TUGAS MPA TK2102

FIRA SALSABILA

13014042

A. HOW TO COUNT MICROORGANISMS BY SPECTROPHOTOMETRY

The increase in the cell size and cell mass during the development of an organism is
termed as growth. It is the unique characteristics of all organisms. The organism must require
certain basic parameters for their energy generation and cellular biosynthesis.
The growth of the organism is affected by both physical and Nutritional factors. The
physical factors include the pH, temperature, Osmotic pressure, Hydrostatic pressure, and
Moisture content of the medium in which the organism is growing. The nutritional factors
include the amount of Carbon, nitrogen, Sulfur, phosphorous, and other trace elements
provided in the growth medium. Bacteria are unicellular (single cell) organisms. When the
bacteria reach a certain size, they divide by binary fission, in which the one cell divides into
two, two into four and continue the process in a geometric fashion. The bacterium is then
known to be in an actively growing phase.
To study the bacterial growth population, the viable cells of the bacterium should be
inoculated on to the sterile broth and incubated under optimal growth conditions. The
bacterium starts utilizing the components of the media and it will increase in its size and
cellular mass. The dynamics of the bacterial growth can be studied by plotting the cell growth
(absorbance) versus the incubation time or log of cell number versus time. The curve thus
obtained is a sigmoid curve and is known as a standard growth curve. The increase in the cell
mass of the organism is measured by using the Spectrophotometer.
The Spectrophotometer measures the turbidity
or Optical density which is the measure of the amount of
light absorbed by a bacterial suspension. The degree of
turbidity in the broth culture is directly related to the
number of microorganism present, either viable or dead
cells, and is a convenient and rapid method of measuring
cell growth rate of an organism. Thus the increasing the turbidity of the broth medium
indicates increase of the microbial cell mass (Fig 1) .The amount of transmitted light through
turbid broth decreases with subsequent increase in the absorbance value. In
spectrophotometry, cultures usually do not need to be diluted, although above a certain
 cell density the results lose reliability. Of all the electrical appliances used for counting cells, a
spectrophotometer is the cheapest and its operation the fastest and most straightforward.
This has made spectrophotometry the methods of choice for quick measurements of bacterial
growth and related applications. There are spectrophotometers in which several cuvettes can
be inserted at one time, reducing work time even more. Additionally, there are
spectrophotometers that require extremely small volumes of culture, as little as 1 microliter .
This, combined with the stochastic nature of liquid cultures, enables only an estimation of cell
numbers. In either case plotting the log of turbidity or number of living cells versus time is
referred to as the growth curve. The generation time is defined as the time required for
bacterial mass to double

SOURCES

Boundless. “Measurements of Microbial Mass.” Boundless Microbiology. Boundless, 06 Sep. 2015.

Retrieved 27 March 2016
from https://www.boundless.com/microbiology/textbooks/boundless-microbiology-
textbook/culturing-microorganisms-6/counting-bacteria-63/measurements-of-microbial-
mass-385-7951/
http://vlab.amrita.edu/?sub=3&brch=73&sim=1105&cnt=1. 26 March 2016 at 21.05 WIB
B. HOW TO DETERMINE PROTEIN CONCENTRATION BY SPECTROPHOTOMETRY

Quantifying protein using absorbance at 280 nm

Considerations for use
Quantifying protein by directly measuring absorbance is fast and convenient, since no
additional reagents or incubations are required. No protein standard need be prepared and
the procedure does not consume the protein. The relationship of absorbance to protein
concentration is linear. Because different proteins and nucleic acids have widely varying
absorption characteristics there may be considerable error, especially for unknowns or protein
mixtures. Any non-protein component of the solution that absorbs ultraviolet light will
intefere with the measurements. Cell and tissue fractionation samples often contain insoluble
or colored components that interfere. The most common use for this method is to monitor
fractions from chromatography columns, or any time a quick estimation is needed and error
in protein concentration is not a concern.This method is recommended for calibrating bovine
serum albumin or other pure protein solutions for use as standards in other methods.

Principle
Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200
nm. Amino acids with aromatic rings are the primary reason for the absorbance peak at 280
nm. Peptide bonds are primarily responsible for the peak at 200 nm. Secondary, tertiary, and
quaternary structure all affect absorbance, therefore factors such as pH, ionic strength, etc.
can alter the absorbance spectrum.

