RRL

I. Starch
- Raw material
Feasibility of starch to provide glucose as substrate
(​Ange [yellow]​, ​Grace [dark berry]​)

II. Acid Hydrolysis

- Purpose and methods H2SO4 and HCl
- Report at least 3 for each acid fermentation
(​Franze [pink]​, ​Jasmin [purple]​)

Manual from SNS (Starch Hydrolysis)

III. Fermentation
-Support 37 degrees Celcius and 48 hours
- at least 5 research
(​Joe [ green]​, ​Maida [gray]​)

IV. Bacteria Count

- Purpose and methods
(​Loriel [dark magenta]​, ​Ysrael [blue]​)

DEADLINE: JUNE 29

Statement of the Problem

Methodology
Title

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<Start>

Plant starches are synthesized inside plastids by coordinated interactions of multiple

biosynthetic enzymes and deposited in storage organs, such as roots, tubers, fruits, grains
and seeds, as well as in leaves and stems (Tetlow, 2011)
Native/ Natural starch is highly variable in structure functionality between and within plant
species. This variability is evident in granule morphology, amylose content, amylopectin
architecture, and the way these two macromolecular constituents are arranged into
crystalline and amorphous regions within granules. The variability in starch functionality
derives from variability of structure, which is due to diversity in the genes that encode the
starch biosynthetic enzymes and environmental factors that act on the genes and enzymes
concerned during plant growth. (Copeland et al., 2013)

However, native starches are not always suitable for specific industrial applications due to
functional limitations such as low resistance to shear stress, poor thermal properties, and a
high tendency towards retrogradation (Hermannsson and Svegmark, 1996). These natural
limitations can be improved greatly by starch modification, which generally involves physical
(heat-moisture treatment, annealing, pre-gelatinization, high pressure treatment, radiation,
sonication), chemical (cross-linking, substitution, acid hydrolysis, oxidation/bleaching) or
enzymatic processes (BeMiller and Huber, 2011; Miyazaki et al., 2006; Jobling, 2004).
Starch functionality is determined by starch structure, and expanding our understanding of
this relationship could lead to improvements in processing methods and the quality of both
new and traditional food products (Hermansson and Svegmark, 1996; Delcour and Hoseney,
2010).

What is acid hydrolysis:

-----
The oldest but still commonly used chemical modification method is acid hydrolysis, which
dates back to when Nägeli in 1874 and Lintner in 1886 used sulphuric and hydrochloric
acids, respectively, to produce hot water “soluble starch”. Acid hydrolysis modifies the
granular structure of starches, making them display a different behaviour upon heating in
water and produce pastes with lower intrinsic viscosity values, reduced hot paste viscosity,
increased gel strength, increased water solubility, and better film forming capabilities.
Acid-hydrolyzed starches have been used widely for many years in many industries.

Importance of Acid Hydrolysis

As an important modification method, acid hydrolysis of native starch is of high interest due
to its wide industrial applications. Recently, the increasing interest in preparation of
biodegradable nanocomposites with enhanced mechanical and barrier properties and
bio-hydrogen production from acid-hydrolyzed starch highlights the importance of acid
hydrolysis of starch in the production biomaterials and bioenergy (Kapdan and Kargi, 2006;
Kapdan et al., 2009; Le Corre et al., 2010).

</End>
 1. Tetlow, I. J. (2011). Starch biosynthesis in developing seeds. Seed Sci. Res. 21:
5-32. Thirathumthavorn, D., and Charoenrein, S. (2005). Thermal and pasting
properties of acid treated rice starches.
2. Wang, S. J., Blazek, J., Gilbert, E.P., and Copeland, L. (2012). New insights on the
mechanism acid degradation of pea starch.
3. Hermansson, A.-M., and Svegmark, K. (1996). Developments in the understanding of
starch functionality. Trends Food Sci. Technology
4. BeMiller, J. M., and Huber, K. C. (2010). Starch (7th ed.). In: Ullmann’s Encyclopedia
of Industrial Chemistry
5. Miyazaki, M., Huang, P. V., Maeda, T., and Morita, N. (2006). Recent advances in
the application of modified starches for breadmaking. Trends Food Sci. Technology
6. Jobling, S. (2004). Improving starch for food and industrial applications. Curr. Opin.
Plant Biology
7. Delcour, J. A., and Hoseney, R. C. (2010). Principles of Cereal Science and
Technology. (3rd ed.). St. Paul: MN, USA: AACC International.
8. Kapdan, I. K., and Kargi, F. (2006). Bio-hydrogen production from waste materials.
Enzyme Microbio Technology
9. Kapdan, I. K., Kargi, F., Oztekin, R, and Argun, H. (2009). Bio-hydrogen production
from acid hydrolysed wheat starch by photo-fermentation using different Rhodobacter
sp. Int. J. Hydrogen Energy
10. Le Corre, D., Bras, J., and Dufresne, A. (2010). Starch nanoparticles: A review.
Biomacromolecules

