BIOLS 404

BIOTECHNOLOGY
Lab manual
2016
Prof. ESSAM Ghanem Dr. Salwa Al-Thawadi

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Table of content

1- Isolation of Industrially Important Microorganisms

a. Isolation of Actinomycetes for Antibiotic Production from Soil (3-4 weeks
i. Enrichment of Actinomycetes (week 1)

ii. Purification Actinomycetes (week 3)

iii. Activity Test of Actinomycetes against test organisms (week 4)

2- Isolation of Proteolytic Bacteria from Soil (1 – 2 weeks)

a. Protease Production (week 1)
b. Measuring the activity of proteolytic bacteria

3- Biosynthesis of Nanoparticles (Nickle and Cobalt)

a. Enrichment of nanoparticles producing bacteria
b. b. Isolation of pure strain which precipitate Nickle and Cobalt
nanoparticles
c. Measuring the activity of nanoparticles producing bacteria

4- Precipitation of Calcium Carbonate Particles by Bacteria (BioCementation

reaction)
a. Enrichment
b. Precipitation of calcium carbonate particles on microscopic slides
c. Measuring the activity of Calcite
d. BioGrout technology (Demo)

5- Long term preservation by Lyophilizer and glycerol method

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 Isolation of Industrially Important Microorganisms
Isolating microorganisms from nature is the microbiologist's first step in screening
for natural products such as secondary metabolites and enzymes. It is possible to
isolate many different microorganisms by employing enrichment techniques or
even single-cell isolation by the use of capillary methods. However, for industrial
screening, such enrichment techniques usually require an inordinate amount of
time, labor, and money, since only a few species of particular genus arise from any
one sample. Also, the industrial screen's assay procedures may require
modification to suit the growth and metabolism of every different genus. Thus a
more scientific approach for isolation is required. One successful approach for the
discovery of new antibiotics and enzymes involves:

1. Considering the desired product characteristics and process development.

2. Using ecological approaches for isolation and screening.

Consideration of the desired product characteristics, process development, or

both may aid in answering the first major question: What do I isolate and from
where?

Using ecological approaches to isolate microorganisms can provide a screen with

both a large number and a wide variety of microorganisms to examine the product
of interest.

The present exercise is therefore designed to enable students to understand and

practice isolation of industrially important microorganisms for example
actinomycetes and bacteria from a particular ecosystem. The first experiment
describes selective collection and isolation procedures and media for
actinomycetes and bacteria. After isolation, the culture is tested for its ability to
produce new products or activities. This experiment will be addressed as
"Screening for New Products from Microorganisms".

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 Experiment 1
Isolation of Actinomycetes for Antibiotic Production from Soil
(Weeks one, two and three)

a. Isolation of Actinomycetes (week one)

Materials:

A. 1 ml sterile pipette
B. Soil (agriculture, non-agriculture)
C. Incubator 30oC and 55oC.
D. Balance
E. Oven 100-120oC.
F. Cork borer.
G. Microfilter and syringe (1 ml).
H. Sterile pestle and stamped.
I. Sterile 10 ml test tubes (5).

Media (per one group)

A. 9 ml sterile dilution tubes

B. 90 ml sterile water in a bottle
C. AV plates (10)
D. ST +0.2 g/L yeast extract + antibiotic (4)
E. 100 ml ST broth in 250 ml flask
F. 100 ml agar (45oC)
G. Test organisms
a. B. subtillus,
b. Staphylococus aureus,
c. E. coli, and
d. Pseudomonas aeruginosa

Methods

1. All soils are air dried in sterile glass Petri dishes at room temperature 3 to
10 days (depending upon moisture content) to aid in reducing the bacterial
population.

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2. The dried soils are then gently powdered with a sterile pestle and
“stamped”.
3. Heat the soil at 100 to 120oC for one hour to kill the vegetative cells.
4. Weight 10 g soil and suspend in 90 ml sterile distilled water.
5. Shake the suspension vigorously for 10 min and let it stand for another 10
min.
6. Using a sterile pipette, remove 1 ml from suspension, add it to 9 ml sterile
water. Vortex well, remove 1 ml and add it to next tube. Thus make serial
dilutions until you reach 10-4 (Fig.1).
7. Plate 0.1 ml of the suspension of 10-3 and 10-4 using L-shaped sterile
glass rod using a turn table on AV agar (Arginine vitamin agar), and SN
(starch nitrate media+ antibiotic+ yeast extract).
8. Take 1 ml from the dilution factor (10-3) in 90 ml ST broth (starch nitrate
broth supplemented with 0.2 glL yeast extract) in 250 ml flask.
9. Incubate the media and broth for 1-30 days at 30oC and 55 oC.
10. Observe the different types of actinomycetes after one week. Take
photographs of your plates for data analysis.