Equipment
In addition to standard liquid handling supplies a spectrophotometer with UV lamp
and quartz cuvette are required.

Procedure
Carry out steps 1-4 (280 nm only) for a very rough estimate. Carry out all steps if nucleic
acid contamination is likely.

1. Warm up the UV lamp (about 15 min.)

2. Adjust wavelength to 280 nm
3. Calibrate to zero absorbance with buffer solution only
4. Measure absorbance of the protein solution
5. Adjust wavelength to 260 nm
6. Calibrate to zero absorbance with buffer solution only
 7. Measure absorbance of the protein solution

Analysis
Unknown proteins or protein mixtures. Use the following formula to roughly estimate protein
concentration. Path length for most spectrometers is 1 cm.

Concentration (mg/ml) = Absorbance at 280 nm divided by path length (cm.)

Pure protein of known absorbance coefficient. Use the following formula for a path length of
1 cm. Concentration is in mg/ml, %, or molarity depending on which type coefficient is used.

concentration = Absorbance at 280 nm divided by absorbance coefficient

To convert units, use these relationships:

Mg protein/ml = % protein divided by 10 = molarity divided by protein molecular weight

Unknowns with possible nucleic acid contamination. Use the following formula to estimate
protein concentration:

Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260)

Comments
Cold solutions can fog up the cuvette, while warm solutions can release bubbles and interfere
with the readings. For concentrated solutions (absorbance greater than 2) simply dilute the
solution.

Absorbance coefficients of some common protein standards:

 Bovine serum albumin (BSA): 63

 Bovine, human, or rabbit IgG: 138
 Chicken ovalbumin: 70

Advantages

This technique is quick and doesn’t require any special reagents, except for the
guanidinium, which you may have on hand anyway.

Disadvantages
 This method relies on having an accurate extinction coefficient for your protein, which
depends on the number of aromatic residues. If there aren’t a decent number of aromatic
residues, your extinction coefficient will be quite low, and you will need a fairly concentrated
sample to get a reasonable absorbance (generally an absorbance between 0.1 and 1.0 is
considered within the “linear range”). Also, ProtParam warns that there may be at least a 10%
error in the extinction coefficient if there are no tryptophans in your protein. Therefore, if
your extinction coefficient is low, which is likely the case if there are no tryptophans in the
sequence, a 10% error could significantly throw off your assessment of the final protein
concentration.

SOURCE

Layne, E. Spectrophotometric and Turbidimetric Methods for Measuring Proteins. Methods in

Enzymology 3: 447-455. 1957.

Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69. 1990.

http://www.ncbi.nlm.nih.gov/pubmed/18228395. 26 March 2016 at 20.59 WIB

http://bitesizebio.com/10178/how-to-measure-protein-concentration-more-accurately/ . 26
March 2016 at 21.21 WIB

http://www.ruf.rice.edu/~bioslabs/methods/protein/abs280.html. 25 March 2016 at 23.33

WIB
C. HOW TO MEASURE PHENOL CONCENTRATION IN WASTE BY SPECTROPHOTOMETRY

Phenol produced as petrochemical waste, oil, coke industry, and other chemical
industries. Normally, phenol consisted in water is less than 1 µg/L, yet the concentration is up
to 20 µg/L in other place. Phenol concentration from 10 upto 100 µg/L can be tested by taste
and smell.

Phenol analysis can use SNI 06-6989.21-2004. This method can be used to determine
the concentration of phenol in water and wastewater by using aminoantipirine with
spectrophotometer. Phenol concentration measured 0,005 mg/L upto 0,1 mg/L use
wavelength of 460 nm and for higher concentration more than 0,1 mg/L use wavelength of
500 nm.

The metod principal is that phenol in water will react with 4-aminoantipirin in pH of
7,9 ± 0,1 in potassium ferri cyanide solution and will produced brownish red color from
antipirin and the absorbance of color produced will be measured in wavelength of 460 nm
atau 500 nm.

Following chemical reaction :

SOURCE

https://environmentalchemistry.wordpress.com/tag/fenol/

www.hach.com

SNI 06-6989.21-2004. Air dan air limbah – Bagian 21: Cara uji kadar fenol secara
Spektrofotometri