Enzymes and inorganic and organic acids can be used to hydrolysed starch. The use
of acids in hydrolysis of starch to glucose must be followed by neutralization and subsequent
production of large amount of salts (Woiciechowski et. al., 2002). Starch is a polymer of
glucose and contains amylase and amylopectin as building blocks that needs to be broken by
different approaches to release the free glucose. The use of enzyme-catalyzed starch
hydrolysis has an advantage like milder reaction condition, however factors such as the high
cost of the initial investment and enzymes, as well as the requirements for specialized labor
and sophisticated laboratories limits the use of enzymes. Acid hydrolysis disadvantage such
as ​the neutralization of hydrolyzates before fermentation, the possible inhibitory effect of the
by-products on yeast growth, and the expensive constructional material for equipment, are
not drawbacks for its industrial use. Important advantages of acid hydrolysis includes a cheap
and easily available acid catalyst, a fast reaction rate, a simple pretreatment for starch
feedstocks, and a relatively low reaction temperature with high acid concentration (​Tasic​ et
al., 2008).
Chavan, et al., (2015) considered two acids, namely concentrated HCl and
concentrated H​2​SO​4, and
​ followed three approaches; cold acid treatment, hot acid treatment
and acid treatment combined with moist heat under pressure (autoclave) for the optimization
of acid hydrolysis process to release the maximum amount of free glucose molecules. The
outcome of the study shows that during the three acid hydrolysis methods used 1 % (V/V)
H​2​SO​4 ​is more efficient than 1% (V/V) of HCl. Results also shows that as the V/V%,
temperature and heating time increases resultant glucose concentration also increases. These
results are similar to the study conducted by ​Ayoola, et al., (2013). Ayoola, et al., (2013)
studies the effect of variation in acid concentration, temperature and time of operation on
glucose obtained during acid hydrolysis of cassava starch. In the study ​50g of cassava starch
was hydrolyzed by dispersing in 150ml of H​2​SO​4​ with solution strength ranging from 0.2M –
1.0M. The slurries obtained were kept in water bath set at different temperatures between
60°C and 100°C within the time range of 30mins and 4hrs. After the specified time, 50ml of
0.1M NaOH was used to neutralize the activity of any trace of H​2​SO​4 that ​ may be present.
The sugar brix and concentration of clear glucose syrup obtained were determined using
DNSA reagent (Ranken method 1984), with spectrophotometer operated at 540nm
wavelength. Results in this study shows that during the 30 minutes of operation the highest
glucose concentration was observed at 100​o​ C while lowest concentration was obtained at 60​o
C. Similar results were observed at 1hour and 4hrs of acid hydrolysis, at 2 hours of acid
hydrolysis maximum glucose concentration was obtained at 0.8M acid concentration and
100​o​C. Thus, during acid hydrolysis, increasing the acid concentration, time and temperature
increases the production of glucose. The complete breaking of α – 1,4 – and α – 1,6 –
glycosidic bonds to glucose (fermentable sugar) required 4 hours of operation at acid
concentration of 1.0M H​2​SO​4 at
​ 100​o​C. ​Woiciechowski et. al., (2002) also employed an
experimental factorial design in optimizing the physical conditions for acid hydrolysis, where
reducing sugar concentration was the response variable. In the experiment 5g of cassava
bagasse and 50ml of the acid solution (0.5, 1 and 1.5% HCl) were used. Cassava bagasse in
acid concentration underwent a different temperatures (100, 120 and 130° C) for 5, 10 and 15
minutes. Results in the researchers experiment shows that for batch process, conditions for
