Serial Dilution and counting method for microorganism:
A serial dilution is a process were the
concentration decreases by the same quantity in each successive step and concentration
of microorganism in different dilution factor. We used three methods in microbiology
for enumeration of microorganisms or counting of microorganisms. Pour plate
method, direct microscopic count method, spread plate method. In all three
methods described in lab require to serially dilute sample until you have
30-300 colony forming units (CFU) on the plate.
Plates which contain more than 300 CFU are very difficult to count. And
plates contain less than 30 CFU are not statistically reliable.
There are some diluents used in microbiology which are
given below:
·
Saline
water.
·
Peptone
saline diluents.
·
Buffered
peptone water.
·
Sodium
citrate diluents.
·
Di potassium
hydrogen phosphate diluents.
Where
=10954 CFU /gm or 10954 CFU/ml
Experiments in Microbiology, Plant Pathology
and Biotechnology by K.R. Aneja
www.google.com
Method
for Dilution:
·
Prepare Diluents transfer 9 ml diluents into
each tube and Label all diluents blank as 10-1, 10-2, 10-3,
10-4, 10-5, 10-6etc.
·
Prepare initial dilution adding 10gm sample + 90
mL diluent or 25 gm sample + 90 mL diluent mix by vortex to obtain uniform
distribution of microbes.
·
From first dilution using a sterile 1mL pipette,
transfer 1mL of suspension into 10-1 Test tube
·
And transfer same 10-1 to 10-2,
10-2 to 10-3, 10-3 to 10-4 and up
to 10-6.
·
Repeat this procedure till the original sample
has been diluted up to 10, 000, 00 (10-6) times using every time a
fresh sterile pipette tip.
·
After preparing dilution liable Petri plate 10-1
to 10-6, 1mL suspension of each dilution tube transfer into each
dilution liable sterile glass or plastic Petri plate.
·
Add approximately 15 ml of the palate count agar
or Nutrient agar medium, melted and cooled to 450 C, to each Petri
plate containing the diluted sample.
·
Mix the content clock wise and anti -clock wise and
incubate appropriate temperature for selective time.
·
Observation: Observed all plate for the
appearance of colony.
·
Count number of colonies in the Petriplates that
have colonies in the range 30-300 CFU range count plate by colony counter.
Calculation: according to
Indian standard-
Where
ΣC = the sum of the colonies counted an
all the plates;
n1=the number of plated
counted in the forts dilution;
n2 = the number of plates
counted in the second dilution
D= the dilution from which the first
counts were obtained (for example,10-1)
For Example:
10-3 dilution: 83 and 97 Colonies
10-4 dilution: 33 and 28 Colonies
References: IS 5403 (1999):
Method for Yeast and Mould Count of. Foodstuffs and animal feeds.
