Theo FAO và tham khảo bài báo

Bảng 1: Môi trường nuôi tảo nâu Walne (Laing,1991)

Thành phần L nước biển

Môi trường A 1.0 ml

Môi trường C 0.1 ml

Môi trường A

FeCl3 .6H2O 1.3 g

MnCl2. 4H2O 0.4 g

H3BO3 33.6 g

Na2EDTA 45.0 g

NaH2PO4. 2H2O 20.0 g

NaNO3 100.0 g

Môi trường B 1 ml

Môi trường B / 100ml

ZnCl2 2.1 g

CoCl2 .6H2O 2.0 g

(NH4)6Mo7O24 .4H2O 0.9 g

CuSO4. 5H2O 2.0 g

HCl cô đặc 10 ml

Môi trường C /200ml

Thiamine HCl (Vitamin B1) 0.2 g

Cyanocobalamin (Vitamin B12) 0,01 g

Môi trường D (L)

Na2SiO3.5H2O 40.0 g

Bảng 2: Môi trường nuôi tảo xanh GuiLards F2

Thành Phần Số lượng

NO3 0.075 g

NaH2PO4. H2O 0.005 g

Vi lượng 1 ml

Vitamin 1 ml

Vi lượng

FeCl3. 6H2O 3.150 g

Na2EDTA 4.160 g

MnCl2. 4H2O 0.180 g

CoCl2.6H2O 0.010 g

CuSO4 .5H2O 0.010 g

ZnSO4. H2O 0.022 g

Na2MoO4. 2H2O 0.006 g

Vitamin

Biotin (Vitamin H) 0,0005g

Thiamine HCl (Vitamin B1) 0,1g

Cyanocobalamin (Vitamin B12) 0,0005g

pH = điều chỉnh 8.0 với 1M NAOH hoặc HCl

Phương pháp Haemocytometer (Karlson & cs., 2010)

Mẫu vi tảo được lấy 1 lần/ngày vào lúc 8 giờ sáng, mỗi lần lấy 10 ml. Mẫu vi tảo
được đựng trong hộp đựng mẫu, được cố định bằng dung dịch Neutral Lugol’s 2%.

Lắc đều mẫu vi tảo, dùng pipet hút mẫu vi tảo cho vào buồng đếm đã được đậy
sẵn lamen, chờ lắng, sau đó đưa vào thị trường kính để đếm, đếm ở vật kính x 10, mỗi
mẫu vi tảo được đếm 3 lần.

Mật độ tế bào trung bình/lit = số tế bào được đếm trung bình x 10000

Mật độ tế bào (tế bào/ml) = N/4 x 104

N: số tế bào được đếm trung bình

Phương pháp đếm: Sử dụng buồng đếm hồng cầu hiệu Thomas của Nhật Bản có
diện tích 1 mm2
Buồng đếm

Công thức tính tảo (Karlson & cs., 2010)

Mật độ tế bào (ml) = N*(At/Ac)*1/V

Trong đó:

V: Thể tích buồng đếm (Ml)

At: Tổng diện tích của buồng đếm (mm2)

Ac: Diện tích được đếm của buồng đếm (mm2)