best acid hydrolysis which resulted 62.4 g of reducing sugars from 100 g of cassava bagasse
were at 1% HCl concentration, temperature of 120​o​C and reaction time of 10 minutes. This
amount of the reducing sugars obtained represents 94.5% of recovery of reducing sugars from
the starch available in cassava bagasse.
Sweet potato that undergoes acidic hydrolysis has an optimum parameter at 57​o​C,
10hrs and 0.59M ​H​2​SO​4 for
​ temperature, time and acid concentration respectively as observed
by Ohno et al. (2018) in their study. The optimum parameters were established by preparing
50cm​3​ each of the prepared concentration of the acid (0.2M, 0.4M, 0.6M, 0.8M and 1M) and
added into 250 ml conical flask with dosage of 3g ground sweet potato peels and heated at an
agitation rate of 300 rpm at different temperatures of (30˚C, 40˚C, 50˚C, 60˚C and 70˚C) and
different time intervals (1,4,8,12 and 16 hours). The DNS method and modified DNS
Reagent were used to determine the reducing sugar produced. To 1.0 ml of the hydrolyzed
supernatant, 3.0 ml of the DNS solution and 1ml of sodium acetate buffer were added to the
test tube, boiled for 15 minutes, cooled and diluted appropriately after which their absorbance
were measured at a wavelength of 540 nm using a UV-Visible Spectrophotometer (model).
Outcome of their study shows that the reducing sugar concentration increases with increase in
acid concentration up to 0.6M Sulphuric acid and decreases when the acid concentration
exceeds 0.6M this could be due to the degradation of product monomers (cellulose and
hemicellulose) by acid. At 12 hrs hydrolysis time, maximum reducing sugar yield
(0.124g/100ml) was obtained, having a hydrolyzing time more than 12 hrs decreases the
reducing sugar these could be a result of decrease in cellulose content of the peel. The result
also showed that increasing temperature had a positive effect on reducing sugar (glucose)
yield. Maximum sugar yield of 0.141g/100ml was obtained at 60˚C, after which the yield
decreased. A similar observation was reported by Gewchingduang and Pengthemkeerati,
(2010). Palmarola et al., (2005) confirmed that higher hydrolysis temperature would enhance
the formation of by products that might have adverse effects on hydrolysis process. Hence,
hydrolyzing sweet potato peels with sulphuric acid at 60˚C for 12 hrs gave the best reducing
sugar yield in this study.
Another study conducted by T ​ asic​ et al., (2008) on t​ he hydrolysis of starch from fresh
potato tubers by HCl and H​2​SO​4​ at different ratios of plant material to acid solution shows
that the final reducing sugars concentration in the hydrolyzates depended on the type and
concentration of acid and the ratio of plant material to acid solution but not on the type of
potato. The highest dextrose equivalent of 94%, were achieved using 1 M HCl at the ratio of
plant material to acid solution of 1:2 (w/v). Tasic​ et al., (2008) performed the hydrolyss in a
0.5L ​glass three-necked roundbottom flask, equipped with a stirrer and a reflux condenser,
immersed in a boiling water bath. The acid solution (HCl or H2SO4, with a concentration of
0.5–2 M) was poured into the flask and heated until boiling. Then, the potato tuber mash was
added, so that the total volume of the reaction mixture was 200 ml. The ratio of plant
material to acid solution was 1:0.75, 1:1 or 1:2 (w/v). During the hydrolysis process, samples
of 3–5 ml were periodically taken from the reaction mixture, neutralized with 5 M KOH and
centrifuged at 3000 min​-1​ for 15 min. After appropriate dilution of the supernatant, the
concentrations of reducing sugars were determined. The DE (%) was then calculated from
the concentration of total reducing sugars, calculated as glucose, measured
spectrophotometrically at 580 nm using the color reaction with picric acid.