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 10 g soil 1 ml

90 ml

10-1

10-2 10-3 10-4

0.1 ml 0.1 ml

1 ml

Incubate
for 24 hrs
A
SN VA Milk
media
SN VA Milk
media
Incubate Incubate Incubate
for 1 week for 24 hrs for 1 week

100 ml

Figure 1: Isolation of Actinomycetes (SN and VA media) and proteolytic

bacteria (Milk media). SN broth

b. Purification Actinomycetes (week 2)

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 Figure 2: Morphological identification of actinomycetes

Figure 3: Actinomycetes, G+

1. Examine the different actinomycetes according to cell shape, aerial color,

mycellium color, substrate color and any diffusible pigment.
2. Select new colonies (maximum 4) and streak them on starch nitrate media
(supplemented with yeast extract) and starch nitrate broth (supplemented
with yeast extract) (Fig. 2).
3. Incubate at 30oC and 55oC depending on the microbe.

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 Figure 4: Streaking of actinomycetes on SN o AV plates

c. Activity Test of Actinomycetes against Test Organisms (week 3)

1. Under aseptic technique, use a sterile cork borer to make disks from the
plates of pure actinomycete.
2. To test the endogenic antibiotic activity, dilute the test organisms (B.
subtillus, Staphylococus aureus, E. coli and Pseudomonas aeruginosa)
(Fig. 3) (serial dilution to reach 10-4). Take 1 ml from each test organism
place it in 90 ml (NA) Nutrient agar (45oC), mix well without introducing air
and pour it immediately into plates (100 ml is enough to prepare 5 NA
plates). Let to them solidify.
3. Take the actinomycetes disks for different types (maximum 4) and place
them on the surface of the test organism NA agar plates (Fig.3).
4. Incubate the plates at either 30oC or 55 oC depending on the type of
isolated actinomycete.
5. To test the exogenic antibiotic activity, take the suspended pure
actinomycete broth and get the supernatant by syringe filter in sterile 10
ml test tube (Fig. 4).
6. By cork borer, make 4 wells far away from each other inside the test
organisms NA plates (Fig. 4).
7. By a sterile pipette, take a quantity of the supernatant enough of different
actinomycetes to fill the wells. The quantity of the filter sterilized
supernatant should be equal in the wells. Incubate the plates under the
desired temperature for 24 hours.

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Figure 5: Endogenic antibiotic activity test of actinomycetes against test organism.

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Figure 6: Exogenic antibiotic activity test of actinomycetes against test organism.

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 Experiment 2
Isolation of Proteolytic Bacteria from Soil
(Weeks one, two and three)

• Protease Production (week one)

Materials

J. 1 ml sterile pipette
K. Soil (agriculture, non-agriculture)
L. Balance
M. Incubator 30oC and 55oC.
N. Oven 100-120oC.

Media (per one group):

H. 9 ml sterile dilution tubes

I. 90 ml sterile water in a bottle
J. Milk agar media (5)

Methods:

1. Repeat steps 1,2 and 3 in experiment 1.

2. Heat the soil at 100 to 120oC for one hour to kill the vegetative cells.
3. Weight 10 g soil and suspend in 90 ml sterile distilled water.
4. Shake the vigorously for 10 min and let it to stand for another 10 min.
5. Using a sterile pipette, remove 1 ml from suspension, add it to 9 ml sterile
water. Vortex well, remove 1 ml and add it to the next tube. Thus make
serial dilutions until you reach 10-4 (Fig.1 ).
6. Plate 0.1 ml of the suspension of 10-3 and 10-4 using L-shaped sterile
glass rod using a turn table on milk media.
7. Incubate the media plates at 30oC and 55oC for 24 hours.
8. Observe the different types of bacteria that will form clear zone around
them and measure their diameter (Fig. 4).

b. Purification of Bacteria (week 2)

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 1. Isolate the bacterium that form clear zone when grown on milk media plate
and streak it on a new milk plate.
2. Incubate the plates at 30oC and 55oC for 24 hours.
3. Next day, keep the growing bacteria at fridge to be used next lab.

