Enumeration and isolation
Calculation of the number of microbes (enumeration) contained in the sample
can be done in various ways, including: dilution method, direct calculation
method in count space (hemasitometer), membrane filter method, dry weight
method and cell volume, MPN method, etc. In this lab, you will learn to
determine the number of cells contained in a sample using the dilution method
and direct calculation method using the Haemasitometer tool (Improved New
Bauwer).
Dilution method
This dilution method uses a dilution series from a sample which is then planted
in the medium. After being incubated, the growing colonies can be calculated
by the assumption that one colony that grows comes from one cell. Thus, the
number of cells in the original sample can be calculated by multiplying the
number of colonies that grow with the thinner. Dilution is usually stated with a
negative rank, for example 10-5 for 1: 100,000 dilution. By using this method we
can calculate the total number of microbes contained in water samples or
ground samples.
Tools
a. Water sample dalatan
breakfast. media na
C.. Sterile bottle
d. Petrol
Hey. Sterile water in a test tube
f. Measuring pipette
Work
a. Take water samples aseptic using a sterile bottle.
breakfast. Melt the NA medium in the bath of water and cool until the
temperature of 45 oC.
C.. Mark 6 petri dishes with 10-1, 10-2, ... 10-6.
d. Beat water samples to homogeneous and take aseptic aseptic as much as 1 ml,
then put in a reaction tube that has 9 ml of sterile water to get a dilution factor
of 10 times (10-1).
Hey. Beat this reaction tube well, and take water in itAseptic was 1 mL, and put
it in the Petri dish marked 10-1. From this tube, take 1 ml of the sample and put
it in a second reaction tube that contains 9 ml of sterile water to obtain a
dilution factor of 100 times (10-2)
f. Pour the Na medium into the petri dish marked 10-1 that contains 1 ml of
water samples, then shaken until homogeneous.
g. Take as many as 1 ml of samples from the second reaction tube and enterIn
the second Petri dish (10-2). From this tube too, take 1 ml of the sample and put
it in the third reaction tube that contains 9 ml. Sterile water to obtain a dilution
factor of 1000 times (10-3).
h. Repeat this work to 1,000,000 dilution factors (10-6).
I. To calculate total microbes in soil samples, procedures used exactlysame as
above, but the number of samples that must be weighed are 1 gramSolid
samples or 1 ml of liquid sample (note the attachment !!!!).
a. Incubate all the dishes that have been planted with samples containing
microbes at 25 oC temperatures for 1-7 days.
k. Calculate the growing colony that grows on all Petri dishes whose colonies
range from 30-300 colonies.
l. The number of microbial cells contained in the sample is calculated by
multiplying the number of colonies that grow on the medium with a dilution
factor.
f.Pour the Na medium into Petri's dish marked 10-1which contains 1 ml of
sample water, then shaken until homogeneous.
g. Take as many as 1 mL of samples from the second reaction tube and enter
into the second Petri dish (10-2). From this tube too, take 1 ml of the sample and
put it in the third reaction tube that contains 9 ml. Sterile water to obtain a
dilution factor of 1000 times (10-3).
h. Repeat this work to 1,000,000 dilution factors (10-6).
I. To calculate the total microbes in soil samples, the procedure used exactly the
same as above, but the number of samples that must be weighed are 1
gramSample solid or 1 ml of liquid sample (note the attachment !!!!).
a. Incubate all the dishes that have been planted with samples containing
microbes at 25 oC temperatures for 1-7 days.
k. Calculate the growing colony that grows on all Petri dishes whose colonies
range from 30-300 colonies.
l. The number of microbial cells contained in the sample is calculated by
multiplying the number of colonies that grow on the medium with a dilution
factor.
Total microbial calculation directly with a microscope
The superiority of this method is to calculate the total cell, both alive and dead.
Besides that, the time needed is much shorter. One common way is done is a
direct calculation under a microscope where certain volumes of a suspension
are spread on an area on the glass object.
By calculating the number of microbes at a certain extent known
andmultiplying it with certain factors, it can be calculated the number of
microbes contained in the original sample. In this direct calculation we can use
the counting space (Counting Chamber), such as haemacytometer with a depth
of 0.1 mm.
Tools
a. Yeast suspension
breakfast. haemacytometer
C.. microscope
Work
a. Close the count room with glass cover, and take a yeast suspension with a fine
pipette.
breakfast. Drop this suspension on the edge of the glass cover, so this
suspension will flow down the glass cover and fill the count space.
C.. Observe under the microscope and count the number of cells in 5 large
boxes (with each box consisting of 16 small boxes).
d. Determine the number of cells / ml samples using the following formula:
X = n x 25 x 10 x 103, where n is the average cell count on each box
Note: the area of 25 large boxes on the hemasitometer tool is 1 mm2.The depth
of the count room is 0.1 mm.The number of cells per mm3 in the counting tool
is N x 25 x 10, where n is the average number of cells in each box of 25 boxes
contained in the count room. When the sample is diluted before calculations,
this dilution fator must also be taken into account. For example, if the sample is
diluted by 10-2 or 100 times, the results obtained from the formula above must
be multiplied by 102 or 100.