​References
Chavan, R., Saxena K.,​ ​& Tigote D., (2015) Optimization of Acid Hydrolysis Process for Free
Glucose Recovery From Starch. International Journal of Innovative Science, Engineering &
Technology, vol. 2 Issue 12.

Ayoola, A. A., Adeeyo, O. A., Efeovbokhan, V. E., & Olasimbo, A. (2013). Optimum
hydrolysis conditions of cassava starch for glucose production. International Journal of
Advanced Research in IT and Engineering, 2(1), 93-101.

Woiciechowski, A.L., Nitsche S., Pandey A., & Soccol C.R. (2002) Acid and Enzymatic
Hydrolysis to Recover Reducing Sugars from Cassava Bagasse: An Economic Study.
Brazilian Archives of Biology and Technology vol. 45 no. 3.

Ohno, I., Onyebuchukwu, G., & Ejike, F., (2018). Optimization of Acidic Hydrolysis of Sweet
Potato Peels To Produce Fermentable Sugar. IOSR Journal of Engineering vol. 08, issue 6, pp
20-26.

Tasić, M. B., Konstantinović, B. V., Lazić, M. L., & Veljković, V. B. (2009). The acid
hydrolysis of potato tuber mash in bioethanol production. Biochemical Engineering Journal,
43(2), 208–211.
 PHAs are synthesized in bacterial cells through a metabolic process. The substrates for
biosynthesizing PHAs are usually restricted to small molecules, since bacteria have thick, rigid cell
walls surrounding membranes. Subtrates for PHA synthesis can be generally categorized into sugars
(monosaccharides), triacylglycerol and hydrocarbons (Jiang, 2016).
In the biosynthesis of PHAs, the bacterial cells can be regarded as biochemical catalysts,
carrying out the metabolic reactions to transform the substrates into different PHAs. Just like chemical
catalysts have selectivity to substrates and the same substrate can be converted into different products
by using varying catalysts, the same is true for PHA-producing bacteria due to their substrate
specificity (Chen, 2009).
Simple sugars subtrates are basically derived from carbohydrates which can then be classified
as monosaccharides, oligosaccharides and polysaccharides. Monosaccharides and disaccharides can
be readily fermented to produce PHA, while polysaccharides are not fermentable unless hydrolyzed
first. Polysaccharides largely consists of starch, cellulose and hemicellulose. Starch is a main
constituent of corn, rice, potato, and cassava. Under enzymatic or acid hydrolysis, starch is hydrolyzed
first into maltose and then glucose, which is the most extensively-used carbon source for industrial
production of PHAs.
A study conducted by Kim et. al (1994) shows that using glucose as a carbon source,
productivity of up to 2.42 g/ L-h can be achieved. Yield of PHA can be approximated at 0.3–0.5 g/g of
glucose, while PHA content of 78% was attained.

GRACE
start---PHB is produced as intracellular carbon and energy reserves by a wide variety of
microorganisms, analogous to fat storage in humans. Bacterial PHB synthesis can be
induced by limiting oxygen or an essential nutrient such as nitrogen, phosphate, sulfate,
magnesium, or potassium. In their absence, microorganisms cannot produce amino acids or
proteins, but instead synthesize and accumulate PHB as discrete granules in the presence
of excess carbon. They store this carbon until the limitation is removed, at which time they
may degrade and metabolize it (Hankermeyer & Tjeerdema, 1999). According to Sing et. al.
(2009), there are a lot of prokaryotic organisms that accumulates PHB. Although PHB
accumulates within the bacteria to levels as high as 90% (w/w) of the cell mass (Verlinden et
al., 2007), it hasn’t been commercialized due several factors such as its cost of production.

The cost of production of PHB is greatly affected by the choice of carbon source,
accounting up to 50% of the overall cost (Rata et al., 2018). Carbon source refers to any
carbon containing molecule used by an organism for the synthesis of its organic molecules.
Choosing the carbon source for a specific bacterium is very critical since this would dictate
the rate of growth and the amount of PHB accumulation. This includes carbohydrate, amino
acid, fatty acid, CO2. One of the most common types of carbohydrates is starch.

Starch is the major carbohydrate reserve in higher plants making it renewable and
biodegradable. It is certainly one of the most versatile materials for potential use in polymer
technology. It can be converted, on the one hand, into chemicals like ethanol, acetone, and
organic acids, used in the production of synthetic polymers and, on the other hand, it can
produce biopolymer through fermentative processes or be hydrolyzed and employed as a
monomer or oligomer. Finally, it can be grafted with a variety of reagents to produce new
polymeric materials, used as such or as fillers for other polymers (Carvalho, 2013).