Preparation of Media
Milk agar

A. Skim milk 30 g and Distilled water 300 ml

B. NA (23 g), yeast extract (1.4 g), distilled water (700 ml)
Autoclave each component separately then mix them aseptically near the
flame

Starch Nitrate Agar Media (g/L)

Starch (20 g)
K 2 HPO 4 (1.6 g)
NaNO 3 (2 g)
KCl (0.5 g)
MgSO 4 .7H 2 O (0.5 g)
FeSO 4 .5H 2 O (trace)
CaCO 3 (2 g)
Agar (15 g)
*Antibiotic solution (1 ml)
Distilled water (1 L)
Autoclave the media in the absence of antibiotic which will be added to the media
(45 oC).

*Antibiotic solution
Nystatin (1 g)
Cycloheximide (Actidone) (1 g)
Polymyxin (0.4 g)
Penicillin (0.016 g)
Distilled water (20 ml)
Sterilize the antibiotic solution by filtration

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 Proteolytic bacteria

Part 3: Total Protein Determination “Folin-Ciocalteau Method”

Introduction

The Folin-Ciocalteu or Lowry reagent contains phosphomolybdic complex under

the proper conditions. In a Folin-Ciocalteu colorimetric assay for protein, colour
development depends on the copper-catalyzed reducing power of tyrosine,
tryptophan, cysteine, and perhaps other residues. An additional colour yield is
derived from the formation of a "biuret complex." In an alkaline solution of cupric
ions, protein-copper complexes show a pink to purple colour. This colorimetric

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reaction is named for the compound biuret, H2NCONHNONH2, which gives the
same reaction. Almost any substance containing two or more peptide bonds will
give the biuret colour reaction. The chromophore, or light absorbing centre, seems
to be a complex between the peptide backbone and cupric ions. (For many years
the biuret reagent, an alkaline solution of a cupric salt chelated with tartrates, was
the standard reagent for protein determination. Although the biuret method is
reliable and linear and responds similarly to different proteins, it now is less
commonly used in biochemistry because of its low sensitivity).

Biochemists use protein Folin assay routinely because this assay is sensitive,
rapid, and more accurate than other assays. In this experiment, Folin assay will be
used to monitor the quantity of protein during the purification of protease.

Materials:

• Protein samples (original supernatant, 60% fraction, 75% fraction for both
endogenous and exogenous enzymes)

• Bovine serum albumin

• Reagent C and Reagent E

• 18X150 mm test tubes

• Micropipettes (100-1000 µl)

• Spectrophotometers

• Non-sterile pipette (5 ml)

Procedures

1. The protein content of the original sonicated supernabtant P 1 , 60%

saturation fraction P 2 and 75% saturation fraction P 3 will determined by
Folin-Ciocalteau method.

2. Prepare 1:10 dilution’s of the three enzyme preparations by removing 0.3

ml of each to 2.7 ml of cold distilled water.

3. Set up the following protocol in clean 18X150 mm test tubes.

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 Tube # Amount (ml) H 2 O (ml)

1 __________ 1.0

2 0.5 (original super.) 0.5

3 1.0 (original super.) ____

4 0.5 ( 60% fraction) 0.5

5 1.0 (60% fraction) ____

6 0.5 (75% fraction) 0.5

7 1.0 (75% fraction) ____

Preparation of Standard Curve of Bovine Serum Albumin (BSA)

Tube # Amount of BSA (ml) H 2 O (ml)

8 0.1 0.9

9 0.2 0.8

10 0.3 0.7

11 0.4 0.6

12 0.5 0.5

13 0.7 0.3

14 1.0 ____

4. To each tube, add 5-ml of reagent C, then mix well immediately with by
using vortex.

5. Incubate the mixture for 10 min at room temperature.

6. Add 0.5-ml of reagent E to each tube, mix well immediately.

7. Allow color to develop for 30 min at room temperature.

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 8. Determine OD at 665 nm, using tube #1 to set the spectrophotometer.

9. Prepare standard dilutions of bovine serum albumin covering the range of

50 to 250 µg/ml of protein.

