MUHAMMAD HAMDI IBRAHIM (2110422012)

COUNTING CHAMBER

I. Work Principle
Haemocytometer is tools that can used to counting cell with quick and low
concentration. Composed by 2 counting chamber and each counting chamber have
microscopis lines in glass surface area. This line form squares that composed of 3
types of size: 0,25 x 0,25 mm; 0,25 x 0,20 mm and 0,20 x 0,20 mm with thickness
0,1 mm. Haemocytometer can count living cell or dead cell, depends on the stain that
used. Morphology of cell can observed and also evaluated homogenity and
contaminants detection (Rouge, 2002).

II. Practicum Methods

2.1 Time and Place
The "Counting Chamber" practicum was held on Wednesday, 19 th October 2022
at the Education Laboratory III, Department of Biology, Faculty of Mathematics
and Natural Sciences, Andalas University, Padang.

2.2 Material and Tools

The tools needed are haemocytometer, microscope, object glass, cover glass,
pipette. Meanwhile, the inggredients are tissue, alcohol 70%, and Fermipan
(yeast) and aquadest solution (2 days and 1 day old).

2.3 Work Procedure

Prepare the yeast with solute them in aquadest for 1 and 2 days. Prepare
microscope, turn it on. Sterilize haemocytometer with alcohol 70%. Dilute the
solution 2 times. Use pipette and drip it on object glass. Cover it with cover
glass, observe with microscope. Yeast cell counts in each square and averaged
with formula:

(C 1+C 2+C 3+C 4+ C 5)

∑ cell= 5
×25 ×10 × dilution factor (cfu/mL)

C = Chamber/Square

III. Results and Discussions

Table. 1 Cell Amount in 5 Chamber Observation
No Chamber Cell Amount
1 CI 787
2 CII 1130
3 CIII 1314
4 CIV 840
5 CV 944
Total 5024
 MUHAMMAD HAMDI IBRAHIM (2110422012)

(C 1+C 2+C 3+C 4+ C 5)

∑ cell= 5
×25 ×10 × dilution factor (cfu/mL)

(787+1130+1314+ 840+944)
∑ cell= 5
× 250 ×10 0(cfu/ mL)

= 5024 × 50 × 100 (cfu/mL)

= 25.120.000 cfu/mL

Yeast colony that estimated in 1 drip of potato soaking water is ± 25.120.000 or

2.512 × 104 cfu/mL.

From practicum that already carried out data obtained mean of cell amount is ± 2.512
× 104 cfu/mL. Cell amount calculated from 5 chamber in haemocytometer that
already observed. Before counted, the solution is dilute for 2 times. More dilutes
more decreasing in concentration of microorganism and decrease the rigidness and
easier to counts.

Microorganism is all of the organism that is only can be observed through

microscope. It is include bacteria, fungi, yeast and according to systematics it also
include algae, protozoans, and virus that only can observed through electrons
microscope. Microorganism generally is cosmopolites, it lives on the any surfaces
such as soil, atmosphere, and aquatic (Waluyo, 2005).

Calculating microbes amount can used directly or undirectly. Counting with directly
can sum amount of microorganism in a material without any treatment before.
Instead, microorganism counting undirectly needs treatments before counting.
Directly counting methods include counting chamber and simple preparat (stain or
stainless) (Stober, 2001).

Experiments to determine the amount of microorganism cell is purposed to study

how to counts amount of microorganism cell with counting chamber methods. In the
scope to determine amount of microorganism cells, there is many methods that can
be used. For undirectly methods, can used turbidimetry (spectrophotometer),
metabolic activity methods, and dry weight (Tortora, 2013). Tools that used for
counting chamber itself is haemocytometer. Haemocytometer is glass chamber or
glass that have elevated sides with transparents small cover appropiate to 0,1 mm
above surfaces (Hansen, 2013).

Counting chamber is directly methods in counting microbes with microscope. Other

tools than haemocytometer is Petroff-Hauser counting chamber that the principle is
similar with Haemocytometer (Laboffe, Michael, and Burton, 2010).
Haemocytometer is made from specific optic glass that may forms suspension of
bacteria cell in specific volume and countable with microscope. Even though,
counting chamber methods have several lacks such as cannot difference the living
and dead cells, hard to observe smaller cells, less precision, and just suits for after
dilution solution (Madigan, 2017).
 MUHAMMAD HAMDI IBRAHIM (2110422012)

IV. Closing
4.1 Conclusion
From practicum that already been carried out, obtained conclusions:
1. Average amount of yeast cell in Haemocytometer is 2512 × 104 cfu/mL
2. To observe yeast cell, needs several dilution to reduce the rigidness of yeast
cells, easier to count. And in this practicum used 2 times dilution (10-2).
3. Microbes that contain in fermipan is Saccharomyces cerevisiae.
4. Counting chamber is directly methods that have several lacks because it
cannot difference the living and dead cells.

4.2 Suggestions
Practitioners must be careful so the data is not biased when using formula and
using haemocytometer and microscope. Please make sure the object lens not
press directly at cover glass or haemocytometer that can cracks the glass over.
The laboratory in Biology Department needs upgrade or new microscope, so the
data that obtained is far from bias.
 MUHAMMAD HAMDI IBRAHIM (2110422012)

REFERENCES

Chen, Y.W., and Chiang, P.J. 2011. Automatic Cell Counting for Haemocytometer
through Image Processing. World Academy of Science : Engineering and
Technology.
Hansen, P. J. 2013 Counting of Bacterial Colony Forming Units for direct
Enumeration of Viable and Total Bacteria in Drinking Water. Department of
Civil Engineering Journal of Microbiology Methods. Canada 37 : 77-79
Laboffe, Michael, J., and Burton, E. P. 2010. Microbiology Theory Laboratory and
Applications. United States. Morton Publishing Company.
Madigan, M.T. 2017. Microbiology of Microorganism. 12th edition. San Francisco :
Pearson Benjamin Cummings.
Rouge, M. 2002. Counting Cells with Haemacytometer. Path. Phys. Colorado State
University Publising.
Stober, W. 2001. Monitoring Cell Growth. Immunology. Vol. 5. USA : John Wiley
& Sons
Waluyo, L. 2005. Mikrobiologi Umum. Malang : Universitas Muhammadiyah
Malang Press.
 MUHAMMAD HAMDI IBRAHIM (2110422012)

ATTACHMENT

Figure 55. Fermipan soaked in water Figure 56. Fermipan day 1 upper left
for 1 day square

Figure 57. Fermipan day 1 upper Figure 58. Fermipan day 1 middle
right square square

Figure 19. fermipan day 1 bottom left Figure 20. Fermipan day 1 bottom
square right square