The use of starch for the production of other chemicals was recently reviewed by
scientists. In all of the processes involved, the macromolecular structure of starch is
destroyed and the polymers derived from the ensuing monomers are totally different from it.
It is important to emphasize that the same monomers (e.g. ethylene, sugars, and dextrin)
can be produced from other sources, both renewable, such as cellulose and sugar cane, and
nonrenewable such as oil (Carvalho, 2013).

Starch or amylum is a polymeric carbohydrate consisting of a large number of

glucose units joined by glycosidic bonds. In most conditions, glucose is the best carbon
source for many bacteria including Bacillus subtilis because it provides faster growth than
other sugars, and is consumed first in sugar mixtures (Bren et. al., 2016). According to
Wacker et. al. (2003), Bacillus subtilis utilizes glucose and glutamine as the preferred
sources of carbon and nitrogen, respectively.

There are existing researches which evaluates various materials for PHB production
using glucose as their carbon source. ------ end

A research conducted by Gowda and Shivakumar (2014) identified the innate

enzymatic potential of the PHB producing strain B.thuringiensis and explored the same for
cost-effective production of PHB using agrowastes. Nine substrates (agro and food wastes)
namely; rice husk, wheat bran, ragi husk, jowar husk, jackfruit seed powder, mango peel,
potato peel, bagasse and straw were subjected to two treatments- acid hydrolysis and
enzyme hyrdolysis, the reducing sugars released thereby were utilized for polymer
production. Production studies were carried out in 250 mL flasks containing 50 mL culture
medium and incubated at 37°C on a rotatory shaker at 120 rpm for 48 h. After 48 h
incubation at 37°C, 5.0 mL of the culture was taken and centrifuged at 8000 x g for 15 min.

Another research considered soil as its source for PHB production. Screening and
Isolation of bacterial strains: 1 gm soil samples were inoculated into 250 ml flask containing
100 ml sterile Nutrient broth. Flasks were incubated at 37°C for 24 hrs on rotary shaker at
120 rpm. Complete medium Glucose, Fructose, sucrose ,maltose and lactose were used as
carbon source, whereas Ammonium sulfate, Malt extract, Peptone and Yeast extract were
tested for their ability to utilize nitrogen source. For large-scale growth, Inoculums was
prepared in nutrient broth medium at 37°C and transferred to 500 mL of nutrient broth in a
wide-necked 1 L culture flask, incubated at 37°C for 48h with continuous gentle shaking.

A statistical optimization of fermentation conditions was conducted by Yadav, J. et al (2017)

wherein a Plackett-Burman Design was the initial optimization tool that was utilized for
screening the most effective factors for optimal production of PHB and bacterial growth. A
design of 12 experiments for 11 factors namely (glucose, peptone, sodium chloride,
K2HPO4, 4 KH2PO4, ammonium sulphate, ammonium chloride, sodium sulphate,
temperature, inoculum size and pH) was formulated using PB Design (Design Expert 5.0.9,
StatEase Inc, USA). The 11 factors consisting of medium components and operating
conditions were tested at two levels, low level (-1) and high level (+1). These factors were
selected on the basis of these being C-source (glucose), N-source (Peptone, ammonium
sulphate, ammonium chloride), phosphate (K2HPO4, KH2PO4), inorganic salts (NaCl,
Na2SO4) and culture conditions (T, pH, inoculum size) which all affect the growth and
possibly can affect PHB production. The range of factors were chosen using literature
reports conducted on PHB production in order to study the intriguing interaction between the
high and low values of different factors (Demain, 1958; García-Torreiro et al., 2016). In order
to study the effect of limiting substrate (glucose) on growth of B. subtilis cells studies were
done in LB media with varying initial concentrations of glucose. The kinetic parameters of
PHB production by B. subtilis was determined by batch culture in shake flasks upto 48 hours
using LB media containing 20 g/l of the limiting substrate (glucose). Agitation was done in a
rotating shaker set at 120 rpm and temperature of 37 ˚C.