10. On the graph paper provided, plot standard curve for bovine serum albumin
which is obtained from tubes # 8-14 (amount of protein versus OD), and fit
a straight line to as many points as possible. Extrapolate the protein
concentration of cell hydrolysates.

Determine the concentration of protein content (mg/ml) of the enzyme

preparations from the straight-line portion of the standard curve and
record it in the table below. Since the enzyme assays on all three
fractions will be run with 5-µl preparation, it will be more convenient
to calculate the specific activity (enzyme activity/mg protein) on this
basis. The protein content/5-µl is simply obtained by multiplying the
mg protein/ml by 0.005.

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 Tube
Reading mg protein/sample mg protein/ml µg protein/0.005ml
#

2 /(0.05 ml)

3 /(0.10 ml)

4 /(0.05 ml)

5 /(0.10 ml)

6 /(0.05 ml)

7 /(0.10 ml)

8 0.05/

9 0.10/

10 0.15/

11 0.20/

12 0.25/

13 0.35/

14 0.50/

11. Let a decrease in OD of 0.10 per min=1 unit.

12. Calculate the specific activity of each fraction:

unit / 0.005 ml
specific activity =
mg protein / 0.005 ml

13. Calculate the total activity in each fraction:

units / 0.005 ml x total volume

Total activity =
0.005

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 (Total volume is 10 ml for original sonorate supernatant and 5 ml for the fractions)

14. Calculate the percent recovery:

Total activity in fraction

Percentr e cov ery = 100
Total activity in original

Units/ Mg protein/ Specific Total Percent

Fraction
0.005 ml 0.005 ml activity activity Recovery

Sonorate
Supernatant

Precipitate at
60% saturation

Precipitate at
75% saturation

Specific Total Percent Recovery

Volume Activity Protein content
Treatment activity activity (AxV)/ (A1xV1) x
(ml) (cm) (mg/ ml)
(A/P) (AxV) 100

Original
volume

Precipitate at
60% saturation

Precipitate at
75% saturation

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 Experiment 4

Biosynthesis of Nanoparticles (Nickle and Cobalt)

(3 weeks)

Introduction

Biosynthesis of nanoparticles is the production of nanoparticles by microbes,

Algae, Fungi, Yeast and higher plants. There are lots of nanoparticles produces
by living organisms such as Gold, silver, Manganese nanoparticls…etc.
Biosynthesis of nanoparticles are environmental friendly (eco-friendly) and
completely safe. This biosynthesis approach is close to principles of ‘Nature’.

The aim of this experiment is to enrich and isolate bacteria which produce Co
and Ni nanoparticles.

Materials and Methods

a. Enrichment

• 50ml of growth medium with NiCl 2 .6H 2 O

• 50ml of growth medium with Co(NO 3 ) 2 .6H 2 O
• soil

The students will be provided with the enriched culture

b. Isolation of pure strain which precipitate Nickle and Cobalt nanoparticles

The quantity is determined for each group.

• YEAST EAXTRACT AGAR pH 8.0 (1 Mm NiCl 2 .6H 2 O)- autoclaved

o 250ml pour in plate (at least 4 plates for each group)
o 250ml in bottles for future work, to be used when needed
• YEAST EXTRACT AGAR pH 6.0 (1 mM Co(NO 3 ) 2 .6H 2 O)-autoclaved
o 250ml pour in plate (at least 4 plates for each group)
o 250ml in bottles for future work, to be used when needed

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 Note: Please check the pH should be around 8.0 for NiCl 2 .6H 2 O while pH 6.0
for Co(NO 3 ) 2 .6H 2 O.

• 50ml of 500 mM NiCl 2 .6H 2 O.

• 50ml of 500 mM Co(NO 3 ) 2 .6H 2 O.
• 10ml sterile yeast extract in test tubes (4 for each group)
• 9ml of sterile water in test tubes (10 for each group)
• 1ml sterile pipette
• Gram stain
• Microscopes

Methods

a. Enrichment

11. All soils are air dried in sterile glass Petri dishes at room temperature 3 to
10 days (depending upon moisture content) to aid in reducing the bacterial
population.
12. The dried soils are then gently powdered with a sterile pestle and
“stamped”.
13. Weight 1 g soil and place in 50ml culture medium and incubate in a shaker
350C for 24 hours.

b. Isolation of pure strains

1. Using a sterile pipette, remove 1 ml from suspension, add it to 9 ml sterile

water. Vortex well, remove 1 ml and add it to next tube. Thus make serial
dilutions until you reach 10-4 (Fig.1).
2. Plate 0.1 ml of the suspension of 10-3 and 10-4 using L-shaped sterile glass
rod using a turn table on YE plates with NiCl 2 .6H 2 O and Co(NO 3 ) 2 .6H 2 O
3. Incubate the media and broth for 1-3 days at 35oC.
4. Observe the different types of strains. Take photographs of your plates for
data analysis.