N: Số lượng tế bào tảo

1. Optical Density:
Dilute the sample to appropriate concentrations as needed, and measure the
absorbance of the sample with a spectrophotometer at 550 nm. Other wavelengths
may also be used, but one must be consistent. Generate a calibration curve to
relate the absorbance with cell dry weight. The usual rules of operating a
spectrophotometer apply here, as well. For example, the accuracy of the method is
the highest when the absorbance is between 0.1 and 0.5. For a given culture
sample, a good spectrophotometer should yield a linear relationship between the
number of cells and the absorbance. However, the optical density is also a function
of cell morphology such as size and shape, because the amount of transmitted or
scattered light depends strongly on these factors. Consequently, an independent
calibration curve is required for each condition in accurate research work, as the
cell size and shape depend on the specific growth rate and the nutrient
composition. As a rule of thumb, an optical density of 1 unit corresponds to
approximately 1 g/l of dry cell. This is also commonly referred to as the turbidity
measurement.
2. Cell Counting Chamber:
This is a standard method for counting the number of microorganisms in milk. It is
also widely used in blood counts and vaccine counts; thus, a counting chamber is
also commonly called a hemocytometer. But the technique is not very popular
among biochemical engineers. Two thin rails of a well defined height of 0.02mm
are attached to the surface of a glass slide that is marked with evenly spaced lines
at 0.05mm intervals. Thus, each square represents a volume of 5.0X10^-8ml.
o Add 0.1 ml of methylene blue and 1 ml of the suspended culture to a test
tube. Mix well. Cells are stained for 3 minutes to enhance visualization.
Add water to dilute the sample as needed. This introduces a dilution factor
which must later be incorporated into the calculation of the original cell
density.
o Clean all the grease from the counting chamber with ethanol so that cells
can be clearly counted later.
o Place a piece of reinforced cover glass on top of the rail. There should be a
small space, 0.02 mm to be exact, between the platform and the cover glass.
 o Fill a capillary pipet with the stained sample. Gently touching the tip of the
pipet against the edge of the cover glass will attract the sample to fill the
space under the cover glass solely by capillary actions.
o The number of cells enclosed in each square is counted visually under an
ordinary light transmission microscope. One may need to raise the oil
immersion lens slightly to shift focus at the space under the cover glass.
Repeat for at least 20 squares. Take the average number of cells per square.
o Calculate the number of cells per ml by multiplying the number of cells per
square by the dilution factor introduced in Step 3a by 1/(5X10^-8), or
2X10^7.
3. Standard Plate Count:
The major part of the procedure deals with a series of successive dilutions of the
original culture in sterile bottles with sterile water. The diluted culture is poured
into Petri dishes along with the nutrient agar. The number of colonies is counted
after incubation.
o Shake the flask containing the culture, and pipet 1 ml of the culture
aseptically into a capped sterile bottle marked "A," which contains 99 ml of
sterile water. Vigorously shake the bottle A to mix the culture and break
any flocculating clumps of microorganisms. The dilution factor in bottle A
is 1:100. A dilution factor of 1:101 is often more easy to work with
physically than 1:100; in this case, the subsequent dilution factor is also
similarly modified without any loss in the accuracy.
o With the second sterile pipet, transfer 1 ml from the bottle A into a similar
bottle marked "B," which also contains 99 ml of sterile water. Shake and
mix. The dilution factor in bottle B is 1:10,000.
o With the third sterile pipet, transfer 1 ml from the bottle B to a Petri dish
marked 1:10,000. Thus, the number of colonies from this dish is multiplied
by 10^4 to give the number of cells in 1 ml of the original sample.
o With the same pipet as Step 4c, transfer 0.1 ml from the bottle B to a Petri
dish marked 1:100,000. The number of colonies from this dish is multiplied
by 10^5 to give the number of cells in 1 ml of the original sample.
o With the same pipet as Step 4c, transfer 1 ml from the bottle B into a third
bottle marked "C," which contains 99 ml of sterile water. Shake and mix.
The dilution factor in bottle C is 1:1,000,000.
o With the fourth sterile pipet, transfer 1 ml from the bottle C to a Petri dish
marked 1:1,000,000. Thus, the number of colonies from this dish is
multiplied by 10^6 to give the number of cells in 1 ml of the original sample.
o With the same pipet as Step 4f, transfer 0.1 ml from the bottle B to a Petri
dish marked 1:10,000,000. The number of colonies from this dish is
multiplied by 10^7 to give the number of cells in 1 ml of the original sample.
o Heat the capped culture tube containing 50 ml of agar to boil for 10
minutes. The heating both melts and sterilizes the agar.
 o After the agar is cooled to 45ºC, pour 12 ml to each of the four Petri dishes.
The culture will be killed if the agar is too hot; it will solidify if it is cooled
for too long. Swirl the plates very gently to mix the culture with the agar.
o Allow the agar to solidify.
o Incubate the plates in the inverted position at 37ºC for 48 hours.
o Select those plates that have 30-300 colonies. Count every colony, large
and small. To keep track of the counted colonies, dot the colonies with a
permanent marker pen as one counts them. Different colored pens may be
used for a mixed culture.

Statistically, the most reliable results are given by plates with between 30
and 300 colonies. Only about two significant figures can be obtained from
this method. The accuracy can be improved if multiple plates can be
prepared. This, however, is rarely done due to the cost.

o Some of the plating may be omitted if the number of cells per ml can be
estimated to the order of magnitude. Finally, a cell counting chamber can
be used in conjunction with cell plating to distinguish the number of viable
and nonviable cells.

(Batista & cs., 2015), (Coutteau, 1996, Malakootian & cs., 2015)
Batista, Isabel, Garcia, Ainhoa, Dalen, Pim, Kamermans, Pauline, Verdegem, Marc &
Smaal, A. C. 2015. Culturing Chaetoceros muelleri using simplified media with
different N sources: effects on production and lipid content. European Journal of
Phycology, 50, 92-99.
Coutteau, Peter 1996. Micro-algae. Manual on the production and use of live food for
aquaculture, 7-48.
Karlson, Bengt, Cusack, Caroline & Bresnan, Eileen 2010. Microscopic and molecular
methods for quantitative phytoplankton analysis.
Malakootian, Mohammad, Hatami, Behnam, Dowlatshahi, Shidwash & Rajabizadeh,
Ahmad 2015. Optimization of culture media for lipid production by
Nannochloropsis oculata for Biodiesel production. Environmental Health
Engineering and Management Journal, 2, 141-147.