Gowda, V., Shivakumar, S. (2014). Agrowaste-based Polyhydroxyalkanoate (PHA)

Production using Hydrolytic Potential of Bacillus thuringiensis IAM 12077 Vol.57, n.1: pp.
55-61

Shah, K.R. (2014).Optimization and production of Polyhydroxybutarate(PHB) by Bacillus

subtilis G1S1from soil. International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 3 Number 5 (2014) pp. 377-387

Yadav J., Balabantaray S. & Patra, N. (2017): STATISTICAL OPTIMIZATION OF

FERMENTATION CONDITIONS FOR THE IMPROVED PRODUCTION OF
POLY-β-HYDROXYBUTYRATE FROM Bacillus Subtilis, Chemical Engineering
Communications

Plate Count Method

*** FOR INTRO***
The determination of microbial count has been a major procedure and standard analyses
worldwide. There are several methods to determine the number of microbial cells in a
sample such as direct counting, used to determine bacterial concentration without the need
for advanced equipment and is widely used in natural environments; and viable cell counting
or plate count method, which allows the identification of the number of active cells in a
sample relying on the growth of bacteria in a nutrient medium.
In this research, after gram staining to prove the presence of PHB by fermentation of the
agro wastes using Bacillus Subtilis, it is needed for us to determine which of the raw
materials or the mixture of raw materials having an equal ratio is best by determining the
number of bacteria remaining after fermentation under the standard operating conditions.
Since the viable cell counting method identifies the number of active cell at a certain time
and it is best used as a counting method for nutrient mediums, this was chosen the chosen
method for bacterial count.
One of the major properties of Bacillus subtilis is its self-lysing genes, which means that after
it has synthesized PHB the bacteria will undergo destruction without the need for other
external forces or methods. With the same amount of bacteria to be used in each test
sample, we can determine the best raw material for PHB production. After the fermentation
time, plate count method will be used to determine the number or remaining bacteria from
the fermented samples, results will be tabulated and will be compared on which one has the
least amount of active bacteria. It will be concluded from the data gathered that the sample
having the least amount of live bacteria has produced the greatest amount of PHB.
Plate counting method works in the principle of serial dilutions. A sample to be counted is
diluted in a solution that will not harm the microbe, yet does not support its growth (so they
do not grow during the analysis). In most cases a volume of liquid (or a portion of solid) from
the sample is first diluted 10-fold into buffer and mixed thoroughly. In most cases, a 0.1-1.0
ml portion of this first dilution is then diluted a further 10-fold, giving a total dilution of
100-fold. This process is repeated until a concentration that is estimated to be about 1000
cells per ml is reached. Samples will be poured onto agar plates and then incubated at a
time depending on the bacteria used and the growth medium, the fermentation medium in
this case. After the period of incubation, it is now possible to count the colonies present in
the original sample.

***FOR RRL***
One of the classic ways to determine the concentration of microbes in a sample is to dilute
the sample, grow the microbes on plates and count the colonies. The plated microbes grow
from a colony forming unit consisting of one or more cells into a visible colony that can be
seen and counted. Bacteria are the most common microbe to assess using plate counts.
Colony counts are used to detect and count microbes in soil, water and food. Protocols for
counting colonies emphasize an accurate and methodical approach (Becker, A. 2018).
The Ibers: Institute of Biological, Environmental and Rural Sciences stated that one of the
most fundamental microbiological techniques is plate counting which is used to determine
the number of viable (i.e. living) cells in a sample. There are several steps to the technique
and all must be carried out carefully in order to obtain accurate results. Aseptic technique
must be used throughout. Aseptic technique is the term given to a collection of procedures
that aim to avoid contamination of the sample. This involves holding tubes at an angle,
flaming lids of bottles, using sterile pipettes etc. The plate count method or spread plate
relies on bacteria growing a colony on a nutrient medium. The colony becomes visible to the
naked eye and the number of colonies on a plate can be counted.
The technique is sensitive and has the advantage of only counting living bacteria, which is
often the important issue. Any concentration of microorganism can be easily counted, if the
appropriate dilution is plated. It is even possible to concentrate a solution before counting, as
is often done in water analysis, where bacterial populations are usually at low density. The
equipment necessary for performing viable plate counts is readily available in any
microbiology lab and is cheap in comparison to other methods. Finally, by using a selective
medium it is possible to determine the number of bacteria of a certain class, even in mixed
populations. These advantages have made viable plate counts a favorite of food, medical,
aquatic and research laboratories for the routine determination of cell number. (Sanders,
2012)
In most cases a volume of liquid (or a portion of solid) from the sample is first diluted 10-fold
into buffer and mixed thoroughly. In most cases, a 0.1-1.0 ml portion of this first dilution is
then diluted a further 10-fold, giving a total dilution of 100-fold. This process is repeated until
a concentration that is estimated to be about 1000 cells per ml is reached. In the
spread-plate technique some of the highest dilutions (lowest bacterial density) are then
taken and spread with a sterile glass rod onto a solid medium that will support the growth of
the microbe. It is important that the liquid spread onto the plate soaks into the agar. This
prevents left over liquid on the surface from causing colonies to run together and the need
for dry plates restricts the volume to 0.1 ml or less. (Sanders, 2012)

