
vi. 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.
vii. 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.
viii. 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.
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
Reference
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.

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HARARE POLYTECHNIC

SCIENCE TECHNOLOGY DIVISION

FIRST NAME(S): VALENTINE . SURNAME: MHUTE .

COURSE: ND1 PHARMACY DATE: 23-11-2020 .

NAME OF LECTURER: MR P MOYO .

SUBJECT: Microbiology and Infectious Diseases .

MARK: MODERATE MARK: .

LECTURER’S SIGNATURE: DATE: .

Procedures on how to carry out serial dilution:

Methods of bacteria inoculation Streak pour and Agar stabs

Streak plating technique

Agar stab technique

Goldman, E., Green, L.H. (2008) Practical Handbook of Microbiology.

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