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 10-2 10-3 10-4
Bacterial culture
grown on NiCl2,
1g of soil 0.1 ml
A

Incubate for
1-3 days

Figure 1: Isolation of Nickle nanoparticles producing bacteria

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 10-2 10-3 10-4
Bacterial culture
grown on
Co(NO3)2, 1g of 0.1 ml
soil

Incubate for
1-3 days

Figure 2: Isolation of Cobalt nanoparticles producing bacteria

d. Growing the pure strains (week 2)

4. Select a pure bacterium then inoculate the yeast extract with this
bacterium (maximum 4).
5. Incubate the culture at 350C for 24 h.
6. Examine the bacteria under the microscope after staining the bacteria by
gram stain.
7. Place two drops of bacterial culture on microscopic slide.
8. Add 2 drops of NiCl 2 and mix well with micropipette or inoculums loop
9. Place the coverslip. Seal 2 sides with nail polish.
10. Examine the slide under the microscope to see the precipitation of Ni
nanoparticles.
11. Repeat steps starting with step number 3 using Co(NO 3 ) 2 .

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 Experiment 5

Precipitation of Calcium Carbonate Particles by Bacteria

(BioCementation reaction)

Ureolytic bacteria are used to precipitate calcium carbonate crystals in the presence of
high concentrations of urea and calcium chloride. Ureolytic bacteria hydrolyse urea
producing carbonate and NH 4 + ions causing an increase in the pH. Due to this increase in
pH, the calcium carbonate crystals will be precipitated when reach high saturation level.

Cell + Ca2+ Cell-Ca2+

HCO 3 + NH 3 NH 4 2+ + CO 3 2-

Cell-Ca2+ + CO 3 2- Cell-CaCO 3

Materials and Methods

Chemicals

• 5 M Urea (1L)
• 5 M CaCl 2 .2H 2 O (1L)
• 1.87 M urea (250 ml)
• Nutrient agar (3 per each group)
• Nutrient slant (4 per each group)
• Solution mix

Growth medium

• 100 ml of the growth medium* in 250 ml flask (1 flask for each group)

Tools and equipment

• Microscopic slides and Coverslip

• Lens tissue
• Immersion oil
• Nail polish
• Conductivity meter (1 per each group)

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 • Micropipetters 1000 µl, 200 µl
• 5 ml pippet (1 each group-nonsterile)
• 50 ml test tube (1 each group)
• Tips (sterile)
• Appendrove (sterile)
• Spectrophotometer
• Cuvette (1 ml or 3 ml)

Methods

1. Place 1 ml culture from the enrichment culture in 100 ml growth medium

2. To follow the growth curve of the bacteria, put 1 ml of the culture in the cuvette and
measure the OD at 600 nm, interval of about 1 hour.

3. Measurement of the conductivity as an indicator of urease activity: Add 1ml of the

culture to 4 ml of 1.87 M Urea, mix well then measure the conductivity by measuring the
change in the conductivity within 3 min.

4. Draw the growth curve and the urease activity.

a- Measuring the activity of calcite precipitation bacteria by conductivity

Table 1: OD at 600 and urese activity of the first subculture

Date Time Elapse OD Urease Sp. Urease Note

time (h) activity activity

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b- Precipitation of calcium carbonate particles on microscopic slides

1. Place two drops of bacterial on microscopic slide.

2. Place the coverslip. Seal 2 sides with nailpolish.

3. Add solution mix on one unsealed side and put a piece of tissue to the opposite side to
enhance the diffusion of solution mix.

4. Seal the rest two sides with nail polish and leave the slides.

5. Examine the slide under the microscope to see the calcium carbonate crystals

c- BioGrout technology (Demo)

BioGrout (conversion of lose soil to sandstone of different strength

measuremens) technology will be demonstrated for the students.

Out-flow

Sand filter

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