METHODOLOGY

Step One: Diluting the sample

Depending on the source of the sample used there might be thousands, millions or even
billions of microorganisms per millilitre of sample. This is too many for us to count so we
dilute the sample.
1ml of sample is added to 9ml of a suitable diluent (e.g. sterile buffer). The sample and
diluent are mixed together.
This new sample (Dilution One) has a concentration (number of microorganisms per ml)
1/10th that of the original sample. 1ml of Dilution One is added to another 9ml of diluent to
make Dilution Two. Dilution Two has a concentration 1/10th that of Dilution One and 1/100th
that of the original sample.
This process is repeated until we have a series of dilutions.

​ tep Two: Plating the sample

S
To find out how many viable cells are in each of our dilutions we need to put some of the
sample onto an agar plate. The agar plate is prepared by mixing growth medium with agar
and then autoclaving to sterilise. Once the agar has cooled to ~50°C approximately 15ml is
poured into a sterile Petri dish and left to set.
0.1ml of sample is pipetted onto the agar surface and spread around using a sterile glass
rod. We usually put 0.1ml of one of our sample dilutions onto a plate – if we use more than
this it can make the plates very wet and if we use less than this it is difficult to spread evenly.
This is repeated so that we have 2 or 3 repiclate plates for our original sample and for each
of our dilutions.

Step 3: Incubating the plates

Once all of the plates have been prepared they are left to dry and then moved to an
incubator at a suitable growth temperature for the microorganism being studied. The
incubation time depends on the organism and the growth medium but during the incubation,
each viable cell that was spread to a discrete position on the agar surface will grow and
divide many times to form a visible colony of microorganisms.

After the incubation period we can count the colonies to determine how many
microorganisms were present in the original sample.

Step 4: Counting the colonies

The plates will have different numbers of colonies depending on the dilution of the sample. If
there are too many colonies it can be impossible or very difficult to count them. If there is
only a small number of colonies it is easy to count them but the results are prone to error. As
a compromise we always aim to count plates with between 30 and 300 colonies.
We record our results noting the dilutions that had between 30 and 300 colonies and how
many colonies there were on these plates.

Step 5: Determining how many viable​ organisms were in the original sample
In this step we need to use our results from Step 4. We need to take account of the amount
by which we diluted the sample in Step 1 and the volume that we put onto the plate in Step
2.

For example if the plates prepared from Dilution Two (1/100th the original sample; 10-2
dilution) had an average of 40 colonies on them we need to multiply 40 by 100 to take
account of the dilution and then by 10 because we only plated 0.1ml.

Plate Counting. (n.d.). Retrieved May, 2019, from

http://users.aber.ac.uk/hlr/mpbb/index_files/Page299.html
Ibers: Institute of Biological, Environmental, and Rural Sciences.
Becker, A. (2019, March 02). How to Count Colonies in Microbiology. Retrieved June 03,
2019, from https://sciencing.com/count-colonies-microbiology-17859.html

Sanders, E. R. Aseptic Laboratory Techniques: Plating Methods. J. Vis. Exp. (63), e3064,
doi:10.3791/3064 (2012).